PROBING THE ACTION OF MAGAININ PEPTIDE IN LARGE UNILAMELLAR VESICLES VIA SITE-DIRECTED SPIN LABELING ELECTRON PARAMAGNETIC RESONANCE TECHNIQUE A Thesis Presented to The Faculty of the Department of Chemistry and Biochemistry California State University, Los Angeles In Partial Fulfillment of the Requirements for the Degree Master of Science in Chemistry By Bayan H Alharbi December 2017 © 2017 Bayan H Alharbi ALL RIGHTS RESERVED ii The thesis of Bayan H Alharbi is approved. Yong Ba , Committee Chair Michael Hayes Yangyang Liu Alison McCurdy, Department Chair California State University, Los Angeles December 2017 iii ABSTRACT Probing the Action of Magainin Peptide in Large Unilamellar Vesicles via Site-Directed Spin Labeling Electron Paramagnetic Resonance Technique By Bayan H Alharbi Magainin is found in the antimicrobial peptides family and has been proven to have antibiotic activities in various living things or organisms. Recent researchers have also confirmed an important magainin II antitumor effects against a wide range of cancerous cells tumor mice models and lines. It is believed that Magainin interacts with the cell membrane which is usually rich in phospholipids acid. The interaction between Magainin and cell membranes makes Magainin form channels of ion-permeable in the cell membrane which kills or denatures the cells that are affected. The peptides of Magainin II exert ant proliferative and cytotoxic efficacy through forming pores in the cancer cells of the bladder which do not affect the normal human fibroblasts or normal murine. Furthermore, Magainin II might provide a strategy of novel therapeutic in the bladder cancer treatment with probably weak effects of cytotoxic on the healthy cells. The objective of this study is to identify the mechanism of microbial cell killing peptides; The hypothesize is that magainin peptides undergo a structural change upon interaction with cell membranes through, a secondary or tertiary alteration that neutralizes acidic amino acids exposed on the cell membrane. The structural dynamics, interaction, and topology of a magainin peptide in a bilayer lipid vesicle will be investigated via spectroscopy of electron paramagnetic resonance (EPR) using a spin that is site-directed labeled (a method for examining the structure and local dynamics of peptides) magainin peptide. iv The dire needs for new antibiotics and anticancer drugs require the understanding of multiple mechanisms of potential antimicrobial and anticancer activities. v ACKNOWLEDGMENTS My thanks go to both my family and Dr. Ba for their guidance, direction, assistance, and assistance. I also would like to thank my Country (Saudi Arabia SCAM) for support me financial during my accommodation in the United States of America. Without forgetting my lab/classmates for their support they accorded to me in my research. vi TABLE OF CONTENTS Abstract .............................................................................................................................. iv Acknowledgments.............................................................................................................. vi List of Tables ..................................................................................................................... ix List of Figures ......................................................................................................................x Chapter 1. Introduction .............................................................................................................1 Introduction .......................................................................................................1 Background Information ....................................................................................4 2. Introduction to EPR spectroscopy ..........................................................................7 Theoretical background of EPR .........................................................................7 Zeeman Effect of Spectroscopy .........................................................................7 Hyperfine interaction .......................................................................................10 Signal intensity.................................................................................................11 Experimental Electron paramagnetic resonance spectroscopy ........................11 Spectrometers ...................................................................................................12 3. Types of AMPs .....................................................................................................13 Introduction .....................................................................................................13 Cecropins .........................................................................................................15 Mellitin.............................................................................................................18 Cecropin-Mellitin Hybrids ..............................................................................19 Magainin ..........................................................................................................23 Interactions of peptide-membrane ...................................................................25 vii Importance of the study of interaction of the peptide ......................................31 Formation of Pores and osmotic stress ............................................................23 Interactions of peptide-membrane ...................................................................25 4. The Models in the AMPs ......................................................................................35 Mechanism of the Membrane Disruption ........................................................35 Detergent-Like “Carpet” Model ......................................................................37 The Barrel-Stave Model ...................................................................................39 Toroidal Pore Model ........................................................................................41 Why the Toroidal Pore Model are the predict model for this research ............45 5. Materials, Methods and Conclusion ....................................................................46 Investigating the structure and_ Local dynamics of proteins using spin labeled AMPs .....................................46 EPR line shape analysis ...................................................................................50 6. Results ................................................................................................................52 7. Discussion .............................................................................................................68 8. Conclusion ...........................................................................................................71 References ..........................................................................................................................73 viii LIST OF TABLES Table 2.1 Microwave Frequencies commonly available in EPR spectrometers .....................9 . . . . . 3.1 Representative of Amino Acid Sequence ..............................................................16 3.2 Cecropin A, Amino Acids Sequence .....................................................................19 3. Results for correlation time ......................................................................................52 ix LIST OF FIGURES Figure 2.1. Two different energy levels .............................................................................15 2.2 Variation of two spin state energy vs magnetic field applied ...............................17 . . . . . 2.3 The impact of magnetic field formed by nucleus ..................................................18 2.4 Main components of the spectrometer ...................................................................20 3.1 Distributions of Non-Polar and Polar Amino acid .................................................31 3.2 D Structure of Magainin antimicrobial peptides ....................................................32 3.3 Association of the Amphipathic.............................................................................34 3.4 Single-cysteine analogs of CM15 ..........................................................................37 3.5 Cysteine Labeling ..................................................................................................37 3.6 Bilayer Depth ........................................................................................................38 3.7 The Circular Dichroism Spectra of CM15 .............................................................39 3.8 Localization of Membrane-bound CM15 .............................................................39 4.1 Models of transmembrane channel formation .......................................................45 4.2 Model of membrane disruption by the carpet mechanism .....................................46 4.3 Detergent-like “carpet” model ...............................................................................47 4.4 The barrel-stave model .........................................................................................49 4.5 Toroidal Pore Model ..............................................................................................51 5.1 MALDI-TOF of protein 2K without radical API ..................................................55 5.2 Graphical representation of the data ......................................................................56 5.3 The structure of DOPC .........................................................................................57 6.1 EPR spectra of magainin with membrane cell at 200C…………………………..60 x 6.2 EPR spectra of magainin with membrane cell at 25 0C .........................................61 6.3 EPR spectra of magainin with membrane cell at 27 0C .........................................62 6.4 EPR spectra of magainin with membrane cell at 29 0C .........................................63 6.5 EPR spectra of magainin with membrane cell at 30 0C .........................................64 6.6 EPR spectra of magainin with membrane cell at 32 0C .........................................65 6.7 EPR spectra of magainin with membrane cell at 35 0C .........................................66 6.8 EPR spectra of magainin with membrane cell at 40 0C .........................................67 6.9 EPR spectra of magainin with membrane cell at 43 0C .........................................68 6.10 EPR spectra of magainin with membrane cell at 46 0C ...................................69 6.11 EPR spectra of magainin with membrane cell at 48 0C ...................................70 6.12 EPR spectra of magainin with membrane cell at 50 0C ...................................71 6.13 Photomicrograph of the Membrane .................................................................72 6.13 Photomicrograph of the Membrane .................................................................72 6.14 Photomicrograph of the Membrane .................................................................72 6.15 Photomicrograph of the Membrane .................................................................73 6.16 Detailed Parameters of the Lipids ....................................................................73 6.17 Photomicrograph of the Membrane .................................................................73 6.18 Photomicrograph of the Membrane .................................................................74 xi CHAPTER 1 Introduction 1.1.Conventional treatment Cancer stills a major mortality and morbidity source worldwide. In the U.S, cancer is a major source of death for most people who have not attained eighty-five years and above (Carmieli et al, 2006). Moreover, the numerous cancers rate that includes cancer of the breast, cancer of the skin cancer of the kidney cancer and cancer of the prostate are in continuous trend. Nevertheless, each kind of cancer is featured by abnormal development of cells that result from a fundamentally environmentally-induced mutation of genes or a modest number of inherited genetic mutation (Milov, et al, 2006). It worth noting that cells must possess six unique characteristics to be characterized as cancerous; 1) the abilities of generating their respond signals of growth of less strong growth which the tissues and responsible for healthy body and life ignore; 2) ant proliferative signs insensitivity; 3) cellular suicide impedance mechanism which usually causes the death of aberrant cells by apoptosis; 4) the limit regarding the replication of the boundless; 5) the capability of fortifying fresh recruits of development vessels that allows for the growth of tumor; 6) the ability of the tissues cells and tissues of the attack, initially at local level, but later metastasize or spread all over the bodies (Sato and Feix, 2006). Nevertheless, the treatment of localized cancerous cells is possible can through radiation therapy, chemotherapy, and surgery. However, chemotherapy is still the typical choice treatment for metastatic or advanced cancer. The development of conventional antibiotics resistance issues has been worldwide public care issue and the demands for advanced antibiotics have accelerated the Antimicrobial peptides (AMPs) development as the chemotherapeutic agents of the human 1 therapeutics convectional which develop to target the cancerous cells have impacted negatively to the healthy cells and are harmful to the cells as well (Carmieli et al, 2006). Because of changes of the cellular including drug transporters’ increased expression and drug detoxifying enzymes, alteration of interaction between the drugs and the substrate, increased abilities to repair DNA defects and damage in the machinery of the cellular which mediate the cancerous cells of apoptosis develop chemotherapy resistance that causes antibiotics deactivation. Antimicrobial peptides (AMPs) therefore, would be a major improvement in the treatment of cancer because they do not have conventional chemotherapeutic agents' toxicity and they do not affect the healthy cells and tissues (Milov, et al, 2006). The antimicrobial peptides (AMPs) are short peptides chains which act as a defensive weapon against the invasion of pathogens and microorganisms. Various living organisms such as insects, bacteria, plants as animals, use Antimicrobial peptides (AMPs) to safeguard themselves against the invasions of the pathogens and microorganisms. In the recent past, studies on anticancer techniques like chemotherapy have been characterized by various harmful and negative side effects (Sato and Feix, 2006). The presently used anticancer drugs pay more attention to the cells that are high proliferated that cause destruction even to the healthy cell which similarly grows at high rates. Furthermore, the origination of the Multi-Drug Resistance (MDR) has threatened the cancerous cells features which hinder the efficiency of the anticancer drug (Gordon-Grossman, et al, 2011). The antimicrobial peptides (AMPs) can result in the formation of the pores on the surface of the cancerous cells lipid membranes and therefore triggering their destructions because the cells are not able to produce the resistance (McMahon, Alfieri, Clark, and Londergan, 2010). The 2 cancerous cells are therefore rendered susceptible to the anticancer drugs as well as the mechanisms of the body immune. Antimicrobial peptides (AMPs) are not the only peptides that have the capacity to penetrate the cell membrane, but other peptides like the Cellpenetrating peptides (CCPs) that are as well protein celled, short compounds of transduction domains that comprise of up to thirty residues of amino acids (AA) that can enter nuclear and mitochondrial membranes as well as plasmalemmal, without damaging the membranes (Carmieli et al, 2006). Cell-penetrating peptides (CCPs) and Antimicrobial peptides (AMPs) consist of various peptide groups that can freely move by the use of advanced mechanisms across the cell membrane. The cell membranes such as cytoplasm membrane or plasma membrane are biological membranes which have dynamic characteristics which separate all the cells’ interior for both eukaryotic and prokaryotic from the external environment (Milov, et al, 2006). The cell membranes have multilayered structures with multiplicities of compounds like molecules, lipids, cholesterols, particles of phosphorous among others. The cell membranes are semi-permeable to certain molecules like ions and other molecules from accessing to the cell interior. 1.2.Background Information AMPs (antimicrobial peptides) are chains of short peptides that function as defensive weapons against the invading microorganisms. Different animals, plants, insects, as well as bacteria, use AMPs to protect themselves from the invasions by microbes (Sato and Feix, 2006). It is known that chemotherapy results in several side effects. The currently applied anticancer drugs focus on the high proliferated cells; these drugs do not spare even the healthy cells that grow at similarly high or even lower rates. Moreover, there has 3 originated another yet threatening character of the cancerous cells; the MDR (Multi Drug Resistance) that hinder the effectiveness of the anticancer drugs. It was recently reported that the antimicrobial peptides can create pores in the lipid membranes of the cancerous cells and hence trigger their destructions since they become unable to form the resistance (Carmieli et al, 2006). The cancer cells are hence rendered susceptible to the body immune mechanism as well as the anticancer drugs. AMPs peptides are not only the ones with the ability to enter the membrane cell, other peptides such as the cell-penetrating peptides (CCPs) can also penetrate the plasmalemmal, as well as mitochondrial and nuclear membranes without causing any damage to the membranes. AMPs and CCPs are part of several peptides groups with the ability to move freely using advanced mechanism across the cell membrane (Milov, et al, 2006). The cell membrane (plasma membrane or cytoplasm membrane) is a biological membrane with a dynamic feature that separates the interior of all cells including both prokaryotic and eukaryotic from outside environment (Gordon-Grossman, et al, 2011). The cell membrane has the multilayered structure with a multiplicity of compounds such as lipids, cholesterol, molecules of phosphorous and many others. The cell membrane is selectively permeable to ions and other materials to the interior of a cell. Cell-penetrating peptide (CPP) refers to the short peptide which is intended to promote cellular uptake or intake of different molecular equipment. The relation between cargo and peptides is established either through chemical linkage via covalent bonds or through non-covalent interactions (McMahon, Alfieri, Clark, and Londergan, 2010). The primary purpose of the cell-penetrating peptides is to pass the cargo to cells, a process that usually happens through endocytosis. Nowadays, the use of cell-penetrating peptides is 4 restricted due to the fact of a lack of knowledge and insufficient understanding of the modes of their uptake. Antimicrobial peptide (AMP) commonly known as host defense peptide (HPD) refers to a part of the innate immune response that can be observed in representatives of all classes of life (Carmieli et al, 2006). Such peptides refer to potent and broad-spectrum antibiotics. Antimicrobial peptides are able to kill enveloped viruses and cancerous cells. AMPs may also have the capability to improve immunity by working as immunemodulators. The antimicrobial peptide is a distinct and unique group of molecules divided into some subgroup (Milov, et al, 2006). The division is based on the structure and composition of their amino acid. Antimicrobial peptide includes around fifteen to fifty amino acids. 5 CHAPTER 2 Introduction to EPR Spectroscopy 1.3.Theoretical background of EPR At the beginning of this century, when. scientists began to use theories of quantum . . . . . . . . mechanics to describe atoms or molecules, they found that the molecules or atoms had . . discontinuous states, each with corresponding energy. Spectroscopy is the measurement is . and interpretation of energy differences between atoms or molecular states. By . . . . . . . . understanding these energy differences, you can gain insight into the identity, structure, . . . . . . and dynamics of the sample being studied. We can measure these energy differences (ΔE) . . . . .. . . . . because there is an important relationship between DE and electromagnetic radiation . . . . absorption. According to the Plank’s law absorption of electromagnetic radiation, E=hv . . . where E is absorbed energy, h is Plank’s constant and v is frequency of radiation. Energy .. . . . . . . absorption causes a shift from a lower energy state to a higher energy state. In conventional . . . . spectroscopy, n is varied or scanned, and the frequency at which absorption occurs . . . . .. corresponds to the energy difference of the states. This record is called spectrum. And . . usually, the frequencies changing megahertz range for nuclear magnetic resonance by . . . . ... . visible light to ultraviolet light. Radiation in the gigahertz range is using for EPR . . . experiments. Figure 2.1: Two different energy levels (source: chem.libretexts.org/ introduction to molecular spectroscopy) 1.4.Zeeman Effect of Spectroscopy The energy difference we study in EPR spectroscopy is mainly due to the interaction of . . . . . .. . . . . . unpaired electrons in the sample with the magnetic field generated by the laboratory . .. . . . . . magnet. This effect is called the Zeeman Effect. Because the electrons have a magnetic . .. . . . 6 . . . . . properties at same instance, it is like a compass in a magnetic field, B0. It will have the . .. \. minimum energy state when the magnetic moment of an electron, μ, is aligned with the . . . . . magnetic field and which has the maximum energy state when μ is aligned against with . . . . . . the magnetic field. Above mentioned two states are signed according to the projection of . . . . . electrons spin, MS and it depends on direction of magnetic field. Because the electron is a . . . spin half particle, the parallel state is developed as Ms = - 1/2 and the anti-parallel state is . . . . .. . . . . M s = + 1/2. . . . According to the contemn mechanics, the basic equation of EPR is shown in equation 1 . . . 1 E = g μB B0 MS = E = hv g μB B0 = ± 2 g μB B0 (1) Where g is the g- factor, it is proportionality constant and this g factor of lot of samples are . . . same and it will change with electron configuration of the radical or ion. μB is Bohr . . . . . magneton it is unit of Electronic magnetic moment. According to the above equation we can say that two spin . . . states have the same energy in the absence of a magnetic field and energy of the spin state . . . . . diverges linearly as the magnetic field increases. Also there are two consequences related . . . . to the spectroscopy, there is no energy different to measure when B0 is zero and the . . . . . . relationship between measured energy difference and magnetic field increases. Because . . . . we can change the energy different between two main spin states using magnetic field . . . . . . strength and we can use constant magnetic field to scan the electromagnetic radiation . . . frequency such as conventional spectroscopy. Every other we can measure the magnetic . . . . field when the electromagnetic radiation frequency is constant and when the magnetic field . . . . . . matches the two spin states, absorption peaks will occur such that their energy difference . . . . matches the energy of the radiation, for this situation is called the field for resonance. This . . . technique is used in all Bruker EPR spectrometers (Poole, C. 1983). . . . 7 The better way identify the compound is the method use g factor it is being freelance from . . . the microwave frequency but the method of field of resonance method is not a good method . . . . to identify the compound because spectra can be acquired at several different frequencies. . . . . . . And keep in mind that high values of g occur in lower magnetic fields and vice versa. . . . Figure 2.2: Variation of two spin state energy vs magnetic field applied (Source: cbc. . . . . . wb01x.chemistry.ohio-state.edu) In the table 2.1 show the field resonance for g=2 signal of microwave frequencies commonly available in EPR spectrometers. Table 2.1: microwave frequencies commonly available in EPR spectrometers. (Source: . . . . . cbc-wb01x.chemistry.ohio-state.edu) Microwave band Frequency (GHz) B_res.(G) L 1.1 392 S 3.0 1070 X 9.75 3480 Q 34.0 12000 W 94.0 34000 8 1.5.Hyperfine interaction Using g factor we can obtain the some useful information about our samples but it does . . . not tell much about it. However the unpaired electrons give us the EPR spectrum and it is very sensitive to the local surroundings. Usually nuclei in the atom (molecule or complex) . . . . have a magnetic moment and it produce magnetic field around the electrons therefore there . . . . . . is an interaction between electrons and nuclei, it is called the hyperfine interaction and it . . . . . . . provide useful information about our sample such as distance between unpaired electrons. . . . . . The magnetic moment of the nucleus function as the bar magnet and produce electron field . . . . . . . (B1) around the electron of the atom. This magnetic field is impacted on the magnetic field . . . . . . formed by laboratory magnet and it may be add or oppose. When B1 add to the laboratory . . . . magnetic field the field for resonance is getting lower (poole, 1983). . . . . Figure 2.3: The impact of magnetic field formed by nucleus (Source: cbcwb01x.chemistry.ohio-state.edu) We will observe single EPR absorption signal for the half nucleus such as hydrogen nucleus and it will split into two signals when impacting the B1. If there if a second nuclei the signal further split into pair and it will prepare four signals. Therefore when the number nucleus getting larger the number of signals exponentially increasing, therefore sometimes these signals are getting overlap and make broad signals. 9 1.6.Signal intensity We have been concerned about the EPR signal, but the EPR signal is important if our . sample requires an active EPR species to measure the concentration. The magnitude of the . . . sign language is defined as the intensity measured in the spectrum. The concentration of . the EPR signal is proportional to the intensity of the EPR signal. Signal intensity alone is . . . . . . . not dependent on the concentration. They depend on microwaving power. If you do not . use a heavy microwave power, the square root of the square root force increases the signal . . strength. At higher energy levels, the signal reduces the signal with higher power levels of . microwave signals. This effect is known Saturation. If you want to accurately measure . current lines, line widths, and closely spaced hyperfine splitting should be avoided by using low energy microwave. A quick way to test saturation is to minimize the strength of the . . microwave power and to ensure that the signal intensity decreases with the square root of . . the power of the micro wave (poole, 1983). . . 1.7.Experimental Electron paramagnetic resonance spectroscopy In the previous section, we discussed the theory of continuous wave EPR spectroscopy. . . . . . The practical aspects of spectroscopy should now be considered. The theory and practical . . . . . of this EPR is always independent. The good example for this is, although the Zeeman . . . . . . . . Effect has been widely known in the visual spectrum over the years, it was necessary to . . . . . obtain EPR's first direct exposure to watch radar recovery during World War II. Moreover, . . . . . are there any necessary components for scientists to make enough sensitive spectrometer? . . . . . . Developing the best allergy techniques like Fourier transform and high frequency . . . . . percussion etc. also true for today. .. . .. 10 . . .. 1.8.Spectrometers Figure 2.4: Main components of the spectrometer (Source: cbc-wb01x.chemistry.ohiostate.edu) The simplest spectrum has three essential elements: electromagnetic radiation, sample and . . . . . . . . detector. To monitor spectroscopic observations, change the electromagnetic radiation frequency . . . . . . and calculate the amount of radiation emitted through samples. Despite the complex nature . of any spectrum, it can still be simplified to a slide. Electromagnetic radiation and detector . . are referred to as the source of the Microwave Bridge (Knowles et al, 1976). The sample is in the microwave cavity, which helps to improve the sample's weak signal. According . . . . . to EPR theory, there is a magnet to tune electromagnetic energy levels. In addition, there . . . . is a console that includes signal processing and control electronics and a computer. It uses . . . . . . . computer to analyze data and coordinate all purchases of a spectrum (Weil et al, 1994). . . . 11 CHAPTER 3 Types of antimicrobial peptides (amps) 1.9.Introduction to types of antimicrobial peptides (AMPS) The continuous rise of multi-drug resistant prevalence to pathogens is a worrying health concern worldwide. Recent studies show that 57 percent of infections of Staphylococcus aureus as for 2003, the intensive care units in the United States showed resistant to various antibiotics. Moreover, studies also indicate that there is an emergence of resistance to vancomycin, antibiotics which have always been seen as the defense of last resort (Sahu, et al, 2014). Therefore, the need to develop advanced antibiotics and drugs cannot be underrated. In the last past year, numerous number of antimicrobial peptide (AMPs) has been recognized a large number of both invertebrates as well as vertebrate specie with the potent antifungal, antibacterial and antiviral properties. The large numbers of antimicrobial peptides (AMPs) are host resistance necessary parts “inmate” that serves as the original antibody to protect and defend the body against infections and pathogens (Gordon-Grossman, et al, 2009). Significantly, antimicrobial peptides (AMPs) are believed to contain action mechanisms that are wholly different from the presently clinically-applied drugs and for this reason, there is a lot of interests in the development of the antimicrobial peptides (AMPs) to treat infections that are drug or antibiotics resistant. Antimicrobial peptides (AMPs) may be broadly categorized based on secondary composition and structure. Antimicrobial peptides (AMPs) that are synthesized ribosomal and contain one naturally occurring amino acids can be classified into linear, α-helical peptides for example mellitin, cecropins and magainins, Antimicrobial peptides (AMPs) 12 such as indolicidin, and proline arginine-rich PR39 which are characterized by one or two amino acids enrichments, as well as Antimicrobial peptides (AMPs) that have disulfide bonds such as protegrins and defensins (Butterfield and Lashuel, 2010). There a numerous number of Antimicrobial peptides (AMPs) that have potent antimicrobial activities which contain modifications of post-translation or are extra-ribosomally synthesized such as the lantibiotics and the lipopeptides (dermaseptin, polymyxin) that have structures of nonpeptide backbone and non-native amino acid. Additionally, a wide variety of synthetically modified Antimicrobial peptides (AMPs) have been designed through the modification of the existing Antimicrobial peptides (AMPs) or through the utilization of the combinatorial synthesis techniques (Guaní-Guerra, et al, 2010). Such Antimicrobial peptides (AMPs) include; cyclic peptides that contain a combination of L- as well as D-amino acids, KSL, a potent fungicidal-bactericidal decapeptide, and the ornithine-lipopeptide based MSI-843. Antimicrobial peptides (AMPs) belonging to prokaryotic cell selectivity are on the basis of the recognition of general characteristics of the membranes (Peters, Shirtliff, and Jabra-Rizk, 2010). The explanation has got the support of a number evidence that includes the Antimicrobial peptides (AMPs) sequence diversities as well as the fact all D-amino acids analogs that are synthetically modified usually retain their complete functional activities. Antimicrobial peptides (AMPs) bond strongly to the bilayers of lipids and the bonds are bettered by increasing the numbers of the anionic lipids. Antimicrobial peptides (AMPs) dissipate the ionic gradients of transmembrane as it secretes solutes that entrapped from the liposomes (Hoskin and Ramamoorthy, 2008). The cell membranes of Eukaryotic have a lot of positive potential of the surface than the cell membranes of the prokaryotic that offers at least specificity partial explanation and promote the interactions of 13 electrostatic with the positively charged peptides (Hoskin and Ramamoorthy, 2008). Even though researches have indicated that certain Antimicrobial peptides (AMPs) interact with the targets of the intracellular, the Antimicrobial peptides (AMPs) still need to traverse the cell membranes to get into active sites of actions. Subsequently, the comprehension of the membrane-peptide interactions is necessary for enhanced development and design of the Antimicrobial peptides (AMPs) drugs (Sahu, et al, 2014). In this literature review, the researcher intends to put into considerations the peptide-membrane interactions, the cell membranes bound structures, the action mechanisms for amphipathic α-helical and linear category of Antimicrobial peptides (AMPs), with a specific focus on the category of peptides which are synthetically hybrids of mellitin and cecropins. 1.10. Cecropins Cecropin refers to the original or initial Antimicrobial peptides (AMPs) that were the first to be identified (Gordon-Grossman, et al, 2009). Originally, cecropins were extracted from the silk moth scientifically known as Hyalophora cecropia, the analogs of the cecropin have currently been obtained from various species of insects as well as the version of a mammalian or porcine has also been isolated. Cecropins have thirty-one to thirty-nine amino acids as a family of Antimicrobial peptides (AMPs) with a hydrophobic domain of C-terminal, the domain of C-terminal, an amphipathic as well as the domain of basic N-terminal as illustrated in table 1 (Butterfield and Lashuel, 2010). Wide varieties of the naturally-occurring synthetic analogs of cecropins have been applied in elucidating activity-sequence relationships. 14 Table 3.1: Representative sequence of amino acids of naturally-occurring cecropins Cecropin A: KWKLFKIEK10VGQNIRDGIIKAGP24AVAVVGQATQIAK37CONH2 Cecropin B: KWKLFKIEK10VGQNIRDGIIKAGP24AIAVLGEAKAL35 CONH2 Cecropin D: WNPFKELEK9VGQRVRDAVISAGP23AVATVAQATALAK38 CONH2 Cecropin B1: RWKIFKKIEK10 MGRNIRDGIVKAGP24AIEVLGSAKAI35 CONH2 Cecropin B2: WNPFKELER9AGQRVRDAVTSAAP23AVATVGQAAIAR38 CONH2 Cecropin C: GW-LKKLGKR9IERIGQHTRDATIQGLG28IAQQAANVAATAR39 CONH2 Cecropin P1 SW-LSKTAKK9LENSAKKRISEG21IAIAIQGGR31 CONH2 The cecropins A, cecropins B and cecropins D are isolated in the Hyalophora species, cecropin B1 is found in Bombyx mori, cecropin B2 is found in Manduca sexta, cecropin C is found in melanogaster while cecropin P1 is from in the pigs' intestinal mucosa (Guaní-Guerra, et al, 2010). The domain of amphipathic and cationic N-terminal is indicated in italics, and the predominant domains of hydrophobic C-terminal are underlined. The proline residues substitution in the domain of the N-terminal critically reduces the activities that indicate the amphipathic α-helix importance (Peters, Shirtliff, and JabraRizk, (2010). Aromatic residues conserved at position two are necessary for the complete Antimicrobial peptides (AMPs) activities, with the analogs that contain the phenylalanine being less active as compared to those that contain the native tryptophan. Researches applying constructs of the modular have shown that a helix of the amphipathic N-terminal general organization linked to the terminal helix of C-terminal of the hydrophobic by a region of flexible hinges is needed for wide-spectrum and strong activities of the 15 antimicrobial (Peters, Shirtliff, and Jabra-Rizk, 2010). These researches resulted in the construction of chimeric cecropin A-cecropin D synthesis with improved activities. Cecropins have wide activities of the Antimicrobial peptides (AMPs) activities against fundamentally certain Gram-positive bacteria and basically every Gram-negative bacterium. However, Cecropins are essentially not active on the Staphylococcus aureus. Moreover, they indicate positive activities on the protozoa parasites, the malarial parasites Plasmodium falciparum, Leishmania, and Candida albicans yeasts (Sahu, et al, 2014). Engineering of higher transgenic plants in expressing cecropins has taken place to improve resistances on the fungal as well as bacterial phytopathogen. Cecropin has also indicated vitro specific activities against various lines of the cell. Cecropins have shown essentially weak hemolytic activities and shown less toxicity towards eukaryotic normal cells (Gordon-Grossman, et al, 2009). In bacterial sepsis of Gram-negative in the rat, cecropin B treatment alone or combined with β-lactams crucially decreases the endotoxin, septic shock measure, reduced load of infection, the tumor necrosis plasma levels factor-α (TNFα) and enhance survival. The studies of circular dichroism (CD) reveal that in aqueous solution, cecropins are not structured (Guaní-Guerra, et al, 2010). They, however, create a high α-helical secondary structure percentage in the (HFIP) hexafluoroisopropanol presence; lipopolysaccharide vessels (LPS); liposomes or micelles of SDS. All the synthetic D enantiomers that form helices that are left-handed with the CD spectra of the image of the mirror have been found to have similar range and level of activities of the antibacterial and to be resistant to protease as the L-enantiomers that are naturally occurring (Hoskin and Ramamoorthy, 2008). The studies of solution NMR cecropin A in fifteen percent (15%) 16 revealed that the presence of two helices that are well defined contains residues of 5-21 and residues of 24-37 that corresponds to the domains of C-terminal and the cationic Nterminal respectively. The orientation of the two helices occurred in the presence of hinge of a Gly23–Pro24 hinge at an angle between 700 and 1000. Cecropin P1 solution-phase NMR in thirty percent (30%) also revealed helical secondary structure adoption and in this scenario, the spanning of a single α-helix being almost the peptides’ full length. 1.11. Mellitin Mellitin contains a distinct domain of the hydrophobic and hydrophilic in a reverse sequence as compared to the cecropins. It is found in the honey bee venom scientifically known as the Apis mellifera, and it is 26-residues peptides with four positively charged or cationic amino acids that are sequestered next to the C-terminus as shown in table 2.2. Mellitin shows broad-spectrum and strong antimicrobial activities despite being highly hemolytic in nature. Mellitin attains an equilibrium or state of balance of concentrationdependent between a predominant α-helical tetramer and random-coil monomer in an aqueous solution state. Various researches show that in the bilayers presence, mellitin forms α–helices of C-terminal and N-terminal in the separation of flexible hinges. At high ratios of lipid to the peptide, for example, >200:1, Melletins which are membrane-bounded shall forms monomeric to the surface of the membrane with its parallel helical axis, with the exposure of every lysine residues to the phase that is aqueous (Hoskin and Ramamoorthy, 2008). With the increase of the membrane-bound mellitin concentration, the peptide has the tendency to undergo relative reorientation of the dynamics to the cell membranes with ultimately generates a full membrane bilayer destruction or micellization. 17 Table 3.2: Cecropin A, Amino Acids sequence 1.12. Cecropin-mellitin hybrids Merrifield, Bowman, and co-workers evaluated and synthesized the activities of a large group of antimicrobial peptides to be able to cut the full-length size of cecropin to facilitate the synthesis of their solid-phases which were hybrids of mellitin and cecropin A (Sahu, et al, 2014). The development of chimeric peptides was designed through the combination of the various hydrophobic domain of mellitin with the hydrophilic domain of cecropin. Original researches carried out an examination of peptides that contained about eighteen and thirty-seven amino acids, with cationic charges of +5 to +7. Most of the initial studies carried out indicated that antibacterial activities comparable to full-length of cecropins, which did not have the hemolytic traits connected with mellitin (GordonGrossman, et al, 2009). In contrast, and interestingly to the cecropins full-length, various hybrids showed positive antibacterial activities against Gram-positive living things, which includes Staphylococcus aureus. Such researches were followed by more studies that further reduced the size as it identified various active sites that only had fifteen amino acids. The reduced peptide each contained the initial seven to eight cecropin A’s residues that had varieties of linkage of mainly hydrophobic amino acids next to the mellitin N-terminus (Butterfield and Lashuel, 2010). The concentrations of the lethal against the bacteria panel 18 had a range of 0.1 to 15μM, while the concentration of the hemolytic was greater than 300 μM. Scholars such Hancock and his co-workers have done a lot of research regarding a hybrid of cecropin-mellitin (CM) that consisted of initial eight cecropin A’s residues and the original eighteen mellitin residues, C(1–8)M(1–18) that they designated to CEME (Guaní-Guerra, et al, 2010). The designated CEME showed traits of strong activities of the both Gram-negative bacteria as well as Gram-positive bacteria. CEME has improved the permeability of the cytoplasmic membrane as well as demonstrating lipopolysaccharide (LPS) high affinities. An analog and CEME with two extra positively charged ions next to the inhibition of the C-terminus has induced lipopolysaccharide (LPS) as well as the production of TNF-α by the isolation of macrophages which enhanced the survival in acute septic shock murine model (Peters, Shirtliff, and Jabra-Rizk, 2010). These peptides also combine with the Gram-positive bacteria cell-wall components and lipoteichoic which ensured reduction of a number of effects of physiology such as the production of cytokine and TNF-α associated to the Gram-positive sepsis. Moreover, a much smaller cecropin-mellitin (CM) hybrid that consisted of the two to nine residues of mellitin, and the initial 7 cecropin residues, C(1-7)M(2-9) that was designated to CM15 has also gotten a special attention and focus because of its widespectrum efficacy of the antimicrobial. Both CEME and CM15 have been proved to fight against the protozoa parasites known as Leishmania (Sahu, et al, 2014). The activities of CM15 against the protozoa parasites have improved the substitution of N-terminal with up to twelve carbons fatty acids. However, ε-amino group of the Lysine 7 acylation crucially reduced both the activities of leishmanicidal and bactericidal. CM15-N-octanoyl derivative 19 has been proven to be effective and safe in treating the canine leshmaniasis that are naturally-acquired. Just like cecropin A, each and every CM-15-D enantiomer maintains complete biological activities against wide bacterial species panels, showing that cellular interactions targets take place in the non-stereospecific way (Gordon-Grossman, et al, 2009). More interestingly, the sequences of all-D reverse commonly known as “retroenantio” and retro reverse remained active against certain strains of bacteria such as Streptococcus pyogenes, Bacillus subtilis, and coli, but they remained inactive against Pseudomonas aeruginosa or S. aureus (Butterfield and Lashuel, 2010). Therefore, it consistently appeared that chirality was never an aspect in the determination of the efficacy of the peptide; even in certain scenarios, it can be significant to put into considerations the amide bonds direction and sequence order. AS their parent peptides, cecropin-mellitin (CM) hybrids belong to the group of amphipathic α-helical, linear classification of Antimicrobial peptides (AMPs) (GuaníGuerra, et al, 2010). In solution, cecropin-mellitin (CM) hybrids exist in a configuration of a random coil, and can only adopt in the presence helix-promoting or membranes organic solvents their secondary structures of α-helical. In thirty percent of HFIP , the solution of NMR-26-residue cecropin-mellitin (CM) hybrids, C (1–13) M (1–13), showed the two α-helices formation in separation by a flexible connector, resembling the motif structure found in the cecropin A. A predominant NMR structure of α-helical solution has also been revealed in fifty percent of HFIP of C (1–8) M (1–12). Cecropin-mellitin (CM) hybrid is highly amphipathic upon folding, with almost similar distributions of non-polar and polar amino acids as indicated in figure 2 (Peters, 20 Shirtliff, and Jabra-Rizk, 2010). Cecropin-mellitin (CM) hybrid maintains the full-length cecropins domain structure, with a predominant C-terminal domain of hydrophobic and strong positively charged N-terminal domains. Wide varieties of cecropin-mellitin (CM) hybrids also have glycine that is centrally-located, resembling the gly-pro hinges which have been indicated to be functionally significant in the full-length of the cecropin. Nevertheless, the centrally-located glycine appears not to be needed for the activities of antimicrobial in the cecropin-mellitin (CM) hybrids because of its absence in a wide variety of effective analogs. For sure, the 15-residue C (1–7) M (3–10) domain, shows importantly reduced activities of the Antimicrobial peptides (AMPs) especially when isoleucine eight is substituted by glycine (Hoskin and Ramamoorthy, 2008). Moreover, the replacement of Ile8 with serine also greatly diminished the activities of the Antimicrobial peptides (AMPs) that suggest the hydrophobic amino acids requirements at the position. Ile8 would bury deep in the bilayer in case it is folded in α-helix of a membrane-bound and therefore the replacement of polar amino acids at these sites can prevent the insertion of the cell membrane (Butterfield and Lashuel, 2010). Reducing the CM15 length by a single residue to provide the 14-mer, C (1–7) M (2–8), diminished the activities of Antimicrobial peptides (AMPs) which add extra residues and providing C (1–7) M (2–10), with slightly improved activities. 21 Figure3.1: Distributions of Non-Polar and Polar Amino Acids 1.3 Magainin belongs to alpha-helical anticancer peptides that act through self-association within a membrane rather than through interaction with a chiral center (Gordon-Grossman, et al, 2011). Furthermore, the α-amino acid enantiomers are not sensitive to proteolytic cleavage a property that has been suggested to be favorable for the development of these peptides as therapeutic agents. It was suggested that Magainin forming toroidal pores that have a diameter or length of 2–3 nm that includes peptides’ translocation and induction of lipid flip-flop into the bilayer inner leaflet coupled by permeabilization of the membrane. However, the interactions of AMPs with bacterial membranes are not well-characterized (McMahon, Alfieri, Clark, and Londergan, 2010). For instance, membrane potential assays using bacterial cells reveal the kinetics of the permeabilization but do not give information on pore size, which is essential for discriminating between various proposed models of membrane permeabilization. Electron microscopic observations failed to capture the dynamic process of membrane permeabilization. However, this process can be improved by using electroporation where temporary pores are introduced by the assistance of high external direct current (DC) electrical field or the use of exponentially decaying pulses 22 (Carmieli et al, 2006). When this is done, the dynamic process of membrane permeabilization can be observed. Figure 3.2: D Structure of Magainin antimicrobial peptides (AMP). AMPs and CPPS are very similar. However, there are a few differences that distinguish between them. They have comparable characteristic both are made of short amino acid chains with the ability to pandurate the cell membrane using a different mechanism, both are cationic in nature (Chongsiriwatana, et al, 2008). These peptides have drawn much attention due to their ability to cross cell membrane with low toxicity making the perfect to detect a new therapeutic technique and for understanding the transportation materials inside and outside the cell through the cell membrane.They distinguish between CPPs and AMPs. The first difference is the fact that CPPs transport various macromolecules across the cell membrane while the AMPs fight invading microorganisms. Second, for CPPs to access inside the cell it used endocytosis pathway, but AMPs depends totally on their self-induced action to pandurate the cell membrane (Dürr, Sudheendra, and Ramamoorthy, 2006). Third, by considering the field of research they are useful in. CPPs 23 have mostly used for gene therapies and cellular drugs delivery methods. While the AMPs are useful in research a new way to control bacteria cell. AMPs are also known as multifunctional molecules because they stimulate a myriad of activities in the cells and can be applied in various useful activities in the human life. 1.13. Interactions of peptide-membrane Irrespective of the extensive studies and researches, the precise mechanisms of interactions of peptide-membrane as well as the denaturing of the cells are yet to be firmly established for most of the Antimicrobial peptides (AMPs) (Sahu, et al, 2014). There is a general agreement that Antimicrobial peptides (AMPs) would undergo self-promotion uptakes upon addition to cell suspensions of bacteria to go through the cell wall or the cell membrane which is followed by the cytoplasmic membrane disruption as the lethal events that lead to the denaturing of the cell bacteria. There is a proposal that the original binding of the outer membranes to the Antimicrobial peptides (AMPs) replaces divalent positively charged ions that destabilizes the envelope and results in the consequent peptide uptake (Gordon-Grossman, et al, 2009). Various researches have indicated that the cecropinmellitin (CM) hybrids and magainins disrupt the permeability of outer membranes barriers. Even though the mechanisms of the molecules which are involved in going through the outer cell membrane still remains poorly described at the level of the molecule, many types of research normally pay much attention to the events linked to the cytoplasmic membrane disruption and binding (Butterfield and Lashuel, 2010). This may take place through discrete pores formation, or detergent-like “carpet” mechanism which dissipates the gradient of the ions. 24 It can be assumed that the original connections of a α-helical Antimicrobial peptides (AMPs) with the bilayers of the lipids might take place in any of the three fundamental orientations that include: at an angle of oblique, along the normal membrane, or parallel to the surface of the membrane as illustrated in the figure 3.3. Figure 3.3: Association of the Amphipathic There enough literature reviews for all the three general orientations. Antimicrobial peptides (AMPs) formed on model peptides that have entirely phenylalanine and lysine as well as C-kinase substrate and the protein kinase C of myristoylated alanine-rich bind parallel to the surface of the membrane with a different penetration depth that depends on the overall hydrophobicity (Sahu, et al, 2014). Antimicrobial peptides (AMPs) developed o the model of helices of the transmembrane, the integral membrane proteins of the transmembrane helices plus some peptide ionophores like alamethicin align themselves less or more vertically along the bilayer of the membrane, while the sequences of the membrane-insertion of the viral fusion of the peptides and the SNARE proteins combine and therefore integrating into bilayers at an angle of oblique. In solution, both Cecropin-mellitin (CM) hybrids and cecropins of full length appear to be monomeric (Gordon-Grossman, et al, 2009). No evidence of aggregation is found using cecropin AD spin labeled analog in either solution of low ionic strengths or physiological solutions of low ionic strengths up to 200 μM of peptide concentration. Even though the cecropin AD aggregation was seen on adding HFIP low concentration of about 25 5% to 10%, on titrating with higher concentration of HFIP, the folded monomer was reverted by peptide consistent with the domain where formation secondary structure and binding of the peptide are concerted in the processes (Butterfield and Lashuel, 2010). The studies of sedimentation balance show that in solution, cecropin A is also monomeric. Therefore, the original events in the interactions of the peptide-membrane are the monomeric peptide associations with large and excess phospholipids. This is a scenario which may readily design by use of bilayers of the artificial membranes. In solid-states, the cecropin A, NMR of full length that are selectively labeled as 15n at either Ala27 or Val11, and integrated into the bilayers of the oriented planers, showed that the bonds of amide at both the 2 ends were paralleled oriented to surface of the membrane, which inferred that the two helical domains of the C-terminal and the N-terminal lie paralleled to the bilayer the membrane surface (Peters, Shirtliff, and Jabra-Rizk, 2010). Consistent with such outcomes, Axelson as well as Silvestro applied the spectroscopy of internal reflection Fourier-transform infrared (FTIR) in demonstrating that on original interactions between the membrane and the cecropin A, the cecropin A would adopt secondary structures which were basically α-helical, with helical axis longitudinally and preferentially orientation and parallel to the surface of the membrane (Guaní-Guerra, et al, 2010). The Antimicrobial peptides (AMPs) would then expand the surface of the membrane on insertion without altering the orientation and structure of the membrane surface. The studies of the Attenuated total reflection (ATR)-FTIR reveal that upon original binding to the phosphatidylglycerol (PG) and the phosphatidylethanolamine PE membranes which mimic Gram-negative bacteria inner membranes of, the cecropins P1 would orient almost similarly paralleled to the surface of the membrane (Sahu, et al, 2014). Moreover, various 26 researches have revealed that the magainin family members of the Antimicrobial peptides (AMPs) originally bind to the surface of the membrane through their long helical axis that is paralleled to the surface of the bilayer. The researcher has applied the site-directed spin labeling (SDSL) in characterizing the CM15 original interactions with the liposomes which mimic inner membranes of the bacteria. In the site-directed spin labeling (SDSL) models, Antimicrobial peptides (AMPs) are expressed or synthesized with one residue of the cysteine at basically possible locations or any given desirable location as illustrated in figure 4 (Gordon-Grossman, et al, 2009). The residue of cysteine is then modified covalently with nitroxide spin label of sulfhydryl-specific as shown in figure 5, that allows for the study of the dynamics and structure of the peptides through spectroscopy of electron paramagnetic resonance (EPR) (Peters, Shirtliff, and Jabra-Rizk, (2010). One of the strength of the site-directed spin labeling (SDSL) models as compared to the mechanisms of molecular propping is the fact that the side that is labeled in the chain is essentially small and almost the tryptophan molecular volume as shown in figure 5 in order not to significantly perturb the peptides’ physical characteristics and traits (Butterfield and Lashuel, 2010). Furthermore, electron paramagnetic resonance (EPR) spectroscopy is not affected by properties of the optics of the samples to enable a person to carry out researches across a broad range of concentrations of the lipids without being concerned with the effects of inner filter or scattering of light (Guaní-Guerra, et al, 2010). The site-directed spin labeling (SDSL) models offers accessibility to two specific significant parameters that include accessibility of the nitroxide and intermolecular interactions as well as the labeled side chain of the rational dynamics that can applied in determining the labeled sites’ depth for reviews of the site-directed spin labeling (SDSL) models, applications and methods. 27 Figure 3.4: Single-cysteine analogs of CM15 Figure 3.5: Cysteine Labeling In characterizing the localization and the structure of the CM15-bound membrane, the researcher conducted a study of “nitroxide-scanning” by use of several of the analogs of a single-cysteine as indicated in figure 4 (Sahu, et al, 2014). The Antimicrobial peptides (AMPs) mixed-up with larger unilamellar vesicles (LUVs) with the composition of inner membranes of lipids of E. coli after being spin-labeled at high peptide to lipid ratio modeled to mimic initial conditions of peptide bindings. The result of the electron paramagnetic resonance (EPR) spectroscopy and spectra showed that under the special conditions, the Antimicrobial peptides (AMPs) would adopt a completely bound-membrane as well as a folded conformation (Gordon-Grossman, et al, 2009). The depth of the bilayer was 28 investigated for every analog of spin-labeled that gave the sequence-linked trend indicated in figure 6. As can be recognized, the data of the experiment matched well to an ideal αhelix which has 3.6 turns or residues. This agrees with circular dichroism (CD) results that show in the presence of liposomes CM15 is fundamentally one-hundred percent α-helical as shown in figure 7 (Butterfield and Lashuel, 2010). Additionally, the data in figure 6 indicate that each and every residue along the peptide hydrophobic face that is F5, I8 and L12 residues are about the same depth of approximately 12.5 Å below the surface of the bilayer, while each and every side of chains parallel to the face of hydrophilic lie approximately 2.5 Å over the membrane of the cell (Hoskin and Ramamoorthy, 2008). Such trend and patterns show that on the original binding, CM15 would intercalate paralleled to the membrane surface, with its core helical axis at a depth of ∼5 Å as showed in in figure 8. The localization is well suitable in burying the chains of the polar sides in the membrane's hydrophobic core, as it positions the residues of lysine to have the ability to interact with lipid phosphates (Guaní-Guerra, et al, 2010). Significantly, fundamentally the peptides’ entire volume need to be accommodated by the membranes that might be a significant aspect encouraging destabilization a well as eventual bilayer disruption. Figure 3.6: Bilayer Depth 29 Figure 3.7: The Circular Dichroism Spectra of CM15 Figure 3.8: Localization of Membrane-bound CM15 1.14. Importance of the study of interaction of the peptide The study of peptide-membrane interactions is important for comprehending a broad range of essential molecular functions, including the antibiotic peptide actions, the incorporation of the proteins involved in fusion of the membrane as well as signaling of the cells (Sahu, et al, 2014). This review discusses the applications of site-directed spins labeling in investigating the interactions of peptide-membrane. Site-directed spin labeling involves the incorporation of a spin label in a site-specific fashion into a macromolecule, such as a protein or a peptide, either through synthesis or site-directed mutagenesis. Electron paramagnetic resonance (EPR) spectroscopy is then used to determine molecular 30 structure and dynamics. A number of examples discussed in this review demonstrate how this methodology can be used to determine the conformation of membrane-associated peptides, their position along the bilayer normal, and their state of aggregation (GordonGrossman, et al, 2009). Recent studies have shown that this approach is not highly perturbing, and with new advances in EPR resonator design, it is a highly sensitive technique. A lot of interests have been developed towards Antimicrobial peptides (AMPs) as new antibiotics source with a possibility of treating a wide variety of drug-resistant infections. A significant group of the Antimicrobial peptides (AMPs) is cationic and linear peptides which form α-helices of amphipathic (Butterfield and Lashuel, 2010). Among the most potent of the Antimicrobial peptides (AMPs) include synthetic peptides and cecropins which are bee venom peptides, mellitin and hybrids of cecropins. Both the cecropinmellitin (CM) hybrids and the cecropins themselves exist as unstructured monomers in the solution form and fold into predominant structures of theα-helical on connecting with the membrane using its long helical axis to the surface of the bilayer (Guaní-Guerra, et al, 2010). Researches applying model membrane have indicated that such peptide intercalates into bilayer of the lipid just beneath the phospholipid level of the backbone glycerol which needs the outer leaflets expansion of the bilayers, and results from various techniques and models of experiments shows that thinning and expansion of the bilayers are common features at the initial phases of the interaction of the Antimicrobial peptides (AMPs) (Peters, Shirtliff, and Jabra-Rizk, (2010). Subsequent membrane permeability disruptions of the barrier may take place by various mechanisms that lead to ultimate loss of the integrity of the cytoplasmic membrane and eventually the death of the cell. 31 1.15. Formation of Pores and osmotic stress Irrespective of the precise structure of the channel, upon the formation of the transient pore across the membrane, the osmotic pressure or gradient shall induce the swelling of the cell which facilitates the lipid bilayer thinning (Sahu, et al, 2014). Melittininduced lysis was improved by the hypo-osmotic conditions although the peptide binding was never affected. Funnily, in the vesicles of the lipids, cecropin A low concentration had the ability of dissipating the ion gradients through the cell membrane, while a high concentration of peptides were needed in releasing a marker of encapsulation fluorescent that indicates that the membrane mechanisms disruptions might with ratio of peptide to lipid (Gordon-Grossman, et al, 2009). Low cecropin A concentrations dissipate the potentials of the inner membranes and denature E. coli cell, while high cecropin A concentration is needed in releasing the β-galactosidase. Moreover, magainin 2a, as well as its analog, induces rapid intracellular potassium release at the concentrations of the peptide which are cytotoxic, without intracellular β-galactosidase release (Butterfield and Lashuel, 2010). The data indicate that before the frank cytolysis, the formation of the channel by the Antimicrobial peptides (AMPs) disrupt the potential of the membrane as well as the gradients of pH. This shall, in turn, uncouple the synthesis as well as produce osmotic shocks. Osmotic swellings may enlarge the surface area of the cell by five percent to twenty-five percent and enlarge chain disorder of lipid alkyl that can encourage the interaction of the peptides with the bilayer of the hydrophobic core (Guaní-Guerra, et al, 2010). This could be specifically significant for the small head group phospholipids such as PE and many investigations and experiments have revealed that the composition of the 32 membrane lipid has a significant impact on the interactions of Antimicrobial peptides (AMPs) which extends beyond effects of simple electrostatic (Peters, Shirtliff, and JabraRizk, 2010). Such transformations in the membrane physical properties could also improve the peptides binding that create disruptive cycles which ultimately results to lysis of the cell. It worth noting that, since the cell wall rigidity, the Gram-positive bacterium has the ability to withstand turgor pressures three to twenty-five times greater than the ones Gramnegative bacteria tolerate (Hoskin and Ramamoorthy, 2008). The Gram-positive bacterium is basically much resistant to the Antimicrobial peptide (AMP). 33 CHAPTER 4 The models in the antimicrobial peptides (amps) 1.16. Mechanism of disruption of the Membrane Intense studies have been carried out the mechanisms and models of membrane disruptions by the Antimicrobial peptides (AMPs) (Lai and Gallo, 2009). Generally, the disruption of the membrane is believed to take place either through the barrel-stave model, detergent-like carpet mechanism, discrete pores formations, non-lamellar lipids stages inductions, or toroidal pore model. There is good experimental evidence for all the models, mechanisms and processes (Li, et al, 2012). It is worth noting that various peptides utilize various models, processes or mechanisms to finally disrupt the membrane of the microbial. These processes should not also be exclusively mutual; a single mechanism may be a representation of an original or intermediate phase while the other process may represent the consequences of the model. More aspects like the ratio of lipid to the peptide as well as the composition of the target can also be involved. Intense researches on disruptions of the membrane by the Antimicrobial peptides (AMPs) have used model membranes such as micelles, lipid monolayers, liposomes among others in simulating the bacteria inner membranes that as explained above is a critical Antimicrobial peptide (AMPs) target (Schauber and Gallo, 2008). The utilization of the model or mechanism membranes gives room for experimental manipulation as well as rigorous control of the composition of the membrane and the ratio of the lipid to the peptide. On the Antimicrobial peptides (AMPs) addition to the artificial membranes, the monomers of the Antimicrobial peptides (AMPs) bind to the bilayers of phospholipid outer 34 leaflet as the initial stage in the membrane lysis processes. As explained before, in a large excess presence of lipids, that is to say, a low ratio between the peptide and the lipid, the state of the original binding of many α-helical Antimicrobial peptides (AMPs) is normally paralleled to the surface of the lipid bilayers (Xiao, et al, 2013). For instance, magainin, cecropin P1, cecropin A, and CM15 all form their original linkage with the membranes in the same fashion. Suggestions have been made that such bindings induce positive curvature of the membrane that increases the outer leaflet surface area, therefore decreasing the hydrophobic core thickness as well as the lipid bilayer thinning (Costa, et al, 2011). A magainin analog, NMR research, MSI-78, shown that the binding of the peptide to the surface of the bilayer creates phospholipid acyl chains disorders that can be either a reflection or cause of the thinning of the bilayer. Even though the original amphipathic α-helical Antimicrobial peptides (AMPs) associations with the surface of the membrane increase the surface areas and the volumes of the outer leaflet, there is little effect on the inner leaflet and such misfit can lead to the aggregation or insertion of the peptides. Therefore, changes in the lipid bilayer physical properties and traits introduced through the binding of the peptides appear to be facilitating more Antimicrobial peptides (AMPs) penetrations that may, in turn, result in the transmembrane pores formation (Melo, Ferre, and Castanho, 2009). A model of pore formation of the activity of the Antimicrobial peptides (AMPs) was originally designed by Mueller and Baumann on the basis of their researches of alamethicin. Huang et al have subsequently designed a two-state technique for the formation of the membrane pore through the Antimicrobial peptides (AMPs) of the α-helical. In the Huang’s model, the Antimicrobial peptides (AMPs) originally linked parallel to the surface of the bilayer; on 35 reaching a crucial threshold by the concentration of the peptides, there was reorientation and penetration or insertion of the membrane-bound Antimicrobial peptides (AMPs) into the bilayer of the hydrophobic core with the transmembrane pores formation. Evidence for these general mechanisms have originated mainly from researches which utilize X-ray scattering, neutron as well as oriented CD (Lai and Gallo, 2009). The mechanisms of the pore formations are also reinforced by the researches indicating the differential solutes leakage of various quantities as well as the patch-clamp experiments that demonstrated the ion channels formation. Figure 4.1: Models of transmembrane channel formation 1.17. Detergent-like “carpet” model Carpet mechanisms are a conceptually outstanding technique in the manner in which the Antimicrobial peptides (AMPs) disturbs the cell membrane as illustrated in figure 9 (Li, et al, 2012). In the Detergent-like “carpet” model, the peptides accumulate at the surface of the bilayer in a way resembling a carpet and more than the monomers threshold concentrations, the membranes are disintegrated and permeated in a manner resembling detergent without discrete channels formation (Schauber and Gallo, 2008). The 36 Detergent-like “carpet” model was originally designed by Steiner and his co-workers basing their observations on the concentrations required acquiring fifty percent cell denaturing, with sufficient availability of cecropin A in the right amount to fully cover the surface of the bacterial cell. Subsequent researches about porcine of the cecropin P1 were evaluated too in carpet models terms (Lai and Gallo, 2009). Applying the ATR-FTIR spectroscopy, the researches on the Cecropin P1 showed that it integrated parallel PE/PG membranes surface and never transformed the acyl chains order of the parameters that suggested that the peptides were never translocated into the core of the hydrocarbons. Figure 4.2: Model of membrane disruption by the carpet mechanism In the Detergent-like “carpet” model, the peptides disrupt the membranes through the orientation paralleled to the lipid bilayers surfaces that from an extensive carpet or layer (Schauber and Gallo, 2008). The peptide hydrophilic areas are indicated red, while the peptides hydrophobic areas are indicated blue as illustrated in figure 10. Detergent-like “carpet” models describe effective and efficient processes of membrane permeation used 37 by various Antimicrobial peptides (AMPs) in comparison to the “barrel-stave” models used by the channels of peptides ions (Costa, et al, 2011). Most significantly, the dogma that is acceptable has been disapproved on the specific sequence, chirality and structure role in the biological roles. Moreover, it was indicated that the assembly of the peptides in the aqueous solutions and specifically in the membrane is an important a parameter that controls the specificity of the target cell (Xiao, et al, 2013). In the Detergent-like “carpet” model there are only interactions between the lipid head groups and the peptides Figure 4.3: Detergent-like “carpet” model 1.18. The barrel-stave model Many researchers have given suggestions that some Antimicrobial peptides (AMPs) associate themselves for the formation of the transmembrane pores (Lai and Gallo, 2009). The transmembrane pore behaves as a channel of conductance which disrupts the ion gradients and potentials of the transmembrane that lead to the cell compositions leakage and ultimately the death of the cell. The dissipation of the electrochemical gradient of the transmembrane causes the loss of the ability of the bacterial cells to form energy in form 38 of ATP, as it increases the ion and water flow which accompanies the permeability barrier loss, thus leading to the osmolysis and the swelling of the cell (Li, et al, 2012). In the model of barrel-stave, amphipathic peptide aligns with its hydrophobic side that face the acyl chains of the phospholipids and its hydrophilic surface lining a channel that is water-filled similar to the barrel staves as indicated in figure 9. The barrel-stave model needs the peptide to be effective long to be able to traverse the bilayer of the hydrophobic core and inserts direct contacts between the peptides on the formation of the channels. A prototype required for the barrel-stave channels formations includes the fungal peptide commonly known as alamethicin (Schauber and Gallo, 2008). The alamethicin with twenty residues hydrophobic peptides from the fungi with the scientific name Trichoderma veridae contains eight unusual amino acids residues known as αaminoisobutyric acid or α-methyl alanine. Even though alamethicin does not have enough selectivity for the eukaryotic or bacterial membranes, numerous studies have been carried on alamethicin because of voltage-gated channels formation in the systems of the bilayer (Xiao, et al, 2013). In contrary to the peptides of the amphipathic, the monomers of the alamethicin insert into the bilayer before aggregating within the membrane to create pores of the barrel staves. The insertion of the Alamethicin membranes was well designed by the use of the dichroism of oriented circular as well as the transformations in the orientation of the peptides was realized continuously and reversible. Studies of in-plane neutrons scattering indicated that alamethicin would form aqueous and stable pores at high concentration of peptides, on the basis of the difference in the scattering D2O and H2O (Lai and Gallo, 2009). The studies of the scattering of neutrons provided a suggestion those barrel-staves models of the channels of alamethicin with eight or nine α-helices of 39 monomers per channels as well as pore diameters of ∼18–26 Å, that depend on the composition of the lipids as well as hydration degree. These agree with the black lipid membrane studies that show eight to ten monomers per channel (Li, et al, 2012). In the barrel-stave model, there is aggregation and insertion of the bound peptides into the bilayers of the membranes to enable the areas of hydrophobic peptides to align core areas of the lipids as well as the areas of the hydrophilic peptide from the pore interior regions. Figure 4.4: The barrel-stave model 1.19. Toroidal Pore Model In the toroidal pores mechanism, the class of lipid head of the Antimicrobial peptides (AMPs)-induced expansion leads to the bilayer binding back on itself that is followed by inner and outer leaflets connections, and the compositions of the pores contain both phospholipids and peptides (Schauber and Gallo, 2008). The surface of the membranes positive curvature, that result from the Antimicrobial peptides (AMPs) accumulations at the interface of the bilayers that facilitate the formation of the toroidal pore and phospholipids bending. The insertion and aggregation of the energeticallyfavorable peptides into the membranes can also cause the transformations in the structure of the bilayers as well as facilitate formation of the toroidal pores. 40 The toroidal pore models were originally introduced on the magainin peptides researches. The magainins are positively charged (cationic) peptides of amphipathic αhelical which form the channels of the transient ions (Xiao, et al, 2013). The size of the magainins has been estimated to be about 30 to 50 Å, developed with ninety phospholipids per pore and four to seven peptides. The studies of the calorimetry of the differential scanning of MSI-78 and 31P-NMR, magainin synthetic analog and optimized magainin also reinforce the formation of the toroidal pore (Costa, et al, 2011). Recent studies on the scattering of the neutrons and the orientation of the circular dichroism (CD) of mellitin indicates that it could also develop a type of toroidal-pores channel that has ∼44 Å as the diameter of the pore (Lai and Gallo, 2009). The pores size in diameter induced through the mellitins were approximated to be about 25 to 30 Å on the basis of the release of fluorescence-labeled differential sizes of dextran markers that conquers good with the size of the pore which was approximately 20 to 30 Å by erythrocytes osmotic protections (Li, et al, 2012). The estimation differences are reasonable, provided the disparities in the experimental techniques, the hydration degrees, and the lipids to peptides ratios. The toroidal pores structure models address the facts that most of the Antimicrobial peptides (AMPs) are not big enough to span the bilayers of phospholipids that are unperturbed in the conformation of the α-helical (Schauber and Gallo, 2008). For a helixalpha, there is a requirement of ∼22 amino acids in traversing the bilayers to typically have a width of 32–38 Å. This is for sure beyond many other Antimicrobial peptides (AMPs) and CM15 limits. For instance, trichogin crystal structure has shown that the α-helix length is ∼16 Å. In the model of the toroidal, the composition of the channels is a mixture of lipids and peptides and therefore is never needed in spanning the full bilayer (Costa, et al, 2011). 41 The proposal of the pores of the toroidal in forming subsequent to the thinning of the peptides-induced membranes that can also enable for the short peptides penetrations in forming peptide-lipid pores (Xiao, et al, 2013). In the Toroidal Pore Model, the aggregation and induction of the bound peptides to the monolayers of the lipids bends continuously via the pore in order the core of water that is lined by the groups of the lipid head and insertion of the peptides. Figure 4.5: Toroidal Pore Model Site-directed spin labeling (SDSL) is a method for examining the structure and local dynamics of peptides using electron spin resonance. The theory of labeling of the sitedirected spin is on the basis of the definite reaction of spin labels with amino acids. Sitedirected spin labeling with the spectroscopy of electron paramagnetic resonance (EPR) has arisen as an effective model to explain the structure and the dynamics of proteins (Melo, Ferre, and Castanho, 2009). Site-directed spin labeling is also a beneficial tool in investigations of the protein folding method. The correct models for the Toroidal Pore Model are the model of “toroidal pore.” In the “toroidal pore” models, the toroidal pore mechanism, the class of lipid head of the Antimicrobial peptides (AMPs)-induced expansion leads to the bilayer binding back on itself that is followed by inner and outer 42 leaflets connections, and the compositions of the pores contain both phospholipids and peptides. In certain cases, it may be quite challenging in distinguishing the mechanisms of the toroidal pore and carpet pore model (Lai and Gallo, 2009). For instance, the AMP LL37 in human beings intercalates paralleled to the membrane surface as closely-linked αhelices which do not reorient along the normal bilayer. As it takes place during the formation of the toroidal by magainin and mellitin as explained earlier, the high peptide to lipid ratios may not reorient the normal bilayer. Nevertheless, P-NMR does not indicate any evidence of the composition of the isotropic lipid as would be needed in certain carpet mechanism stage (Li, et al, 2012). Even though the models of channel-forming and carpet have various numbers of dissimilarities, they also share certain similar features. Both kinds of the models start with the association of the Antimicrobial peptides (AMPs) parallel to the surface of the membranes, that is then followed by the accumulations of the peptides to certain crucial concentrations. In the Detergent-like “carpet” model, the peptides remain linked the groups of the phospholipids heads throughout the processes of the membranes disintegrations (Xiao, et al, 2013). This is the same as the transition which takes place in the model of the toroidal-pores, and suggestions have been offered that the toroidal pores or “holes” formation may take place as an initial stage in the disintegration of the membranes that is to say that the pores o0f the toroidal are seen as intermediate states before the actual micellization (Melo, Ferre, and Castanho, 2009). It appears likely that many of the Antimicrobial peptides (AMPs), by virtues of the characters of the amphipathic, shall acts as detergent at effectively high concentrations. However, in most scenarios, for example, MSI-78, magainin it is precise that the formation of the pores is 43 efficient in inducing the lysis of the membrane, without complete progression to the destruction of the membrane. 1.20. Why the Toroidal Pore Model are the predict model for this research? The toroidal pores developed by the Antimicrobial peptides (AMPs) indicate critical disorders. A wide variety of Antimicrobial peptides (AMPs) have been illustrated to at least act in vitro through the lipid membrane poration. However, the nanometer size of the toroidal pores complicates the characterization structure of the pores through experimental models. The Toroidal Pore model allows for the application of the simulations of the molecular dynamics in studying the specific class interactions of the Antimicrobial peptides (AMPs), the mellitin, with the bilayer of the dipalmitoylphosphatidylcholine in the detail of the atom. The Toroidal Pore Model indicates that the pores of the transmembrane spontaneously form a crucial lipid to peptide ratio. 44 CHAPTER 5 Materials and Methods 1.21. Step 1: Investigating the structure and local dynamics of proteins using spin labeled AMPs The original sequence is GIKKFLHIIWKFIKAFVGEIMNS-NH2; Our modified sequence is: GIKKFLHIIWKFIKAFVGEIMNC-NH2 Procedure (i) Investigating the structure of AMPs and local dynamics of the proteins using spin- labelled 5 mg of protein was first weighed before dissolving in a 500mL buffer. 5.09 mg of IAP inhibitor was then weighed and dissolved in 0.25mL ethanol solution, which was then mixed with the buffer solution and shaken. A reaction time of about 4 hours was then allocated before the sample was lyophilized. A dialysis was then performed on the mixture (to separate the dissolved molecules regarding their molecular weight) in deionized water at 40 C, and the water was changed on hourly basis overnight. The sample was again lyophilized and then stored at a temperature of -200 C. The sample was then dissolved in an aqueous solution which contained a mild solvent, then the measurement was directly taken from the mass analyzers for ionization-mass spectrometry, for molecular weight characterization. (ii) Preparing large uni-lamella vesicles 3 mg of lipid was first dissolved in 1mL chloroform. The resultant solution was then heated in a vacuum until it evaporated when the lipid formed a homogenous film on the walls of 45 the reaction vessel. A salt solution was then added, then the lipid film was lyophilized for 1-4 hours in a 700 C water bath, such that it separated from the walls and formed lipid liposomes with ranging diameters. The vessel was then given a gentle shake to disintegrate the lipid spheres to allow the formation of smaller ones. The apparatus was then cleaned after use. (iii) EPR The x and y terminals were connected to the CRO via connecting leads and Helmholtz coils. The current knob was then adjusted to a minimum, then the frequency, sensitivity, and phase were set to ‘center,' ‘maximum' and ‘center' positions respectively. The H-coil power was then turned on, and the current was then set to 150mA. After four peaks appeared on the screen, the phase knob was adjusted such that it is coincided two peaks over the other two. The frequency and sensitivity knobs were then adjusted on the spectrometer along with the sensitivity knob on the CRO to provide sharp peaks. The phase knob was used to merge the peaks. The resonance frequency was then measured and recorded using the RF signal. Figure 5.1: MALDI-TOF of protein 2K without radical API 46 Figure 5.2: Graphical representation of the data Vesicles (made up of lipid bilayer) are cellular organelles. Vesicles are cellular coverings which are used to give a passage to materials from one place to another (GuaníGuerra, et al, 2010). They also function in enzyme storage and metabolism as well. The inverted cell membranes are called inside-out vesicles (E.g.: E. coli), these membranes are used to study the transporter-mediated drug interactions in the in-vitro environment. If the cell membranes are sealed at one surface and by impermeable molecules, right-side-out membrane vesicles can be formed. These are used to study membrane permeability (Dürr, Sudheendra, and Ramamoorthy, 2006). The main purpose of using vesicle as a model system in this prospectus is that the lipids are significant for this type of researches and it is used for transportation. Phospholipid bilayers contain tiny pouches known as vesicles that are involved in the molecules transportation. The main purpose of vesicles is to differentiate small enzymes as well as molecules into one place. The outer covering of vesicles is also phospholipid bilayer (Fjell, Hiss, Hancock, and Schneider, 2012). 1, 2dioleoylo-sn-glycerol-3-phosphocholine (DOPC) lipid is used in this study because it is 47 very common lipid and can be obtained from a variety of readily available sources, such as egg yolk or soybeans (Thomas et al, 2009). The structure of DOPC is given below: Figure 5.3: The structure of DOPC Note: it is important to note that the size and amount of vesicles rely on the time used in incubation, and the time taken to shake the sample at 70℃. As bilayer densities of head reduce and the thinner with the bilayer decrease of the size of the vesicles. The smaller sizes allow for fast movement within the cells hence faster transportation of the nutrients (Dürr, Sudheendra, and Ramamoorthy, 2006). The unilamellar vesicles were prepared by hydrating the lipid films to establish the large sized molecules. These can then be used as a control set up to monitor any other changes. Various preparations techniques of SL AMP vesicles from various cells and cells have been investigated (Nguyen, Haney, and Vogel, 2011). These involve the osmotic shock and the disaggregation of the enzymes, as well as the different techniques of homogenization. Spin-labeled peptides can be incorporated into membrane vesicles by binding titrations which are typically carried out through the addition of lipids that are envisaged to subsequently form vesicles to about 100 to 200 μl sample of a spin-labeled peptide solution (Fjell, Hiss, Hancock, and Schneider, 2012). Thereafter, a small volume of the spin-labeled peptide/lipid mixture, typically about 5 μl, is drawn into a glass or quartz 48 capillary with an inner diameter of between 0.5 and 1.0 mm (or less). The glass or quartz capillary with the mixture is then placed into an EPR resonator, which after the spectra have been recorded the mixture is unloaded back into the bulk spin-labeled peptide/lipid mixture. 1.22. EPR line shape analysis The interelectron distances and relative g-tensor orientations of many biradicals can be described with an analysis of their 9 and 140 GHz continuous-wave EPR line shapes (Chongsiriwatana, et al, 2008). Powder EPR line shape simulations can be adjusted to determine the possibility of a right EPR frequency separation by a randomly oriented biradical. Methods for the determination of the molecular rotational correlation time from EPR spectra have been known for decades. For instance, X-band EPR studies have been an imperative role to successfully obtain these correlation times where spectral density has played a negligible role (Guaní-Guerra, et al, 2010). Rotational correlation time of spinlabeled peptides has also been used to elucidate molecular dynamics of molecules, which have varied adsorption properties, as well as membranes with varying pore sizes. In particular, rotational correlation time has been found that it reflects variations in the molecular dynamics of molecules including peptide adsorption and the subsequent pore plugging on membranes of varied pore sizes. For example, the rotational correlation times of molecules that are highly adsorbent have been found to be significantly higher compared to those of molecules with low adsorbent properties (Dürr, Sudheendra, and Ramamoorthy, 2006). However, rotational correlation time has been found to be increased by steric 49 hindrance attributed to the plugging of the pore even though not as significantly manifested as in the effect of the adsorption. 50 CHAPTER 6 Results Results for probing the action of magainin peptide in large unilamellar vesicles via sitedirected spin labeling electron paramagnetic resonance technique Table 6.1: results for correlation time Temperature (deg.C) Correlation time 21 761 ps 25 829 ps 27 857 ps 29 910 ps 30 902 ps 32 955 ps 35 980 ps 37 1.02 ns 40 1.16 ns 43 1.21 ns 46 1.31 ns 48 1.49 ns 50 2.15 ns 51 Figure 6.1: EPR spectra of magainin with membrane cell at 20 0C 52 Figure 6.2: EPR spectra of magainin with membrane cell at 25 0C 53 Figure 6.3: EPR spectra of magainin with membrane cell at 27 0C 54 Figure 6.4: EPR spectra of magainin with membrane cell at 29 0C 55 Figure 6.5: EPR spectra of magainin with membrane cell at 30 0C 56 Figure 6.6: EPR spectra of magainin with membrane cell at 32 0C 57 Figure 6.7: EPR spectra of magainin with membrane cell at 35 0C 58 Figure 6.8: EPR spectra of magainin with membrane cell at 40 0C 59 Figure 6.9: EPR spectra of magainin with membrane cell at 43 0C 60 Figure 6.10: EPR spectra of magainin with membrane cell at 46 0C 61 Figure 6.11: EPR spectra of magainin with membrane cell at 48 0C 62 Figure 6.12: EPR spectra of magainin with membrane cell at 50 0C 63 Figure 6.13: Photomicrograph of the Membrane Figure 6.14: Photomicrograph of the Membrane Figure 6.15: Photomicrograph of the Membrane 64 Figure 6.16: Photomicrograph of the Membrane Figure 6.17: Photomicrograph of the Membrane Figure 6.18: The size of the membrane the magainin 65 Figure 6.19: The size of the membrane the magainin 66 CHAPTER 7 Discussion Vesicles are organelles that provide a pathway for materials from one point to another, offer storage for enzymes and help in metabolism control. Inside-out vesicles have inverted membranes, and control drug interactions with the outside environment. Molecules that are impermeable may at times block one side of a membrane, creating right-side-out vesicles which are used in the study of membrane permeability. Vesicles used in the transportation of molecules are located within phospholipid layers. Their membranes are composed of DOPC lipids as well. The time is taken to shake and incubate the sample determines the size and number of vesicles that would be formed. Smaller vesicles move faster than bigger ones, hence facilitate the transportation process more effectively. The resultant solution is put in a quartz capillary before being placed on an EPR resonator to record the spectra. EPR spectra are used to determine molecular rotation correlations via the X-band and spectral density (Klug, and Feix 621). Spin-labeled peptides' rotational correlation times are used in the elucidation of molecules' molecular dynamics, which have different adsorptive properties, as well as the size of the pores. Variations in the values of the rotational correlation times are a reflection of how the molecular properties and dynamics vary in the peptides (PinoAngeles e1004570; Xu et al. 2851). The spectral changes that are observed in the graphs are as a result of the geometry of the peptides in the membrane. The EPR spectroscopy results show that magainin does adopt a helical formation that extends over lipid layer. The helical shape shows that the peptide is amphipathic with the polar part of the amino-acid 67 chains that make up part of it. These amino-acids allow for hydrophobic interactions (as well as electrostatic) to take place within the peptide. The presence of the amphipathic lipid bilayers gives magainin the ability to increase their conductivity across lipid layers as well as to increase transmembrane transport of molecules and electrophysiology. EPR membrane alignment determines orientations between the peptides and spin labels. The high-amplitude mid-zone of the EPR graphs indicates a parallel orientation of the peptides, while the low-amplitude outer zones indicate perpendicular orientation. The orientation determines the hyperfine splitting of the peptides, in addition to their molecular axis. Rotational correlation time refers to the time that a molecule takes to make a rotation of one radian. It aids in the investigation of micro-viscosity in peptides, as was done for magainin in this experiment. The graph showing the rotational correlation time for magainin at different temperatures shows that the dynamic properties of spin-labeled magainin molecules vary with temperature at different sites. The value for the rotational correlation time is also dependent on the spin label motion that is the rate of motion of the EPR timescale. Low spin label motion causes broadening of the EPR lines thus leading to reduced amplitudes, and vice versa. Transport via membranes is crucial in the development of nanotechnologies, and the process has been rather too fast about the single-molecule techniques' resolutions that are in use. Great importance in the control of the residence time, direction as well as the sequential order of the spatiotemporal dynamics is displayed by the peptides (Pino-Angeles e1004570). The time-dependent ordering of the peptide structures is revealed by simulations of the molecular dynamics, which produced the graphs shown above. Some biological processes depend on peptide transport via membranes that depend on peptide 68 transport via membranes. They include transport of protein molecules within mitochondria and the endoplasmic reticulum, transport of proteins via chloroplast membranes and across the envelopes (nuclear) of eukaryotic cells, sorting of proteins to peroxisomes, among others (Xu et al. 2853;Toledo et al. 54). Such an experiment does have several sources of error which could affect the final results, and consequently the conclusions that would be made. For instance, the reagents used may be subjected to pre-contamination by accident. This could be through the use of unclean equipment which may have been containing other chemicals from previous experiments, which would then affect the reactions. Another cause of this is exposure to the reagents to atmospheric air long before the experiment, which may initiate decomposition in some. Errors may also occur when sequencing the software, which may result in wrong plots being drawn, etc. Another probable source is due to parallax when taking measurements of the volumes of the reagents. All these errors can be avoided or minimized by cleaning all equipment before use, storing all reagents well, being careful while taking the readings and when setting up the software. 69 CHAPTER 8 Conclusions The probing actions of Magainin Peptide in Large Unilamellar Vesicles were investigated via Site-Directed Spin Labeling Electron Paramagnetic Resonance Technique. The resultant solution was put in a quartz capillary before being placed on an EPR resonator to record the spectra. EPR spectra were used to determine molecular rotation correlations via the X-band and spectral density. The intense studies on the interactions of the antimicrobial peptide–membrane have resulted to development of membrane disruptions of molecular which include both processes of the non-particular detergents-like and discrete channels formation (Fjell, Hiss, Hancock, and Schneider, 2012). The EPR spectroscopy results indicated that magainin does adopt a helical formation that extends over lipid layer. The helical shape indicated that the peptide is amphipathic with the polar part of the amino-acid chains that make up part of it. These amino-acids allowed for hydrophobic interactions (as well as electrostatic) to take place within the peptide and the presence of the amphipathic lipid bilayers gave magainin the ability to increase their conductivity across lipid layers as well as to increase transmembrane transport of molecules and electrophysiology. EPR membrane alignment determines orientations between the peptides and spin labels. The high-amplitude mid-zone of the EPR graphs indicates a parallel orientation of the peptides, while the low-amplitude outer zones indicate perpendicular orientation. The orientation determines the hyperfine splitting of the peptides, in addition to their molecular axis. Even though there have continues arguments concerning the specific mechanisms applied by specific peptides, certain basic principles are starting to crop up. There is a 70 general agreement that, for the antimicrobial peptides, peptides originally links parallel to the surface of the bilayer (Nguyen, Haney, and Vogel, 2011). Moreover, there is a general consensus that many of the orientation of the antimicrobial peptides (AMPs) persist to allow for enough accumulations of the peptides to accomplish crucial concentrations of the membrane-bound. The accumulation of the peptides causes to the bilayer thinning that in turn results in the conditions that the lipid bilayer localized collapse in forming a toroidal pore of a lipid–peptide, insertion, and self-associations of the peptides into the bilayer arrangement of the barrel-staves or simply disintegration of detergent-like of the structure of the bilayer (Chongsiriwatana, et al, 2008). The resultant precise models for sure depends on the specific the antimicrobial peptides (AMPs) that are involved as well as the physical properties and the composition of the target bilayer. The accumulations of the peptides in the bilayer outer leaflet is a crucial stage in the membrane disruption process, and for most of the antimicrobial peptides (AMPs) it is becoming precisely clear that membrane perturbation next to the interface of the hydrophobic-hydrophilic results in the lipid bilayer thinning which precedes the permeability barrier loss (Guaní-Guerra, et al, 2010). More advancement of our comprehensions in the molecular events which results to the disruptions of the membrane shall facilitate the enhancements...
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