ENGINEERING THE E. COLI GLYOXYLATE REDUCTASE A Laboratory Manual for CHEM 603: Introduction to Biochemistry Lab Nicholas R. Silvaggi and David N. Frick Department of Chemistry & Biochemistry 2nd Edition, September 2020 ii TABLE OF CONTENTS GRADING & SCHEDULE ............................................... 1 Grading ...................................................................................... 1 Safety Training ........................................................................ 1 Lab Note Book ......................................................................... 1 Attendance/ Participation .......................................................... 1 Schedule: ................................................................................... 2 Safety Training ........................................................................... 3 Safety Rules ............................................................................... 4 Glossary ..................................................................................... 5 THE BIG PICTURE ....................................................... 6 LAB 1: DETERMINING CONCENTRATIONS................. 10 Purpose..................................................................................... 10 Learning goals ...................................................................... 10 Background ............................................................................... 10 Procedure .................................................................................. 15 Materials.............................................................................. 15 iii Determining the concentration of NADPH ................................. 15 Is it OK to use old NADPH stocks? ........................................... 18 .......................................................................................... 18 Testing an “aged” NADPH stock .............................................. 18 Results...................................................................................... 19 Discussion Questions .................................................................. 21 LAB 2: PROTEIN ASSAYS .......................................... 22 Purpose..................................................................................... 22 Learning goals ...................................................................... 22 Background ............................................................................... 22 Procedure .................................................................................. 25 Materials.............................................................................. 25 Determining the Concentration of EcGR ................................... 25 EcGR Specific Activity ............................................................ 28 Results...................................................................................... 29 Discussion Questions .................................................................. 31 LAB 3: PCR ............................................................... 32 Purpose..................................................................................... 32 Learning goals ...................................................................... 32 Background ............................................................................... 32 Procedure .................................................................................. 35 Preparation of LB Agar Plates for Cloning ................................. 35 PCR..................................................................................... 36 iv In silico cloning .................................................................... 37 Results...................................................................................... 38 Discussion Questions .................................................................. 39 LAB 4: DNA DIGESTION & PURIFICATION ................ 40 Purpose..................................................................................... 40 Learning goals ...................................................................... 40 Background ............................................................................... 40 Procedure .................................................................................. 44 Digestion of PCR Products ...................................................... 44 Agarose Gel Electrophoresis of Digested PCR Products ............... 45 Results...................................................................................... 47 Discussion Question .................................................................... 50 LAB 5: PURIFICATION, LIGATION, AND TRANSFORMATION ................................................... 51 Purpose..................................................................................... 51 Learning goals ...................................................................... 51 Background ............................................................................... 51 Procedure .................................................................................. 52 Materials.............................................................................. 52 Purification of PCR Product ..................................................... 53 Ligation ............................................................................... 54 Transformation of Competent Cells ......................................... 55 Results...................................................................................... 57 Discussion Questions .................................................................. 57 v LAB 6: SELECTING A CLONE...................................... 58 Purpose..................................................................................... 58 Learning goals ...................................................................... 58 Background ............................................................................... 58 Procedure .................................................................................. 60 Preparation of LB for Next Week ............................................. 60 Streak to single colonies ........................................................ 60 Finding a recombinant clone, step 1: Plasmid mini-preps ........... 61 Step 2: Compare plasmid sizes ............................................... 63 Results...................................................................................... 63 Discussion Questions .................................................................. 64 LAB 7: CELL GROWTH ............................................... 65 Purpose..................................................................................... 65 Learning goals ...................................................................... 65 Background ............................................................................... 65 Procedure .................................................................................. 67 Inoculation........................................................................... 67 Preparation of Buffers for Next Week....................................... 68 Results...................................................................................... 69 Discussion Questions .................................................................. 72 LAB 8: PROTEIN PURIFICATION ............................... 73 Purpose..................................................................................... 73 Learning goals ...................................................................... 73 vi Background ............................................................................... 73 Procedure .................................................................................. 75 Lyse Bacteria ....................................................................... 75 NiNTA Chromatography (IMAC) .............................................. 76 Gel Filtration Chromatography ................................................ 77 Results...................................................................................... 78 Discussion Questions .................................................................. 81 LAB 9: ACCOUNT OF YIELD & ACTIVITY.................... 83 Purpose..................................................................................... 83 Learning goals ...................................................................... 83 Background ............................................................................... 83 Procedure .................................................................................. 84 SDS-PAGE ........................................................................... 84 Determining Protein Concentration (Bradford Assay) ................. 87 EcGR Specific Activity Assessment .......................................... 88 Results...................................................................................... 89 Discussion Questions .................................................................. 92 LAB 10: ENZYME KINETICS ....................................... 93 Purpose..................................................................................... 93 Learning goals ...................................................................... 93 Background ............................................................................... 93 Materials.............................................................................. 98 Determining the kinetic parameters for glyoxylate .................... 98 Determining the kinetic parameters for NADPH ....................... 100 vii Results.................................................................................... 102 Discussion Question .................................................................. 106 LAB 11: ANALYSIS OF GR PROMISCUITY AND THE EFFECTS OF THE W45F MUTATION ......................... 108 Purpose................................................................................... 108 Learning goals .................................................................... 108 Background ............................................................................. 108 Procedure ................................................................................ 109 Materials ................................................................................. 110 viii GRADING & SCHEDULE All meetings are held in Chem283, Tuesdays or Thursdays at 11:00 am. Instructor: Nicholas R. Silvaggi (Office: Chemistry 372a; email: silvaggi@uwm.edu, phone: x2647) Teaching Assistant: Jon Mielke (Office: Lapham 441; email: jwmielke@uwm.edu) GRADING Safety Training • 10 pts/Course = 30 pts Lab Note Book • 10 pts/Week = 110 pts Each week you will record your observations in an electronic lab notebook in MS Word format. This facilitates inclusion of data (e.g. gel images, UV-vis spectra, or chromatograms) in your reports. This will be a record of all that you do each week in the laboratory. You will hand in your experimental notes from the previous week by the beginning of the next lab period via file upload in Canvas. Only .doc or .docx files can be uploaded. Lab reports will be graded on the following criteria: • Title & Date of experiment………………………………………………………… 1 • Data Quality ………………………………………………………………………………. 3 • General neatness and organization…………………………………………… 1 • Discussion Question Answers……………………………………………………… 3 • Attention to detail……………………………………………………………………… 2 Attendance/ Participation • 5 pts/Week = 55 pts This part of your grade is based on your individual lab performance. This class requires that you contribute to your experiments and the activities of your group. 1 Points will also be deducted if you arrive late, leave early, or just generally do not perform at a satisfactory level in the laboratory. SCHEDULE: Tues./Thurs. Introduction January 25/27 Lab 1 Making Dilutions January 25/27 Lab 2 Enzyme kinetics I February 1/3 Lab 3 PCR Amplification February 8/10 Lab 4 Gene Cloning I February 15/17 Lab 5 Gene Cloning II February 22/24 Lab 6 Gene Cloning III March 1/3 Lab 7 Protein Expression March 8/10 NO CLASS March 15/17 SPRING BREAK March 22/24 Lab 8 Purification I March 29/31 Lab 9 Purification II April 5/7 Lab 10 Substrate specificity April 12/14 Lab 11 Assay Development April 19/21 Open Week (For contingencies) April 26/28 Open Week (For contingencies) May 3/5 2 SAFETY TRAINING All students must complete online safety training Register here: https://about.citiprogram.org/en/homepage/ • Register under UW-Milwaukee. • Do not register as an “independent learner Once you have registered, log in, add and complete three courses: • Basic Introduction to Biosafety • Initial Biosafety Training • NIH Recombinant DNA Guidelines Procedure: • Select Add a Couse • Select Biosafety/Biosecurity then • Introduction to Biosafety • Basic Biosafety training • NIH Recombinant DNA Guidelines Print Each Diploma as a pdf and upload them through Canvas. All three diplomas are required to start lab work! 3 SAFETY RULES • Lab coats are available at UWM bookstore but are not absolutely required. • Face masks are absolutely required and should be worn in the lab except when working with open flames. • Follow social distancing rules as much as possible during the laboratory sessions. • Report all accidents, injuries, and breakage of glass or equipment to instructor immediately. • Keep pathways clear by placing extra items (books, bags, etc.) on or under the work tables • Long hair (chin-length or longer) must be tied back to avoid catching fire. • Wear sensible clothing including footwear. Loose clothing should be secured so they do not get caught in a flame or chemicals. • Do not taste or smell chemicals. • Wear safety goggles to protect your eyes. • Keep solids out of the sink. • Leave your workstation clean and in good order before leaving the laboratory. • Do not lean, hang over or sit on the laboratory tables. • Learn the location of the fire extinguisher, eye wash station, first aid kit and safety shower. • Do not lift any solutions, glassware or other types of apparatus above eye level. • Learn how to transport all materials and equipment safely. • No eating or drinking in the lab at any time! 4 GLOSSARY 5 mL milliliter(s) µL microliter(s) s seconds min minute(s) h hour(s) SN supernatant PLT pellet SDS sodium dodecyl sulfate M molar EtOH Ethyl alcohol EDTA ethylenediamine tetraacetic acid (disodium salt) vol volumes prep preparation THE BIG PICTURE The Silvaggi lab studies enzymes of unknown function from bacterial biosynthetic pathways. In some cases, a pathway produces an interesting compound like an antibiotic, and in other cases, the biosynthetic pathway is totally uncharacterized, and nobody has any idea what the end product is. In either case, the exact series of chemical steps catalyzed by the enzymes in the biosynthetic pathway are often partially or totally unknown. Take, for example, the biosynthesis of the nonproteinogenic amino acid L-enduracididine (L-End, Scheme 1) in the soil-dwelling bacterium Streptomyces wadayamensis. L-End is generated from L-arginine (Arg) by three enzymes, MppP, MppQ, and MppR. NB: The “Mpp” part of the names refers to the antibiotic mannopeptimycin, which is abbreviated “MPP”. All of the proteins in the MPP biosynthetic pathway are named “Mpp” plus a single letter according to the order of occurrence of the genes in the genome (e.g. MppA, MppB, etc). It turns out that MppP is a very unusual pyridoxal 5’-phosphate (PLP)-dependent enzyme that catalyzes the first step in L-End biosynthesis, a 4-electron oxidation of Arg to 4-hydroxy-2-ketoarginine (4HKA) (Scheme 1). MppP is unusual, because it stabilizes an electron-rich quinonoid intermediate that is capable of reducing molecular oxygen to generate a radical species. This is not something that PLPdependent enzymes were known to do until quite recently. MppP is a pretty sloppy enzyme, at least in vitro, and some amount of the time it releases a half-baked, 2electron-oxidized product, 2-ketoarginine (2KA, Scheme 1). The next enzyme in the process, MppR, takes the fully oxidized 4HKA and catalyzes an intramolecular cyclization reaction to create the iminoimidazolidine ring of L-End. The product of MppR is thus the ketone form of L-End that we will call 2-ketoenduracididine (2KE). Finally, MppQ, a typical PLP-dependent aminotransferase catalyzes Scheme 1. The steps in the L-End biosynthetic pathway and the enzymes that catalyze them. 6 the transamination of 2KE and an as-yet-unidentified amino acid to generate L-End (the amino acid) and a new ketoacid. Obviously, a key question in the workings of the L-End biosynthetic pathway is the identity of MppQ’s amino donor substrate. Previous data had suggested that alanine (L-Ala) and glycine (Gly) were both good candidates for the amino donor substrate. To test whether or not these amino acids were viable substrates, we performed steady state enzyme kinetics to determine kcat (turnover number, see below), KM, and the specificity constant kcat/KM. The reaction with L-Ala and 2KA produces L-Arg and pyruvate. The rate of pyruvate production, which cannot be monitored spectrophotometrically, can be coupled to the oxidation of NADH to NAD+, which can be monitored on the spec. The enzyme lactate dehydrogenase (LDH, EC 1.1.1.27) can be run “backwards” so that it reduces pyruvate back to lactate and, in the process, oxidizes NADH to NAD+. This type of enzymatic assay, called a “coupled assay,” allows one to measure the rates of reactions that otherwise have no measurable signal. We will discuss this in more detail later. For now, it is enough to say that the steady state kinetics done using the lactate dehydrogenase assay showed that L-Ala was not at all a good substrate for MppQ. The literature suggested that LDH would also accept glyoxylate as a substrate, which would also allow the LDH assay to be used to follow the reaction of MppQ with 2KA and Gly (after donating its amino group, Gly becomes glyoxylate). However, we found that LDH was not able to reduce glyoxylate at all. This led us to look for other potential coupling enzymes that would be able to reduce glyoxylate. The glyoxylate/hydroxypyruvate reductase from E. coli (EcGR; EC 1.1.1.79) is known to reduce glyoxylate or hydroxypyruvate to glycolate or glycerate, respectively. The reduction occurs with the concomitant oxidation of NADPH to NADP+. On this basis, we subcloned EcGR from E. coli genomic DNA and expressed it recombinantly in E. coli (we will cover the details of expression and purification during the laboratory experiments below). The purified EcGR, to our surprise and dismay, accepted both glyoxylate and 2KA as substrates! Obviously, one cannot use a coupling enzyme that competes with the enzyme of interest for the substrate. The rate one would measure in this case would be the combination of the two and it would be very difficult to separate the relative contributions of each enzyme to the measured rate. To get around this difficulty, we decided to try mutating the EcGR active site to reduce the efficiency of the undesirable reaction with 2KA, while (hopefully) leaving the reaction with glyoxylate unchanged. In other words, we are trying to make an EcGR mutant that is more selective for glyoxylate. 7 The first step in this protein engineering endeavor was to determine the structure of EcGR with 2KA bound in the active site. This let us understand how the undesirable substrate was interacting with the enzyme (Figure 1). Figure 1. Schematic representation of the active site in the X-ray crystallographic structure of EcGR with 2KA bound. The ketoacid portion of 2KA aligns well with the nicotinamide ring of NADP, ensuring efficient hydride transfer. The guanidinium group is too far from any active site residue to make strong hydrogen bonding interactions. The two closest residues to the guanidinium group whose side chains contained hydrogen bond acceptors, W45 and H46, were targeted for mutation. In contrast to the ketoacid portion of 2KA, which is held tightly by a number of strong hydrogen bonds and possible a salt bridge to R227, the guanidinium side chain of 2KA make remarkably little contact with the enzyme. There is one watermediated contact with E256. The next nearest side chains capable of making hydrogen bonding interactions with 2KA are those of W45 and H46. Since these are the closest side chains even capable of participating in hydrogen bonding interactions with 2KA, they were targeted for mutagenesis. Site-directed mutagenesis was used to change W45 to Phe (W45F) and H46 to Ser (H46S). The mutations were made independently, and also combined in a W45F/H46S double 8 mutant. All three of the variant proteins were tested for activity against the “real” substrate, glyoxylate, and the “spurious” substrate, 2KA. Surprisingly, all of the variants (W45F, H46S, and W45F/H46S) increased the selectivity of EcGR for glyoxylate. In the case of the double mutant, the reaction with 2KA was too slow to measure steady state kinetic parameters. Ultimately, the engineered W45F/H46S variant of EcGR allowed us to determine that Gly is not a substrate for MppQ. That’s how science goes sometimes, but we now at least have a variant that is more specific for glyoxylate, making it a potentially more useful enzyme to use when studying the kinetics of other glyoxylate-producing enzymes. 9 LAB 1: DETERMINING CONCENTRATIONS PURPOSE Learn to use UV-Vis spectroscopy to measure the concentration of NADPH in fresh, and aged samples. Learning goals • How to deliver small volumes with mechanical micro-pipettes • Learn to use the UV-Vis spectrophotometer • • Single wavelength mode • Spectrum (wavelength scan) mode Understand the Beer-Lambert law and extinction coefficients BACKGROUND NAD(P)H-dependent oxidoreductases are one of the largest groups of enzymes. Because of the variety of redox reactions catalyzed by these enzymes, and the broad range of substrates represented, NAD(P)H-dependent oxidoreductases are prime candidates for metabolic engineering and for directed evolution of novel biocatalysts. The common thread in these enzymes is that a substrate is reduced with the concomitant oxidation of NAD(P)H or oxidized with concomitant reduction of NAD(P)+ (Figure 1). The substrate can be an organic molecule like an amine, alcohol, or ketone, or an inorganic one like sulfite or mercury. Whatever the substrate being oxidized or reduced, the reaction with respect to NAD(P)H is the same: a hydride group (H-) is transferred from carbon 4 (C4) of the nicotinamide ring of NAD(P)H to the substrate or from the substrate to NAD(P)+. This chemistry makes NAD(P)H-dependent oxidoreductases interesting targets for engineering into useful industrial biocatalysts. More importantly from our point of 10 view, NAD(P)H absorbs UV light at 340 nm, while NAD(P)+ does not. This means that turnover of NAD(P)H-dependent oxidoreductases is accompanied by a clear signal that can be monitored by UV-vis spectrophotometry. So, a very common strategy for studying enzyme kinetics is to find an NAD(P)H-dependent enzyme that will oxidize or reduce the product of some enzyme of interest. These are known as coupled assays, and we will discuss these is more detail below. For now, we will focus on determining the concentration learning of NAD(P)H about and UV-vis spectrophotometry. The main goal of today’s lab is to learn how to use the spectrophotometer, since this will be one of the most-used and most important tools this semester. Figure 1. The structures of the oxidized and reduced forms of NAD(P). Spectrophotometry is a widely used technique in biochemistry, mainly for detecting or determining the concentrations of compounds of interest (like the products of an enzymatic reaction). The spectrophotometer we will use examines ultraviolet light (190320 nm) and visible light (320-700 nm), hence the term ultraviolet-visible (UVvis) spectrophotometry. As can be seen in Figure 2, NAD(P)H absorbs UV light with an absorption maximum at 340 nm. Figure 2. UV-vis absorption spectra of oxidized and reduced NAD. Note that both Since this absorption maximum falls forms absorb strongly at 260nm due to the close adenine ring. Only the dihydronicotinamide 11 to the UV part of the ring of NAD(P)H absorbs at 340 nm. electromagnetic spectrum, solutions containing NAD(P)H are colorless. Quantitative measurements of concentration by spectrophotometry rely on the Beer-Lambert Law (aka Beer’s Law) of light absorption by solutions as shown in Figure 3. The amount of light absorbed by the sample is defined as the ratio of the intensities of the incident and the transmitted light. From the degree of absorption, the concentration of the absorbing component can be deduced using the following equation, called the Figure 3. Schematic representation of a spectrophotometric measurement. Beer-Lambert Law: 𝑬𝑬 = 𝒍𝒍𝒍𝒍𝒍𝒍 𝑰𝑰𝟎𝟎 = 𝜺𝜺 ∗ 𝒄𝒄 ∗ 𝒍𝒍 𝑰𝑰 The law states that there is a linear relationship between the intensity of light, I0, diminishing to I after passing through a path of length, l, in a medium containing a chromophore (the molecule absorbing the light) at some concentration, c. The expression log I0/I is formally called extinction (E), but it is more commonly referred to either as absorbance (A) or optical density (OD). Absorbance, therefore, is proportional to the concentration of the solution (c) times ε, the extinction coefficient, times l, which is the path length through the cuvette. An extinction coefficient is defined as the absorbance of a specific chromophore in a solution of some set concentration. For example, if the concentration is given in molar (mol/L), and the path length (l) is measured in centimeters (cm), then ε is a “molar extinction coefficient” and its units would be M-1 cm-1. An extinction coefficient’s value is characteristic of the material in question at a specific wavelength, but it also depends on the solvent and the temperature, both of which can affect absorption of light. For example, the molar extinction coefficient for NADH at 340 nm (ε340nm) in neutral (~pH 7) aqueous solution is 6.22 x 103 M-1 cm-1. This means that a 1 molar solution of NADH in a 1 cm cuvette would have an absorbance of 6,220. NB: Remember that absorption is unitless (look at the Beer-Lambert Law above to prove it to yourself!). 12 This also means that you can always determine the concentration of a substance if you know the absorbance, which you can measure, and its extinction coefficient, which can often be obtained from published sources or determined empirically: 𝑐𝑐 = 𝐴𝐴 𝜀𝜀 ∗ 𝐿𝐿 The spectrophotometer is an apparatus designed to measure absorbance. It can produce a beam of light of a specific wavelength, direct it at a sample (usually in solution in a cuvette) and measure the intensity of the transmitted light. All this requires is a light source, a monochromator, a sample holder, a detector and a display (Figure 4). In the wavelength range of 200 to 320 nm, the light source can be either a deuterium, a high- pressure hydrogen or a high-pressure Figure 4. Schematic of a simple spectrophotometer. xenon lamp. To generate light in the visible spectrum, tungsten filament lamps are typically used. The role of the monochromator is to select light of a given wavelength from the continuous spectrum of the light source. Light passes through the sample placed in a holder (cuvette) made of plastic, glass, quartz or another transparent material. Plastic and glass cuvettes are cheap but cannot be used at wavelengths under 320 nm, because they interfere with absorbance of short wavelength light. I will say this again: plastic or glass cuvettes cannot be used to measure absorbance in the UV range, because they both absorb UV light! Quartz cuvettes must be used to measure absorbance of UV light (Figure 5). Figure 5. A selection of cuvettes used for routine UV-vis absorption measurements. The first 8 are quartz, while the last two are disposal plastic cuvettes used only for the visible range. 13 There are 3 optical configurations for UV/vis spectrophotometers: • Single Beam: These are the simplest and most economical instruments. In a single beam spectrophotometer, all of the light passes through the sample holder. Measurements are made by placing a reference in the sample holder, which is measured to standardize the instrument. This reference value is subtracted from subsequent sample measurements to remove effects from the solvent and the cell. • Split Beam: Also known as dual beam spectrophotometers or split beam spectrophotometers with Reference Beam Compensation (RBC). The light from the source is split into two paths with approximately 70% of the energy from the monochromator passing through the sample and the rest going to a separate feedback detector. This corrects for variations in energy and improves measurement stability and reproducibility. As with single beam instruments, all the light passes through the sample holder and measurements must be made first by placing a reference (aka blank) in the sample holder to correct for solvent and cell effects. • Double Beam: In a double beam spectrophotometer, there are two beams from the light source. One beam illuminates the reference cell holder and the other illuminates the sample. This configuration enables reference correction to be applied continually throughout a measurement and is commonly used when the sample and reference change over time e.g. kinetics measurements. Although advances in electronics have greatly improved the reliability of single and split beam instruments, double beam instruments still provide the greatest accuracy. The instruments in our lab are advanced double beam spectrophotometers, meaning the there are two positions for cuvettes. Always place your samples in the front 14 position. The back position is for the reference cuvette. You will place a cuvette containing only solvent (water or buffer) in the reference (back) position and leave it there for the course of the experiment. Today’s Experiment: You will make dilutions from NADH and NADPH stocks, and then measure the concentration of NAD(P)H in each one to assess how accurately you pipetted the stock and water. You will measure absorption spectra for fresh and aged samples to NADPH to determine whether or not there are any differences between the two. PROCEDURE Materials • 20 mM NADPH stock (prepared fresh by the TA) • “22 mM NADPH” stock (prepared many moons ago and stored at 4 °C) • milliQ H2O • Quartz cuvettes (2) • 1.7ml Microfuge tubes (4) • 5.0ml Snap-cap tubes (4) Determining the concentration of NADPH • Turn on your group’s spectrophotometer. • Once the spectrophotometer has warmed up (~15 min) you can place 1.0 mL of milliQ water (mqH2O) in each of two semi-micro quartz cuvettes and place these in the sample (FRONT) and reference (BACK) compartments. • Create a set of 4 “independent dilutions” of the NADPH stock in 4 clean 1.7ml Eppendorf tubes (aka microfuge tubes) as detailed in the Results Tables. • Create a “serial dilution” of the NADPH stock in 4 clean 5.0ml snap-cap tubes as detailed in the Results Tables. • At this point, you should have a total of 8 tubes, each with 1ml of NADPH solution (except for the last tube of the serial dilution, which will have 2ml). These dilutions are calculated to deliver four concentrations of NADPH that will have 15 peak absorbances in the linear range of the spectrophotometer (0.01 to 2 Abs). Remember, a 50 mM NAD(P)H solution should have peak absorbances of: A260nm = 0.050 M * 17,400 M-1 cm-1= 890 A340nm = 0.050 M * 6220 M-1 cm-1 = 311 • Record a baseline spectrum from 200-500nm. • Discard the water in the sample compartment cuvette and replace it with 1 mL of your first independent NADPH dilution. Keep the reference cuvette in place. • Record a spectrum (200500 nm) of your NADPH sample and repeat with the 7 remaining NADPH samples. BE SURE TO NAME AND SAVE EACH SPECTRUM IN AN ORDERLY MANNER IN THE CORRECT DIRECTORY FOR YOUR LAB SECTION • Record the peak absorbances at 260nm and 340nm in Results Tables. The peak pick function automatically detects peaks and valleys on spectrum data and displays the wavelength and photometric values as tabulated data (Peak Pick Table). The peak pick operation is executed automatically when data are created after measurement, and the obtained results are saved with the measured data (see the screen shots on the following page). 16 • Plot A340nm (Y-axis) vs the volume (µl) of NADPH stock added. • Calculate actual concentration of NADPH in each dilution from the extinction coefficient of NADPH, then calculate the concentration of the stock solution using the dilution factors. 17 Is it OK to use old NADPH stocks? In a word, “NO!” Even when NAD(P)H solutions are aliquoted and immediately stored at -20°C, some of the NAD(P)H molecules will become oxidized, and in turn will be rereduced by the remaining NAD(P)H. A significant amount will be re-reduced at the “wrong” carbons – either C2 or C6 rather than C4. Both 2-dihydro-NAD(P)H and 6dihydro-NAD(P)H can be inhibitors of NAD(P)H-dependent enzymes. The NAD(P)H can also undergo anomerization – switch from the β- to the α-anomer of the ribose. The αNAD(P)H is not a substrate from most NAD(P)H-dependent enzymes. This is a problem because it is vitally important for kinetics assays to know the concentrations of all assay components as precisely as possible. If some of the β-NAD(P)H is converted into something else, then there could be significantly less β-NAD(P)H in the assay. This could have a dramatic effect on the kinetics data obtained from such a flawed assay. Testing an “aged” NADPH stock When rummaging through the refrigerator in the lab, I found an old (undated and minimally labeled!!!) tube of “22 mM NADPH”. Using something like this in an experiment is just the sort of thing an inexperienced and/or pathologically lazy researcher would do. To prove to everyone that being lazy is especially bad in the laboratory, test this old solution and see if there is anything off about it. • Obtain 20µl or so of this aged NADPH stock from the TA and make an appropriate dilution. • Measure the absorption spectrum of your dilution on the spectrophotometer and calculate the concentration of NADPH from both the absorbance at 340nm and the absorbance at 260nm. 18 RESULTS Pipet NADPH and water into 1.7ml Eppendorf tubes using a P10, P20, or P200 micropipet as appropriate. Independent dilutions of the NADPH stock µL of NADPH stock dH2O ___ µM 20 980 1.856 ___ µM 10 990 1.709 1.014 1:100 ___ µM 5 995 1.150 0.520 1:200 ___ µM 2.5 997.5 0.630 0.291 1:400 A260nm A340nm [NADPH] in cuvette (µM) Dilution Factor [NADPH] in stock 1:50 1.837 Pipet NADPH and water into 5.0ml snap-cap tubes using a P10, P20, or P200 micropipet as appropriate. Serial dilutions of the NADPH stock µL of NADPH stock 40 µl of dH2O previous dilution -1960 -- 1000 1000 -- 1000 1000 -- 1000 1000 19 A260nm 1.843 1.727 1.052 0.620 A340nm 1.611 [NADPH] in cuvette (µM) Dilution Factor 1:50 1.006 1:100 0.484 1:200 0.283 1:400 [NADPH] in stock Plot A340nm (left Y-axis) vs µL of NADPH (x-Axis) for each type of dilution (independent or serial). Use different symbols and/or colors to differentiate the two plots. Remember to add proper labels and title. Use a straight edge to draw a line of best fit for each data set. Preparation of aged 22mM NADPH sample: _____µl stock _____µl mqH2O, nominal [NADPH] = ______mM 0.721 A340nm of aged NADPH dilution: ________ and calculated [NADPH]: ________ A260nm of aged NADPH dilution: ________ and calculated [NADPH]: ________ 1.910 20 DISCUSSION QUESTIONS Examine the plots of A340nm vs µL of NADPH. Which method of preparing dilutions gave the most accurate results? Calculate the concentration of the NADPH stock solution. Assuming that your pipetting was perfect, why else might this value be different from the expected 20 mM? Why does NADPH absorb light at 260 nm? At 340 nm? Was anything amiss with the aged NADPH stock? How do you know? 21 LAB 2: PROTEIN ASSAYS PURPOSE To measure the concentration of a purified protein in preparation for enzyme kinetics experiments. Learning goals • Understand how to quantify the amount of a protein in solution and determine its concentration using: • • The Bradford protein reagent and a standard curve • Protein extinction coefficient at 280 nm Determine the specific activity of an EcGR preparation BACKGROUND Determining the amount of an enzyme in solution is not as simple as what we did last week. Unlike small molecules, like NADPH, proteins are fragile and frequently denature (unfold) when stored in cell-free extracts. They also tend not to have handy chromophores like the nicotinamide group of NADPH. In an investigation of an enzyme, it is critically important to know the concentration of active enzyme. This is easier said than done. If you are lucky enough to work on a heme-containing protein, for example, you can use the absorbance of the heme to tell you how much properly folded protein you have. In the vast majority of cases, however, there is no such “native” reporter molecule bound up in the protein. Most of the time it is necessary to find a reporter molecule that will irreversibly bind to some active site residue (e.g. a catalytic cysteine or lysine, or a general acid/base catalyst). For example, most enzymes with a nucleophilic lysine residue can be irreversibly labeled with 5-nitro-salicylaldehyde (5NSA). This is a bright yellow compound with a known extinction coefficient; the concentration of 5NSA attached to the protein equals the concentration of active protein. Unfortunately for us, the glyoxylate reductase mechanism proceeds by direct hydride transfer from NADPH to the substrate; the enzyme is essentially just holding the substrate at the proper distance and orientation with respect to the coenzyme to make this happen. 22 Since there is really no easy way to label our active EcGR, the best we can do is measure the specific activity. Specific activity is a relative measure of the purity of active enzyme. Put another way, specific activity does not tell you the concentration of active protein directly, but it does tell you whether your new preparation of the enzyme is more or less pure than some reference batch (often the first preparation). Each time a preparation of the enzyme is made, it is compared back to the reference batch. If the specific activity is below the expected value, it means that either there are more contaminating proteins from the expression host (proteins are most commonly purified from bacteria; it is impossible to remove 100% of the cellular proteins) or some of the enzyme has been denatured or otherwise inactivated (e.g. by proteolysis). To calculate specific activity, you need to measure two things: (1) the rate of the enzyme-catalyzed reaction and (2) the amount of total protein. The specific activity is the ratio of these two quantities. The enzyme activity is expressed as enzyme units (U) per ml of the preparation (U/ml). An enzyme unit is defined separately for each enzyme. For EcGR, 1 U is defined as the amount of enzyme required to convert 1 µmole of glyoxylate to glycolate in 1 min at pH 6.4 and 25 °C. In this laboratory, we will measure how many µmoles of NADPH are consumed during some arbitrary period of time (e.g. 10 s) where the reaction rate is constant (the plot of [NADPH] vs time has a constant slope). If, for the sake of argument, 1 µl of the enzyme stock consumes 3 µmoles of NADPH in 10 s, then it will have consumed 18 µmoles in 1 min. So, that would be 18 U. To get our activity in U/ml, we need to multiply that by 1000, since we only had 1 µl of the enzyme solution in the cuvette. Our final activity measurement, then, is 18,000 U/ml. The handy thing about EcGR is that, since it uses NADPH as a cosubstrate, we can directly monitor the reaction by watching the decrease in absorbance at 340 nm as the cosubstrate is consumed. There is a 1-to-1 correlation between the consumption of NADPH and the production of product (glycolate). In the procedure below, you will make a reaction mix containing the enzyme and a saturating amount of NADPH in a suitable buffer, and then initiate reactions by adding a saturating amount of glyoxylate. This will give us our activity measurement. The other thing we need is the concentration of total protein. To determine the total protein concentration, we will use a colorimetric assay called the “Bradford Assay” (see Analytical Biochemistry 72, 248-254 (1976) for the original reference). This assay is based on the observation that the absorbance maximum for an acidic 23 solution of Coomassie Brilliant Blue G-250 dye shifts from 465 nm to 595 nm when it is bound to protein. The method is simple, reliable, and rapid. The assay involves quantitation by spectrophotometry. The binding of the dye to an unknown protein is compared to that observed using varied amounts of a protein of known concentration. That is, accurate determinations of an unknown protein concentration require the generation of a “standard curve” of absorbance versus protein amount (µg), using protein of a known concentration. Our known (or “standard”) protein is bovine serum albumin (BSA). By determining the amount of protein in your unknown, it is straightforward to determine the concentration of total protein (just like we did last week for NADPH). For example, let us say you assay 10 µL of an unknown protein sample and interpolate from the standard curve (which you have generated) that this aliquot contains 20 µg of protein. The concentration of protein in your unknown sample is: = 20 µg/10 µL = 2 µg/µL (commonly expressed as 2 mg/mL) The concentration of a purified protein can also be determined quite accurately from the extinction coefficient of the protein at 280 nm. Since there are only three amino acids with significant absorbance at 280 nm*, it is possible to sum the absorbance contribution of the total number of each of these amino acids in a peptide and determine arithmetically the extinction coefficient. *Note: Actually, there is one more group found in proteins that can also absorb UV light at 280 nm: the disulfide bond (extinction coefficient = 125 M-1cm-1) There are many online tools available to calculate protein extinction coefficients. For example, the ProtParam server (https://web.expasy.org/protparam/) is a tool that computes various physical and chemical parameters for a given protein sequence. The computed parameters 24 include the molecular weight, theoretical isoelectric point (pI), amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity. The fact that an extinction coefficient is specific to one particular protein sequence means that using A280nm to determine the protein concentration is discounting the presence of other contaminating proteins. With highly purified protein (95-98% pure) that is typically used for structural and enzymology experiments, this difference is negligible. However, if one were using crude cell extracts, then determining the concentration of one specific protein by A280nm would be nonsensical. Note that a wavelength scan is more useful than single A280 measurements, since the shape of the curve can give clues about the quality of the protein preparation. For example, a large peak around 260 nm that “swallows up” the 280 nm peak indicates significant DNA contamination. A long, “tailing” 280 nm peak that approaches the baseline slowly is indicative of denatured/precipitated protein. Now that we have our two measurements, activity (U/ml) and total protein concentration (mg/ml), we can calculate the specific activity (U/mg): 𝑈𝑈𝑈𝑈𝑈𝑈𝑈𝑈𝑈𝑈 𝑚𝑚𝑚𝑚 𝑈𝑈𝑈𝑈𝑈𝑈𝑡𝑡𝑠𝑠 ∗ = 𝑚𝑚𝑚𝑚 𝑚𝑚𝑚𝑚 𝑚𝑚𝑚𝑚 PROCEDURE Materials • 0.1 mg/ml BSA standard • Bradford protein reagent • Assay buffer stock (0.5 M HEPES, pH 7.5) • 20 mM β-NADPH stock • 10 mM Na glyoxylate stock Determining the Concentration of EcGR Bradford assay • Using the 0.1 mg/mL BSA solution provided, prepare a set of six 0.5 ml BSA standards ranging from 0-16 µg of BSA (see Results table for details). 25 • Add 0.5 mL of Bradford solution to each tube. Mix by inverting 5 times (avoid foaming; do not shake the tubes). • Measure the absorbance at 595 nm using a clean plastic cuvette. After “blanking” the spec with the 0 mg/mL (water only) standard, read the BSA samples from the lowest amount to the highest amount. Use care to avoid spills in and on the specs! • Make a standard curve in Excel by plotting the A595nm against the µg of BSA in the standards. (You blanked with the 0µg standard, so it is appropriate to include this sample as “0” in your standard curve.) Add a trendline to the data, which should be linear; show the line equation on your plot. • Make three samples with different amounts of the EcGR stock. Start with 1, 2, and 5 µl. If none of these fall on the standard curve, you will need to dilute the stock and make new unknown samples (if the first 3 are too high) or use more stock (if the first 3 are too low). Plug the A595nm values of the unknown samples into the line equation from the standard curve and solve for the X intercept, which is the µg of protein in the sample. Direct quantitation by A280nm • This is the sequence of the wild-type EcGR protein you are using: MDIIFYHPTFDTQWWIEALRKAIPQARVRAWKSGDNDSADYALVWHPPVEMLAGRDLKAVFALGAGVDSILSKLQA HPEMLNPSVPLFRLEDTGMGEQMQEYAVSQVLHWFRRFDDYRIQQNSSHWQPLPEYHREDFTIGILGAGVLGSKVA QSLQTWRFPLRCWSRTRKSWPGVQSFAGREELSAFLSQCRVLINLLPNTPETVGIINQQLLEKLPDGAYLLNLARG VHVVEDDLLAALDSGKVKGAMLDVFNREPLPPESPLWQHPRVTITPHVAAITRPAEAVEYISRTIAQLEKGERVCG QVDRARGY • Use the information given below to calculate the extinction coefficient of EcGR. • Put 990 µl of water in a cuvette and add 10 µl of the EcGR stock. Measure the absorbance spectrum of EcGR from 240 to 340 nm. • Calculate protein concentration in both µM and mg/ml (hint: the extinction coefficient (units of M-1cm-1) will give you the concentration in M (mol/L); pay attention to the units to figure out how to convert the molar concentration to mg/ml. 26 Selected ProtParam output: Number of amino acids: 312 Molecular weight: 35343.42 Da (g/mol) Theoretical pI: 6.32 Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val (A) (R) (N) (D) (C) (Q) (E) (G) (H) (I) (L) (K) (M) (F) (P) (S) (T) (W) (Y) (V) 27 23 8 17 3 18 19 20 9 15 34 10 6 11 21 18 12 10 8 23 8.7% 7.4% 2.6% 5.4% 1.0% 5.8% 6.1% 6.4% 2.9% 4.8% 10.9% 3.2% 1.9% 3.5% (200 M-1cm-1) 6.7% 5.8% 3.8% 3.2% (5,690 M-1cm-1) 2.6% (1,280 M-1cm-1) 7.4% Formula: C1594H2473N445O449S9 Total number of atoms: 4970 Extinction coefficients: Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water. Ext. coefficient: ___________ (Assuming all pairs of Cys residues form cystines [disulfides]) Ext. coefficient: ___________ (Assuming all Cys residues are reduced) 27 EcGR Specific Activity • Prepare 50 ml of assay buffer by diluting 5 ml of the 0.5 M HEPES stock with 45 ml of water in a 50 ml conical tube. • • Make serial four-fold dilutions of EcGR as follows: • Transfer 30 µL of 50 mM HEPES, pH 7.5 into 3 tubes labeled 4, 16, 64. • Add 10 µL of EcGR to tube “4” and mix by inverting. • Transfer 10 µL from tube “4” to tube “16”, mix. • Transfer 10 µL from tube “16” to tube “64”, mix. • Store dilutions on ice. Prepare four reactions by adding 975 µl of assay buffer (50 mM HEPES, pH 7.5) to four clean, labeled 1.7 ml centrifuge tubes. Add 10 µl of 20 mM NADPH stock to each one and mix. Add 5 µl of EcGR stock to the first tube, 5 µl of dilution “4” to the second tube, 5 µl of dilution “16” to the next tube, and 5 µl of dilution “64” to the last tube. Incubate the enzyme and NADPH together at room temperature (~22 °C) for at least 1 min. • While the reactions are incubating, set the spec to “Kinetics” mode. Set the time to 5 min (you can always stop it early) and set the wavelength to 340 nm. • Transfer the contents of the first tube to a quartz cuvette and zero the spectrophotometer. Leave the cuvette with the reaction mixture in the spec. • The next step is to add the substrate to start the reaction. You want to mix and start recording the A340nm as close to simultaneously as possible, since during mixing you are missing the reaction. In other words, you don’t have a lot of time to mess around mixing things up. Having one person add the substrate and mix and a second person pressing “pause/continue” on the spec is a good way to do this quickly. The “hockey stick” mixer also helps. This is a tiny block of ppolycarbonate with two vertical channels through the middle attached to a long stick. You should be able to add the substrate and get back to recording in
Purchase answer to see full attachment