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INTRODUCTION
Lysozyme is a protein that is an important factor in the innate immune response of higher organisms. It
cleaves sugars of the outer cell walls of bacteria, breaking down their integrity and the bacteria’s
viability. Information on its thermal stability and folding intermediates may be medically relevant as
the human form of the protein has been shown to be involved in the formation of amyloid fibers
associated with familial amyloidosis, which has severe pathogenic consequence. Although a great deal
is known from crystal structures at low or ambient tempera-tures, knowledge about the hightemperature structure and thermally induced unfolding intermediates and pathways is much more
limited in the absence of crystallographic data.
Hen egg white lysozyme (HEWL) is a 129-residue protein that has been classified as ‘‘mostly a’’ in
the CATH structure classification database (2) and as (a þ b) protein by the SCOP classification
system. One of its two domains consists of four a-helices and a 310 helix, and the other has a triplestranded antiparallel b-sheet, an irregular loop, and a 310 helix.
Studies have investigated the reversible and irreversible thermal denaturation processes of this
structure using a variety of experimental techniques (e.g., small-angle x-ray scattering (SAXS), quasi
elastic neutron scattering (9), differential scanning calorimetry, nuclear magnetic resonance (NMR),
Fourier transform infrared (FTIR) (15), circular dichroism (CD) , and Raman spectroscopies. Despite
all of these studies, however, the mechanism of this process and the forces that drive it remain unclear.
In addition, the results of molecular-dynamics (MD) simulations have not always been consistent or in
agreement with experimental observations. In this study, we used synchrotron radiation CD (SRCD)
spectroscopy, which yields highly accurate values of the helical content of proteins, and NMR to
determine the secondary structure, and SAXS measurements to examine the tertiary structure and the
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role of water (and in particular the hydration shell) in this transition. SAXS and FTIR spectroscopy
were also used to verify the absence of aggregation.
These methods were combined to examine details of the unfolding/refolding processes and define the
structural nature of the intermediates. The crystallographic structures at low temperature were
compared with the quantitative SRCD, NMR, and MD results. They all yielded similar results, leading
to confidence in the accuracy of the methods. In the absence of a crystallographic structure, the
combined experimental and computational methods provide a view of the high-temperature form of
the protein. Confidence in this view is reinforced by the correspondence between the experimental
SRCD and NMR results and the MD calculations for the high-temperature structure.
Hence, despite the large differences in the sensitivities and physical bases of these techniques, the
results of all of the methods and the computational studies are remarkably consistent.
RESULTS AND DISCUSSION
In addition to a-helices and b-sheet, HEWL contains a large amount of non-helical, non-sheet structure
in the turns and loops, as well as regions classified as ‘‘other’’ types of secondary structure, usually
considered to be disordered. The CD spectrum is typical of a protein with a significant helical content
but also some b-sheet structure. The peak at 22...