Conformational stability and disassembly of Norwalk virus-like particles. Effect of pH and temperature

Greater than 99% of the Norwalk virus (NV) capsid consists of 180 copies of a single 58-kDa protein. Recombinantly expressed monomers self-assemble into virus-like particles (VLPs) with a well defined icosahedral structure. NV-VLPs are an appropriate vaccine antigen since the antigenic determinants...

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Bibliographic Details
Published in:The Journal of biological chemistry 2006-07, Vol.281 (28), p.19478-19488
Main Authors: Ausar, Salvador F, Foubert, Thomas R, Hudson, Mary H, Vedvick, Thomas S, Middaugh, C Russell
Format: Article
Language:English
Subjects:
Animals
Capsid - chemistry
Circular Dichroism
Hydrogen-Ion Concentration
Insecta
Microscopy, Electron, Transmission
Norwalk virus
Norwalk virus - metabolism
Protein Conformation
Protein Structure, Quaternary
Protein Structure, Secondary
Scattering, Radiation
Temperature
Ultraviolet Rays
Viral Proteins - chemistry
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Summary:Greater than 99% of the Norwalk virus (NV) capsid consists of 180 copies of a single 58-kDa protein. Recombinantly expressed monomers self-assemble into virus-like particles (VLPs) with a well defined icosahedral structure. NV-VLPs are an appropriate vaccine antigen since the antigenic determinants of the parent virion are preserved. They also constitute very simple models to study the mechanisms of assembly and disassembly of viral capsids. This work examines the inherent stability of NV-VLPs over a range of pH and temperature values and provides detailed insight into structural perturbations that accompany disassembly. The NV-VLP structure was monitored using a variety of biophysical techniques including intrinsic and extrinsic fluorescence, high resolution second-derivative UV absorption spectroscopy, circular dichroism (CD), dynamic light scattering, differential scanning calorimetry, and direct observation employing transmission electron microscopy. The data demonstrate that NV-VLPs are highly stable over a pH range of 3-7 and up to 55 degrees C. At pH 8, however, reversible capsid dissociation was correlated with increased solvent exposure of tyrosine residues and subtle changes in secondary structure. Above 60 degrees C NV-VLPs undergo distinct phase transitions arising from secondary-, tertiary-, and quaternary-level protein structural perturbations. By combining the spectroscopic data employing a multidimensional eigenvector phase space approach, an empirical phase diagram for NV-VLP was constructed. This strategy of visualization provides a comprehensive description of the physical stability of NV-VLP over a broad range of pH and temperature. Complementary, differential scanning calorimetric analyses suggest that the two domains of VP1 unfold independently in a pH-dependent manner.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M603313200