Structural characterisation and comparison of the native and A-states of equine lysozyme.
Abstract: Native state 1H NMR resonance assignments for 125 of the 129 residues of equine lysozyme have enabled measurement of the hydrogen exchange kinetics for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state. Native holo equine lysozyme hydrogen exchange protection factors are as large as 10(6), the most protected residues being located in elements of secondary structure. High exchange protection in the domain interface correlates with the binding of Ca2+ in this region. Equine lysozyme differs from most non-Ca2+ binding lysozymes in forming a highly populated partially folded state at low pH. The protein in this A-state at pH 2.0 has been found to bind 1-anilino-naphthalene-8-sulphonate with the enhancement of fluorescent intensity and blue shift in the spectral maximum characteristic of molten globules. NMR spectra indicate that the A-state is globally much less ordered than native equine lysozyme but does not contain significant regions of random coil structure. The amides most protected against hydrogen exchange in the A-state (protection factors up to 10(2) at 5 degrees C) correspond to residues of three of the four alpha-helices of the native state; the side-chains of these residues form a hydrophobic cluster that includes five aromatic residues. Circular dichroism and tryptophan fluorescence indicate that these residues are substantially more constrained than similar residues in "classical" molten globules. Taken together, the data suggest a model for the A-state of equine lysozyme in which a more ordered core is surrounded by a less ordered but still compact polypeptide chain.
Publication Date: 1997-05-23 PubMed ID: 9180380DOI: 10.1006/jmbi.1997.0996Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study examines the structural characteristics of equine lysozyme in its native state and in a partially folded state known as the A-state. The research utilised nuclear magnetic resonance (NMR) measurements and hydrogen exchange kinetics to study the behaviour of the protein in these different states and proposes a model for the A-state structure.
NMR Resonance Assignments
- The team used nuclear magnetic resonance (NMR) to assign resonances to 125 of the 129 residues of equine lysozyme. This assignment technique allowed the researchers to identify individual atoms within the protein, which is a key step in understanding its three-dimensional structure.
- The hydrogen exchange kinetics measurement for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state was also enabled by these assignments. Hydrogen exchange kinetics is a technique used to study the dynamics, stability, and folding of proteins. The slow exchange of some hydrogen atoms indicates that they are part of the stable, folded core of the protein.
Native Versus A-State
- The difference between the native and A-state of the protein is most evident in their level of order. The native state is highly ordered, with high hydrogen exchange protection factors up to 10(6), especially in regions participating in secondary structure elements like alpha-helices and beta-sheets.
- However, the A-state, which appears at low pH, is globally much less ordered but does not contain significant random coil structure, indicating some level of residual structure. The protection factors in this state are only up to 10(2) at 5 degrees Celsius, far lower than in the native state, indicating a higher level of dynamic motion and reactivity.
A-State Structure
- The findings, including the binding characteristics of a fluorescent probe, a differential hydrogen exchange rate, and spectral data, suggest that the A-state maintains some of the secondary structural elements, namely three out of four alpha-helices.
- Furthermore, side-chains of these residues form a hydrophobic cluster, including five aromatic residues. This suggests a complex structure in the A-state with a more ordered core and a less ordered but compact periphery, rather a simple random coil or molten globule.
Overall, the research reveals the dynamic differences between the different states of equine lysozyme and sheds some light on the structural characteristics of its low-pH A-state.
Cite This Article
APA
Morozova-Roche LA, Arico-Muendel CC, Haynie DT, Emelyanenko VI, Van Dael H, Dobson CM.
(1997).
Structural characterisation and comparison of the native and A-states of equine lysozyme.
J Mol Biol, 268(5), 903-921.
https://doi.org/10.1006/jmbi.1997.0996 Publication
Researcher Affiliations
- Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, England.
MeSH Terms
- Amides / chemistry
- Animals
- Circular Dichroism
- Horses
- Hydrogen / chemistry
- Magnetic Resonance Spectroscopy
- Muramidase / chemistry
- Protein Conformation
- Spectrometry, Fluorescence
Grant Funding
- Wellcome Trust
Citations
This article has been cited 10 times.- Kuwajima K, Yagi-Utsumi M, Yanaka S, Kato K. DMSO-Quenched H/D-Exchange 2D NMR Spectroscopy and Its Applications in Protein Science.. Molecules 2022 Jun 10;27(12).
- Woods KN, Pfeffer J. Using THz Spectroscopy, Evolutionary Network Analysis Methods, and MD Simulation to Map the Evolution of Allosteric Communication Pathways in c-Type Lysozymes.. Mol Biol Evol 2016 Jan;33(1):40-61.
- Honda RP, Yamaguchi KI, Kuwata K. Acid-induced molten globule state of a prion protein: crucial role of Strand 1-Helix 1-Strand 2 segment.. J Biol Chem 2014 Oct 31;289(44):30355-30363.
- Watanabe M, Kobashigawa Y, Aizawa T, Demura M, Nitta K. A non-native alpha-helix is formed in the beta-sheet region of the molten globule state of canine milk lysozyme.. Protein J 2004 Jul;23(5):335-42.
- Van Dael H, Haezebrouck P, Joniau M. Equilibrium and kinetic studies on folding of canine milk lysozyme.. Protein Sci 2003 Mar;12(3):609-19.
- Polverino de Laureto P, Frare E, Gottardo R, Van Dael H, Fontana A. Partly folded states of members of the lysozyme/lactalbumin superfamily: a comparative study by circular dichroism spectroscopy and limited proteolysis.. Protein Sci 2002 Dec;11(12):2932-46.
- Kobashigawa Y, Miura K, Demura M, Nemoto N, Koshiba T, Nitta K, Tsuda S. Assignment of 1H, 13C, and 15N resonances of canine milk lysozyme.. J Biomol NMR 2001 Apr;19(4):387-8.
- Bai P, Song J, Luo L, Peng ZY. A model of dynamic side-chain--side-chain interactions in the alpha-lactalbumin molten globule.. Protein Sci 2001 Jan;10(1):55-62.
- Li R, Woodward C. The hydrogen exchange core and protein folding.. Protein Sci 1999 Aug;8(8):1571-90.
- Kikuchi M, Kawano K, Nitta K. Calcium-binding and structural stability of echidna and canine milk lysozymes.. Protein Sci 1998 Oct;7(10):2150-5.
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