1. Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme
- Author
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Valerie Laux, V. Trevor Forsyth, Michael Haertlein, Elisabetta Boeri Erba, Katherine McAuley, Estelle Mossou, Sine Larsen, Joao Ramos, Annette Eva Langkilde, Institut Laue-Langevin (ILL), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), DIAMOND Light source, IT University of Copenhagen (ITU), Department of Drug Design and Pharmacology [Copenhagen] (ILF), Faculty of Health and Medical Sciences, University of Copenhagen = Københavns Universitet (UCPH)-University of Copenhagen = Københavns Universitet (UCPH), MS platform of the Grenoble Instruct-ERIC center (ISBG, UAR 3518 CNRS-CEA-UGA-EMBL), Diamond Light Source for beamtime at I03 and MAX IV for beamtime at BioMAX, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), European Project: GR/R99393/01, European Project: EP/C015452/1, ILL, IT University of Copenhagen, and University of Copenhagen = Københavns Universitet (KU)-University of Copenhagen = Københavns Universitet (KU)
- Subjects
biophysical characterization ,perdeuterated lysozyme ,protein refolding ,010402 general chemistry ,Q1 ,01 natural sciences ,Biochemistry ,Pichia pastoris ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,structural biology ,General Materials Science ,Thermal stability ,protein structure ,030304 developmental biology ,X-ray crystallography ,chemistry.chemical_classification ,0303 health sciences ,Crystallography ,biology ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Protein dynamics ,HEWL ,General Chemistry ,Condensed Matter Physics ,biology.organism_classification ,Research Papers ,R1 ,0104 chemical sciences ,Enzyme ,chemistry ,Structural biology ,QD901-999 ,Biophysics ,Protein folding ,Lysozyme ,QD415 ,isotope effect - Abstract
Perdeuteration and in vitro refolding of hen egg-white lysozyme impact protein thermal stability and activity. Deuteration appears to primarily affect enzymatic function through changes in protein dynamics, while refolding contributes to a small decrease in protein thermal stability., This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.
- Published
- 2021