scholarly journals The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme

Author(s):  
Joao Ramos ◽  
Valerie Laux ◽  
Michael Haertlein ◽  
V. Trevor Forsyth ◽  
Estelle Mossou ◽  
...  

The biological function of a protein is intimately related to its structure and dynamics, which in turn are determined by the way in which it has been folded. In vitro refolding is commonly used for the recovery of recombinant proteins that are expressed in the form of inclusion bodies and is of central interest in terms of the folding pathways that occur in vivo. Here, biophysical data are reported for in vitro-refolded hydrogenated hen egg-white lysozyme, in combination with atomic resolution X-ray diffraction analyses, which allowed detailed comparisons with native hydrogenated and refolded perdeuterated lysozyme. Distinct folding modes are observed for the hydrogenated and perdeuterated refolded variants, which are determined by conformational changes to the backbone structure of the Lys97–Gly104 flexible loop. Surprisingly, the structure of the refolded perdeuterated protein is closer to that of native lysozyme than that of the refolded hydrogenated protein. These structural differences suggest that the observed decreases in thermal stability and enzymatic activity in the refolded perdeuterated and hydrogenated proteins are consequences of the macromolecular deuteration effect and of distinct folding dynamics, respectively. These results are discussed in the context of both in vitro and in vivo folding, as well as of lysozyme amyloidogenesis.

Background: Glycation causes severe damage to the protein structure, instigating different diseases like cataracts, nephropathy, vasculopathy, retinopathy, atherosclerosis, neurodegenerative disease, diabetes, and age-dependent complications. Formaldehyde, a pollutant present in human habitation, is produced endogenously or exogenously during cooking or incinerating wood, paints, furniture, chipboards, fabric etc. Its higher concentrations can cause cell damage that promotes the formation of DNA/Protein cross-links. The present study aimed to evaluate the glycating effects of formaldehyde on hen egg white lysozyme in comparison with known glycating agent D-ribose. Methods: In this, in-vitro study, hen egg white lysozyme (HEWL) glycation with different concentrations of formaldehyde (0.25mM, 0.5mM, 1mM and 2mM) and D-ribose (0.01mM, 0.05mM, 0.1mM and 0.5mM) was examined using two different experimental conditions: concentration and time duration. Further cross-linking of protein was also analysed using SDS-PAGE technique. Results: Glycation of HEWL treated with formaldehyde increased with increasing concentrations (0.25mM, 0.5mM, 1mM and 2mM) and time duration (1, 3, 7 and 15 days). Cross linking of HEWL showed visible glycation at 2mM concentration. Cross-linked HEWL products gave dimer at 0.25mM and 0.5mM and trimers at 1mM and 2mMat 3, 7 and 15days. However, compared to formaldehyde, D-ribose glycation at different concentrations (0.01mM, 0.05mM, 0.1mM and 0.5mM) did not show the prominent cross linking of protein. Conclusion: Formaldehyde was found to be a more potent glycating agent compared to D-ribose. Compared to D-ribose, formaldehyde can produce protein misfolding and can be used in clinical research to establish the role of formaldehyde in patients with diseases.


IUCrJ ◽  
2021 ◽  
Vol 8 (3) ◽  
Author(s):  
Joao Ramos ◽  
Valerie Laux ◽  
Michael Haertlein ◽  
Elisabetta Boeri Erba ◽  
Katherine E. McAuley ◽  
...  

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.


2020 ◽  
Vol 17 ◽  
Author(s):  
Nandini Sarkar ◽  
Vidyalatha Kolli ◽  
Taraka Prabhu MP ◽  
Arbin Basak ◽  
Hitesh Mandal

Background: Amyloids are a class of ordered protein aggregates which have been implicated in the onset of several degenerative diseases such as Alzheimer's disease, Parkinson's disease, Type II diabetes and so on. Despite extensive research, the exact mechanism and the driving factors for the amyloidogenesis process remain elusive. Identifying molecules which can effectively inhibit and/or disaggregate the fibrils may be one effective therapeutic strategy against amyloidosis Objectives: In the current study, few hydroxy-benzoic phytochemicals were selected to study their effects on formation as well as disaggregation of hen egg white lysozyme (HEWL) amyloids, namely gallic acid, syringic acid, vanillic acid and iso-vanillic acid. Method: Amyloidogenesis was monitored using methods like the thioflavin T assay, field emission scanning electron mi-croscopy (FESEM) and dynamic light scattering (DLS) studies. Further protein conformational changes were monitored us-ing methods like 8-Anilino-naphthalene-1-sulfonate (ANS) fluorescence, circular dichroism (CD) spectroscopy and guani-dine hydrochloride mediated stability studies. Computational approach was also employed to get an insight on the interac-tion(s) between the selected compounds and HEWL using docking studies Result: The selected compounds exhibited significant inhibitory as well as disaggregation effects on HEWL amyloids. In-teraction with the phytochemicals was also associated with considerable conformational changes in HEWL. Docking studies show role of hydrogen bonding between HEWL and the phytochemicals. Conclusion: Thus the current study throws light on the key factors that drive amyloid formation and hence will be helpful for development of effective therapeutics against amyloidosis.


2016 ◽  
Vol 219 ◽  
pp. 28-37 ◽  
Author(s):  
Vishwanath Sivalingam ◽  
Nalla Lakshmi Prasanna ◽  
Neetu Sharma ◽  
Archana Prasad ◽  
Basant K Patel

2019 ◽  
Vol 129 ◽  
pp. 1015-1023 ◽  
Author(s):  
Mohammad Furkan ◽  
Mohammad Khursheed Siddiqi ◽  
Syed Mohammad Zakariya ◽  
Faez Iqbal Khan ◽  
Md. Imtaiyaz Hassan ◽  
...  

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