Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

Abstract

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.

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

“…In the case of the Asn103 side chain, significant disorder is evident from the lack of density in the 2F o À F c electron-density map (contoured at 1). Mean-while, for Asp101 the electron density suggests a different side-chain position compared with other HEWL variants (Ramos et al, 2021), leading to the disruption of a hydrogenbond crystal contact with Glu7.…”

“…The enzymatic activity was measured using the method originally reported by Shugar (1952), which estimates the activity rates by following the decrease in absorbance at 450 nm when HEWL is added to a cell suspension of Micrococcus lysodeikticus. The conditions used were identical to those used in our previous study (Ramos et al, 2021). The results presented in Supplementary Fig.…”

“…The backbone disorder of the Lys97-Gly104 region was shown to be associated with the occurrence of an Asn103 peptide-plane flip (for example in PDB entry 2vb1; Wang et al, 2007). While a partial Asn103 peptide-plane flip has been described in H-HEWL with a refined occupancy of 33%, it was found that this alternate conformation was present in 46% of the molecules of D-HEWL EC (Ramos et al, 2021).…”

“…While hydrogenated and perdeuterated variants of a protein are usually close to identical in structure, several studies of the macromolecular H/D isotope effect have reported decreases in the thermal stability of perdeuterated proteins compared with their hydrogenated analogs (Berns, 1963;Hattori et al, 1965;Brockwell et al, 2001;Meilleur et al, 2004;Koruza et al, 2018;Nichols et al, 2020;Ramos et al, 2021). Observations have also been made regarding solvent H/D isotope effects which suggest that both perdeuterated and hydrogenated proteins are thermally more stable in D 2 O than in H 2 O (Hattori et al, 1965;Harrington & von Hippel, 1961;Makhatadze et al, 1995;Freyman et al, 2001;Kuhlman & Raleigh, 1998;Sasisanker et al, 2004;Efimova et al, 2007).…”

Secondary Structure of Human De Novo Evolved Gene Product NCYM Analyzed by Vacuum-Ultraviolet Circular Dichroism

“…Furthermore, the analysis of results combined with a neural network can predict the locations of the secondary structure of proteins at the amino acid sequence level (15). We carried out VUVCD measurements on both hydrogenated NCYM and perdeuterated NCYM, because some perdeuterated proteins have been reported to change their local structure and to have decreased protein stability compared with their hydrogenated counterparts, affecting their function/activity (16)(17)(18). A comparison of the possible differences in the secondary structures between these molecules may provide insights into regions that contribute to molecular stability and function.…”