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

Joao Ramos,V Trevor Forsyth,Sine Larsen,Michael Haertlein,Annette E Langkilde,Estelle Mossou,Valerie Laux

doi:10.1107/s2059798321010950

Joao Ramos, V Trevor Forsyth + Show 5 more

Open Access

https://doi.org/10.1107/s2059798321010950

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Abstract

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.

Highlights

Secreted eukaryotic proteins produced in vivo typically pass through a process that starts at the ribosome
The results from the differential scanning fluorimetry (DSF) experiments show clear trends regarding the effects on protein thermal stability of in vitro refolding, protein perdeuteration, H/D solvent substitution and the pH of the buffer solution
The observations suggest that in vitro refolding has a stronger impact on hen egg-white lysozyme (HEWL) thermal stability than protein perdeuteration, with the respective decreases in Tm being greater than 3.5C compared with variations of smaller than 1.6C

Summary

Introduction

Secreted eukaryotic proteins produced in vivo typically pass through a process that starts at the ribosome (for example, at the endoplasmic reticulum). The unfolded peptide passes through the membrane, whereupon the pre-sequence is cleaved, with folding subsequently occurring through a pathway involving multiple chaperones (Shikano & Colley, 2013). The in vitro folding mechanism of lysozyme was later shown to involve intermediate states (Radford et al, 1992; Miranker et al, 1993; Wildegger & Kiefhaber, 1997). The unfolding process of an amyloidogenic variant of human lysozyme, highly homologous to HEWL, seems to involve local cooperativity (Canet et al, 2002). Understanding protein folding and the impact of different chemical environments on folding pathways is essential for current efforts in predicting three-dimensional structure using in silico methods and for the study of amyloidogenic pathologies. In the case of human lysozyme, several amyloidogenic mutations have been identified, in -helix C (Ile88–Asp101) and the -domain (Pepys et al, 1993; Gillmore et al, 1999; Valleix et al, 2002; Yazaki et al, 2003; Wooliver et al, 2007; Girnius et al, 2012; Jean et al, 2014; Sperry et al, 2016; Nasr et al, 2017)

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