TY - JOUR

T1 - The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme

AU - Ramos, Joao

AU - Laux, Valerie

AU - Haertlein, Michael

AU - Forsyth, V. Trevor

AU - Mossou, Estelle

AU - Larsen, Sine

AU - Langkilde, Annette E.

PY - 2021/12/1

Y1 - 2021/12/1

N2 - The biological function of a protein is intimately related to its structure anddynamics, 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 proteinsthat are expressed in the form of inclusion bodies and is of central interest interms of the folding pathways that occur in vivo. Here, biophysical data arereported for in vitro-refolded hydrogenated hen egg-white lysozyme, incombination with atomic resolution X-ray diffraction analyses, which alloweddetailed comparisons with native hydrogenated and refolded perdeuteratedlysozyme. Distinct folding modes are observed for the hydrogenated andperdeuterated refolded variants, which are determined by conformationalchanges to the backbone structure of the Lys97–Gly104 flexible loop.Surprisingly, the structure of the refolded perdeuterated protein is closer tothat of native lysozyme than that of the refolded hydrogenated protein. Thesestructural differences suggest that the observed decreases in thermal stabilityand enzymatic activity in the refolded perdeuterated and hydrogenated proteinsare consequences of the macromolecular deuteration effect and of distinctfolding dynamics, respectively. These results are discussed in the context of bothin vitro and in vivo folding, as well as of lysozyme amyloidogenesis.

AB - The biological function of a protein is intimately related to its structure anddynamics, 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 proteinsthat are expressed in the form of inclusion bodies and is of central interest interms of the folding pathways that occur in vivo. Here, biophysical data arereported for in vitro-refolded hydrogenated hen egg-white lysozyme, incombination with atomic resolution X-ray diffraction analyses, which alloweddetailed comparisons with native hydrogenated and refolded perdeuteratedlysozyme. Distinct folding modes are observed for the hydrogenated andperdeuterated refolded variants, which are determined by conformationalchanges to the backbone structure of the Lys97–Gly104 flexible loop.Surprisingly, the structure of the refolded perdeuterated protein is closer tothat of native lysozyme than that of the refolded hydrogenated protein. Thesestructural differences suggest that the observed decreases in thermal stabilityand enzymatic activity in the refolded perdeuterated and hydrogenated proteinsare consequences of the macromolecular deuteration effect and of distinctfolding dynamics, respectively. These results are discussed in the context of bothin vitro and in vivo folding, as well as of lysozyme amyloidogenesis.

U2 - 10.1107/S2059798321010950

DO - 10.1107/S2059798321010950

M3 - Journal article

C2 - 34866613

VL - 77

SP - 1579

EP - 1590

JO - Acta Crystallographica Section D: Biological Crystallography

JF - Acta Crystallographica Section D: Biological Crystallography

SN - 2059-7983

IS - 12

ER -