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.