Mapping the folding pathway of the transmembrane protein DsbB by protein engineering.
The four-helical transmembrane protein DsbB (disulfide bond reducing protein B) folds and unfolds reversibly in mixed anionic/non-ionic micelles, consisting of an unfolding intermediate I and a rate-limiting transition state (TS) between I and the denatured state D. Here, I describe the analysis of the folding behavior of 12 different alanine-scanning mutants of DsbB. For all mutants, TS is as compact as D and there is an accelerating increase in compaction as the protein proceeds to I and the native state. This unusual pattern of consolidation may reflect significant amounts of secondary structure in D, analogous to a classical folding intermediate. Unexpectedly, an increase in apolar surface area upon mutation is stabilizing whereas an increase in polar surface area is destabilizing. This effect is probably dominated by the effect of the mutations on the structure of the denatured state. I observe clear Hammond postulate behavior, in which a destabilization of I moves it closer to D. Φ-Value analysis indicates that in TS, a folding nucleus consisting of two to three residues with Φ-values of > 0.5 forms at one end of the transmembrane helices, which expands to include residues closer to the middle of the protein in I. Thus, folding proceeds from a highly polarized starting point.
Department of Molecular Biology, Interdisciplinary Nanoscience Centre, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark. dao@inano.au.dk
Daniel E
DE
Otzen
D000409:Alanine / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D055550:Protein Stability; D012997:Solvents; D013816:Thermodynamics
D001426:Bacterial Proteins; C079503:DsbB protein, Bacteria; D008565:Membrane Proteins; D050505:Mutant Proteins; D012997:Solvents; D000409:Alanine