An on-pathway hidden intermediate and the early rate-limiting transition state of Rd-apocytochrome b562 characterized by protein engineering.
The folding pathway of Rd-apocytochrome b562, a four-helix bundle protein, was characterized using Trp and Ala/Gly pair mutations. We found that the Trp mutants (F65W) of both the fully folded Rd-apocytochrome b562 and a partially unfolded intermediate with the N-terminal helix (helix I) unfolded, fold with identical folding rates, providing direct evidence for the conclusion that the rate-limiting transition state folds before the partially unfolded intermediate; and that this hidden intermediate is an on-pathway intermediate. We further characterized the helical structures formed in the rate-limiting transition state by measuring the folding/unfolding rates for Ala/Gly pair mutations at solvent-exposed positions. Little change in folding rates occurred for the Ala/Gly pair mutations at positions in helix I and the C-terminal regions of helix II and IV. In contrast, a significant difference in folding rates was observed for the Ala/Gly pair mutations in helix III and the N-terminal regions of helix II and IV, suggesting that helix III and the N-terminal regions of helix II and IV are formed in the rate-limiting transition state. These results complement those obtained from earlier studies and help to define the folding pathway of Rd-apocytochrome b562 in more detail.
Zhou
Zheng
Z
Laboratory of Biochemistry, Center for Cancer Research, National Cancer Institute, NIH, Building 37, Room 6114E, Bethesda, MD 20892, USA.
Huang
Yingzi
Y
Bai
Yawen
Y
D000409:Alanine / Q000378:metabolism; D045303:Cytochromes b / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000378:metabolism
D014364:Tryptophan; D045303:Cytochromes b; D000409:Alanine; D005998:Glycine