The folding transition state of the cold shock protein is strongly polarized.
The cold shock protein CspB from Bacillus subtilis consists of a three-stranded (beta1-beta3) and a two stranded (beta4-beta5) sheet, which form a closed beta barrel structure. CspB folds and unfolds rapidly in a two-state reaction, and the unfolded and the folded molecules interconvert with a time constant of 30 ms at the midpoint of the urea-induced transition (at 25 degrees C). The transition state of folding is native-like, as judged by the Tanford betaT value of > or =0.9. By using a mutational approach and Phi value analysis, we find that the folding transition state of CspB is energetically polarized. Despite the high betaT value, most Phi values are low. Values close to 1 were found for only a few residues, particularly in strand beta1 (Lys5, Val6, Lys7, Asn10). The interactions of the Asn10 side-chain with the backbone at positions 12 and 13 define the turn that connects the strands beta1 and beta2. Lys5 and Val6 in beta1 interact with residues in beta4, and their high Phi values indicate that an energetic linkage between beta1 and beta4 and thus between the two sheets exists already in the transition state. We compared our experimental Phi values with theoretical predictions of the folding pathway of cold shock proteins. Several of them suggest that the entire first sheet is formed in the transition state, and some identify the beta1-beta4 pairing as a crucial step in folding. Alternative paths that involve formation of the second sheet and beta3-beta5 pairing reactions were, however, suggested as well. The calculations gave coarse-grained pictures that are limited in resolution to the two sheets of CspB or to the elements of secondary structure. They did not identify the key residues with the high Phi values within these structural elements.
Garcia-Mira
Maria M
MM
Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, D-95440 Bayreuth, Germany.
Boehringer
Daniel
D
Schmid
Franz X
FX
D001412:Bacillus subtilis / Q000737:chemistry; D001426:Bacterial Proteins; D002942:Circular Dichroism; D006360:Heat-Shock Proteins / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; C407094:cold shock protein, Bacillus