Folding of the disulfide-bonded beta-sheet protein tendamistat: rapid two-state folding without hydrophobic collapse.
We investigated the reversible folding and unfolding reactions of the small 74 amino acid residue protein tendamistat. The secondary structure of tendamistat contains only beta-sheets and loop regions and the protein contains two disulfide bonds. Fluorescence-detected refolding kinetics of tendamistat (disulfide bonds intact) comprise of a major rapid fast reaction (tau = 10 ms in water) and two minor slow reactions. In the fast reaction 80% of the unfolded molecules are converted to native protein. The two slow reactions are part of a parallel slow folding pathway. On this pathway the rate-limiting step in the formation of native molecules is cis to trans isomerization of at least one of the three trans Xaa-Pro peptide bonds. This reaction is catalyzed efficiently by the enzyme peptidyl-prolyl cis-trans isomerase. Comparison of kinetic data with equilibrium unfolding transitions shows that the fast folding pathway follows a two-state process without populated intermediate states. Additionally, various sensitive tests did not detect any rapid chain collapse during tendamistat folding prior to the acquisition of the native three-dimensional structure. These results show that pre-formed disulfide bonds do not prevent efficient and rapid protein folding.
Schönbrunner
N
N
Department of Biophysical Chemistry, Biozentrum der Universität Basel,Switzerland.
Koller
K P
KP
Kiefhaber
T
T
D000591:Amino Acid Isomerases / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D002352:Carrier Proteins / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D007700:Kinetics; D010455:Peptides / Q000737:chemistry*; D019696:Peptidylprolyl Isomerase; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D012995:Solubility; D013816:Thermodynamics; D014867:Water
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004220:Disulfides; D006146:Guanidines; D010455:Peptides; D011994:Recombinant Proteins; D014867:Water; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase; D019791:Guanidine; C030526:tendamistate