Shape of the free energy barriers for protein folding probed by multiple perturbation analysis.
The characterization of the free energy barriers has been a major goal in studies on the mechanism of protein folding. Testing the effect of mutations or denaturants on protein folding reactions revealed that transition state movement is rare, suggesting that folding barriers are robust and narrow maxima on the free energy landscape. Here we demonstrate that the application of multiple perturbations allows the observation of small transition state movements that escape detection in single perturbation experiments. We used tendamistat as a model protein to test the broadness of the free energy barriers. Tendamistat folds over two consecutive transition states and through a high-energy intermediate. Measuring the combined effect of temperature and denaturant on the position of the transition state in the wild-type protein and in several mutants revealed that the early transition state shows significant transition state movement. Its accessible surface area state becomes more native-like with destabilization of the native state by temperature. To the same extent, the entropy of the early transition state becomes more native-like with increasing denaturant concentration, in accordance with Hammond behavior. The position of the late transition state, in contrast, is much less sensitive to the applied perturbations. These results suggest that the barriers in protein folding become increasingly narrow as the folding polypeptide chain approaches the native state.
Schätzle
Manuela
M
Department of Biophysical Chemistry, Biozentrum der Universität Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland.
Kiefhaber
Thomas
T
D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013696:Temperature; D013816:Thermodynamics
D004791:Enzyme Inhibitors; D010455:Peptides; C030526:tendamistate