Effects of mutations on hydrogen exchange kinetics, structure, and stability suggest that the slow exchange core is a key element in protein folding. Single amino acid variants of bovine pancreatic trypsin inhibitor (BPTI) have been made with glycine or alanine replacement of residues Tyr 35, Gly 37, Asn 43, and Asn 44. The crystal structures of Y35G and N43G are reported [Housset, D., Kim, K.-S., Fuchs, J., & Woodward, C. (1991) J. Mol. Biol. 220, 757-770; Danishefsky, A. T., Housset, D., Kim, K.-S., Tao, F., Fuchs, J., Woodward, C., & Wlodawer, A. (1993) Protein Sci. 2, 577-587; Kim, K.-S., Tao, F., Fuchs, J. A., Danishefsky, A. T., Housset, D., Wlodawer, A., & Woodward, C. (1993a) Protein Sci. 2, 588-596]. NMR chemical shifts indicate few changes from the wild type (WT) in G37A and N44G. Stabilities of the four mutants were measured by calorimetry and by hydrogen exchange. Values of delta delta(WT-->mut), the difference in delta G of folding/unfolding between the wild type and mutant, estimated by both methods are in good agreement and are in the range 4.7-6.0 kcal/mol. There is no general correlation between stability and hydrogen exchange rates at pH 3.5 and 30 degrees C. Exchange occurs by two parallel pathways, one involving small noncooperative fluctuations of the native state, and the other involving cooperative, global unfolding. In the mutant proteins, the rates for exchange by the unfolding mechanism are accelerated by a factor corresponding to the increase in the unfolding/folding equilibrium constant.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 272, 273, 274, 275, 276, 11766, 11767, 11768, 11769, 11770 Extra Details: protein folding; BPTI; calorimetry; hydrogen exchange kinetics;,fluctuation; unfolding/folding equilibrium constant
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:14 p.m.
|Number of data points||30|
|Proteins||Pancreatic trypsin inhibitor ; Pancreatic trypsin inhibitor ; Pancreatic trypsin inhibitor ; Pancreatic trypsin inhibitor|
|Assays/Quantities/Protocols||Experimental Assay: dCp ; Experimental Assay: dG ; Experimental Assay: dHcal ; Experimental Assay: Tm ; Derived Quantity: ddG ; Derived Quantity: dTm|
|Libraries||Mutations for sequence RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA|