Prolyl isomerase as a probe of stability of slow-folding intermediates.
Catalysis of slow folding reactions by peptidyl prolyl cis-trans isomerase (PPI) provides estimates of stabilities of intermediates in folding of normal and mutational variants of yeast iso-2 cytochrome c. A two-state model postulating a rapid preequilibration of intermediates with the unfolded protein is employed to calculate the stabilization free energy of the intermediate from the catalytic efficiency (kcat/Km) of PPI toward slow folding species. Stability measurements have been made for two distinct slow-folding intermediates: the absorbance-detected (IIS) and fluorescence-detected (IIIS) intermediates. Mutation-induced changes in the stability of the intermediates and in the activation free energy for slow folding are compared to changes in equilibrium thermodynamic stability. The results show that (1) for iso-2 the absorbance-detected intermediates (IIS) are slightly more stable than the fluorescence-detected intermediates (IIIS), (2) most mutations have different effects on equilibrium stability and the stability of the IIS or IIIS intermediates, and (3) for both slow folding reactions the mutation-induced changes in the activation free energy are small compared to the magnitude of the activation free energy barrier. Differential effects of mutations on equilibrium stability and the stability of intermediates provides a means of assessing the sequence-encoded structural specificity for folding. Mutations with different effects on intermediate stability and equilibrium stability change the encoded folding information and may alter folding pathways and/or lead to different three-dimensional structures. Identification of mutations which stabilize a folding intermediate relative to the native conformation provides an empirical approach to the design of thermodynamically stable forms of folding intermediates.
Veeraraghavan
S
S
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Rodriguez-Ghidarpour
S
S
MacKinnon
C
C
McGee
W A
WA
Pierce
M M
MM
Nall
B T
BT
D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D019791:Guanidine; D006146:Guanidines; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed; D019696:Peptidylprolyl Isomerase; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
D002352:Carrier Proteins; D006146:Guanidines; D015335:Molecular Probes; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase; D019791:Guanidine