Search PubMed⌕ Search

Biomedical subjects

S Veeraraghavan

Publications and source records attributed to S Veeraraghavan.

27 records · Page 2Linked to original sources
PubMed1996

Autocatalyzed protein folding.

Proline isomerization, an intrinsically slow process, kinetically traps intermediates in slow protein folding reactions. Thus, enzymes that catalyze proline isomerization (prolyl isomerases) often catalyze protein folding. We have investigated the folding kinetics of FKBP, a prolyl isomerase. The main conclusion is that FKBP catalyzes its own folding. Altogether, the FKBP refolding kinetics are resolved into three exponential phases: a fast phase, tau 3; an intermediate phase, tau 2; and a slow phase, tau 1. Unfolding occurs in a single phase, the unfolding branch of phase tau 2. In the presence of native FKBP, both the intermediate (tau 2) and slow (tau 1) phases are faster, suggesting that folding phases tau 1 and tau 2 involve proline cis-trans isomerization. In the absence of added native FKBP, autocatalytic folding of FKBP is detected. For refolding starting with all the FKBP unfolded initially, the slowest folding phase (tau 1) is almost 2-fold faster at a final concentration of 14 microM FKBP than at 2 microM FKBP, suggesting that catalytically active FKBP formed in the fast (tau 3) or intermediate (tau 2) folding phases catalyzes the slow folding phase (tau 1). Moreover, autocatalysis of folding is inhibited by FK506, an inhibitor of the FKBP prolyl isomerase activity. The results show that the slow phase in FKBP folding is an autocatalyzed formation of native FKBP from kinetically trapped species with non-native proline isomers. While the magnitude of the catalytic effects reported here are modest, FKBP folding may provide a prototype for autocatalysis of kinetically trapped macromolecular conformational changes in other systems.

Amino Acid Isomerases↗
PubMed1995

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.

Amino Acid Isomerases↗
PubMed1994

Characterization of folding intermediates using prolyl isomerase.

Structure-reactivity relationships of human peptidyl prolyl cis-trans isomerase (PPI) toward the two slow folding reactions of yeast iso-2 cytochrome c have been used to characterize the structure of folding intermediates in the vicinity of critical prolines. We propose that the relative catalytic efficiency of PPI for the protein substrate relative to a peptide substrate, (kcat/Km)rel, is a measure of structure in folding intermediates. The structural stability of slow-folding intermediates as detected by changes in (kcat/Km)rel was investigated using two structural perturbants: guanidine hydrochloride and site-directed mutagenesis. Neither of the two slow folding reactions for wild-type cytochrome c is catalyzed at low denaturant concentrations. However, both phases are catalyzed at moderate concentrations of guanidine hydrochloride. A mutation in cytochrome c enhances catalysis of the fluorescence-detected slow folding phase. For protein substrates destabilized by denaturants or mutation, we suggest that increases in (kcat/Km)rel result from a loosening of the substrate structure, providing better access of peptidyl prolyl isomerase to critical proline(s).

Amino Acid Isomerases↗
PubMed1989

A form of protease nexin I is expressed on the platelet surface during platelet activation.

A protein that has several similarities to protease nexin I, a fibroblast thrombin and urokinase inhibitor, has been detected on platelets (Gronke RS, Bergman BL, and Baker JB: J Biol Chem 262:3030, 1987). On incubation of platelets with 125I-thrombin, this platelet protein forms complexes with 125I-thrombin that are found both in the incubation medium and, as demonstrated here, associated with purified platelet plasma membranes. The present results indicate that interaction with the platelet surface may modulate the conformation and function of this platelet form of protease nexin I (PNIp) because: (a) an antibody against protease nexin I inhibited released PNIp, but not platelet-bound PNIp from complexing 125I-thrombin, and (b) whereas PNIp extracted from platelets bound both thrombin and urokinase, platelet-bound PNIp bound only thrombin. In experiments using several different platelet isolation methods, PNIp accounted for a large fraction of the rapid high affinity binding of 125I-thrombin to platelets. However, platelets isolated and maintained in the presence of metabolic inhibitors failed to take added thrombin into 125I-thrombin-PNIp complexes. This finding suggests that PNIp is released from inside platelets during activation, and thus does not function to transmit the primary activating signal that is generated by thrombin binding to platelets.

Amyloid beta-Protein Precursor↗