Kinetic Mechanism of a Partial Folding Reaction. 1. Properties of the Reaction and Effects of Denaturants

Abstract

The bimolecular association rate constant (k on ) and dissociation rate constant (k off ) of the complex between fluorescein-labeled S-peptide analogues and folded S-protein are reported. This is the first kinetic study of a protein folding reaction in which most of the starting material is already folded and only a small part (one additional helix) becomes ordered; it provides a folding landscape with a small conformational entropy barrier, and one in which kinetic traps are unlikely. Refolding and unfolding are measured under identical strongly native conditions, and the reaction is found to be two-state at low reactant concentrations. The dissociation constant (K d ) of the complex and the properties of the transition state may be calculated from the rate constants without extrapolation. The folded complex is formed fast (k on ) 1.8 × 10 7 M -1 s -1 ) and is very stable (K d ) 6 pM) at 10°C, 10 mM MOPS, pH 6.7. Charge interactions stabilize the complex by 1.4 kcal mol -1 . The charge effect enters in the refolding reaction: increasing the salt concentration reduces k on dramatically and has little effect on k off . Urea and GdmCl destabilize the complex by decreasing k on and increasing k off . The slopes (m-values) of plots of ln K d vs [cosolvent] are 0.75 ( 0.04 and 2.8 ( 0.3 kcal mol -1 M -1 for urea and GdmCl, respectively. The ratio m on /(m on + m off ) is 0.54 ( 0.04 for urea and 0.57 ( 0.1 for GdmCl, where m on is the m-value for k on and m off is the m-value for k off , indicating that more than half of the sites for interaction with either cosolvent are buried in the ensemble of structures present at the transition state.The refolding kinetics of numerous small proteins obey simple exponential kinetics, indicating that intermediates are not populated and that these polypeptides cross a free-energy barrier before reaching their native states (for recent examples see refs 1 and 2). This free energy barrier may be the wall of a kinetic trap or a decrease in conformational entropy before the formation of stabilizing protein-protein interactions (3, 4). There may also be an enthalpic barrier in simple refolding reactions (5). Both the depth of kinetic traps and the height of the conformational entropy barrier to refolding are expected to decrease dramatically with decreasing chain length, but the shortest stable proteins are of the order of 60 residues. We present data for the reaction where p is a fluorescently labeled S-peptide analogue, N is the folded form of S-protein, and pN is the S-peptide analogue/S-protein (RNaseS* 1 ) complex. Our aim is to characterize the free energy barrier on a folding landscape which has a low conformational entropy barrier and is unlikely to contain deep kinetic traps. The conformational entropy lost during folding is chiefly that of the 15 residue S-peptide analogue.Protein folding reactions with exponential time courses may be well described by the transition state theory (6). The observed rate constant of such a reaction is the product K q k q , where K q ...

Mapping backbone and side-chain interactions in the transition state of a coupled protein folding and binding reaction

“…the final folding reaction from the encounter complex to native RNase S. Partly contradictory results on the structure of the transition state for association were obtained from a fluoresceinlabeled S-peptide, which binds 40-times faster than the wild-type S-peptide in a process that is accelerated by electrostatic interactions (12,34,35). In contrast, electrostatic contributions slightly slow down association of the unlabeled peptide as indicated by the slightly decreased k on at low ionic strength (Table 2).…”

A Universal Strategy for Proteomic Studies of SUMO and Other Ubiquitin-like Modifiers

“…The resulting cell lysate was clarified and incubated with an IMAC matrix, the IMAC beads were washed extensively, and the bound proteins were eluted by the use of a low pH buffer. For the second affinity purification stage, different buffer conditions were tested as the interaction between the S-peptide and the S-protein is affected by pH, salt, and urea (43)(44)(45)(46). The use of a low urea, low salt, and high pH buffer proved to yield the best recovery of tagged proteins, and was therefore incorporated into the TAP protocol.…”

Osmolytes Stabilize Ribonuclease S by Stabilizing Its Fragments S Protein and S Peptide to Compact Folding-competent States

“…S pro has also not been crystallized. Native state concentrationdependent hydrogen exchange studies using two-dimensional NMR have been used to get indirect information on the structure of S pro in its free state (10).The binding of S pep to S pro is one of the last steps in the folding pathway of RNase A (16) and the S pro⅐S pep interaction is a model system to study protein folding and stability (12,17,18). In the present work we have examined the effects of * This work was supported by grants from the Council of Scientific and Industrial Research, the Department of Science and Technology, and the Department of Biotechnology (India) (to R. V.).…”

“…RNase S is a complex of two fragments, the N-terminal S peptide (S pep; residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and the C-terminal S protein (S pro; residues 21-124). The fragments form a 1:1 complex in solution, and RNase S has properties similar to its parent molecule RNase A, in terms of structure (9), activity as well as dynamics (10).…”

“…The binding of S pep to S pro is one of the last steps in the folding pathway of RNase A (16) and the S pro⅐S pep interaction is a model system to study protein folding and stability (12,17,18). In the present work we have examined the effects of osmolytes on the structure and stability of RNase S. There are several advantages of using a bimolecular fragment complementation system such as RNase S, as opposed to a monomeric protein for these studies.…”