Jean-Renaud Garel

Test of the extended two-state model for the kinetic intermediates observed in the folding transition of ribonuclease A☆

Abstract A test has been made of the proposal that: (a) the extended two-state model describes the kinetic intermediates seen in the folding transition of RNAase A, i.e. that the only species present in folding experiments are the native protein and multiple forms of the completely unfolded protein; and (b) that the interconversion between the two known unfolded forms of RNAase A (the U1 U2 reaction) is described solely by the cis-trans isomerization of the proline residues. The test is to measure the rate of the U1 U2 reaction in a wide range of refolding conditions and to compare these data with the kinetic properties of proline isomerization. The main results are as follows. (1) The activation enthalpy of the U1 U2 reaction in refolding conditions (pH 6, 20 ° to 40 °C) is less than 5 kcal/mol. This is much too small to be explained as proline isomerization. (2) Both the rate and the activation enthalpy change sharply at guanidine hydrochloride concentrations below 2 m . There appear to be two pathways for the U1 U2 reaction in refolding conditions, and the slower pathway is favored by adding guanidine hydrochloride. (3) The rate and activation enthalpy for proline isomerization in l -alanyl- l -proline are unaffected by 2 m -guanidine hydrochloride. The results show that the proline isomerization hypothesis and the extended two-state model cannot both be correct for RNAase A. They suggest that partial folding occurs rapidly in refolding conditions and that the extended two-state model is invalid. They leave open the question of whether or not proline isomerization is the rate-limiting step in the U1 U2 reaction. Another possible source of slow configurational reactions in the unfolded state is mentioned. The three major, overlapping, disulfide-bonded loops of RNAase A can exist in two isomeric configurations. Interconversion of these isomers requires pulling one loop, or one end of the polypeptide chain, through a second loop and this is likely to be a slow process. In some conditions, heat-unfolded but not guanidine-unfolded RNAase A shows a second slow-refolding process. It may result from aggregates of the heatunfolded protein which are formed and broken up slowly. Conditions are given for eliminating this reaction.

The heat-unfolded state of ribonuclease A is an equilibrium mixture of fast and slow refolding species.

A pH-dependent equilibrium between different species of heat-unfolded ribonuclease A has been studied by measuring the dependence on initial pH of the ratio of fast to slow refolding material; refolding is induced by stopped-flow pH jumps in which the final pH is held constant. These different unfolded forms are shown to be responsible for the fast- and slow-refolding reactions of ribonuclease A studied previously. Their interconversion is slow compared with the stopped-flow mixing time (3 ms). The results fit a simple kinetic mechanism for refolding U1slow U2fast, in which U1 and U2 are slow- and fast-refolding species. The fast-refolding form is not “melted out” by raising the temperature above the transition zone for thermal unfolding.

A physical difference between the fast- and slow-refolding forms of nitrotyrosyl ribonuclease A: the pK values of the nitrotyrosyl groups.

Abstract In the preceding paper we present kinetic evidence for a slow equilibrium between two conformational forms of heat-unfolded ribonuclease A whose rates of refolding differ 100-fold. In a search for physical differences between these two forms, we undertook a study of the p K changes during refolding of a specific set of freely ionizing surface groups. By use of a standard procedure the three freely ionizing tyrosine groups (p K ∼- 10) have been nitrated by tetranitromethane, yielding three nitrotyrosine groups (p K ∼- 6.8). Nitrotyrosyl ribonuclease A closely resembles the unmodified enzyme as regards: (1) enzymatic activity; (2) thermal unfolding transition at neutral pH; and (3) kinetics of refolding. In particular, stopped-flow measurements of 2′CMP binding during refolding show that the fast-refolding reaction is unchanged by nitration and yields fully folded enzyme able to bind 2′CMP. The p K change of the nitrotyrosyl groups upon refolding is quite different in the fast- and slow-refolding reactions. In the slow reaction it is small (− 0.046 ± 0.006 pH unit) but easily measureable, whereas in the fast-reaction it is too small to be detected (− Δp K less than 0.02 pH unit). This difference in p K change upon refolding can be attributed to different p K values of the nitrotyrosyl groups in the slow-refolding and fast-refolding forms of the heat-unfolded protein. Presumably the same structural differences between these two heat-unfolded forms are responsible both for the p K difference and for the 100-fold difference in rates of refolding. These results support the simple three-species mechanism for refolding discussed in the preceding paper. (a) They demonstrate a physical difference between the fast- and slow-refolding species. (b) They do not show any additional kinetic complexity when refolding is measured by a property that distinguishes between the fast- and slow-refolding species.

Binding interactions between two ligands and a monomeric protein: Study on indole, protons and chymotrypsin☆

Abstract The binding of indole and protons to chymotrypsin has been studied by polarimetry and potentiometry. δ-Chymotrypsin is able to assume two discrete conformations E and E′. The conversion of E′ to E can result from the binding of a proton to the α-amino group of the N-terminal isoleucine number 16, from the binding of a molecule of indole to the active site of the enzyme, or from the binding of both ligands. These two conformations are in equilibrium; the equilibrium is completely shifted by the binding of these ligands to their specific site. In the absence of indole the E conformation is maintained by the NH3staggered +COO− interaction between isoleucine 16 and aspartic acid 194. In the absence of the proton on this isoleucine residue the E conformation is maintained by indole. The protonation of the isoleucine 16 residue decreases the binding constant of indole and the presence of indole decreases the binding constant of the proton. The pK of isoleucine 16 is shifted from less than eight in the E′ state to 10.5 in the E state and the conversion of E′ to E upon binding indole is accompanied by a proton uptake. These results are correlated with the crystallographic data available for chymotrypsin and with the similar transition observed at acidic pH.

Early steps in the refolding reaction of reduced ribonuclease A

Abstract The early part of the reaction of refolding of reduced ribonuclease A has been studied using the reappearance of enzymatic activity as an index of refolding. It is found that a low level of activity, about 0.04% of that of native enzyme, can be measured early after refolding has been initiated. This low level of activity is apparently not due to a contaminant or to incompletely reduced RNAase A molecules, but rather seems to be a property of the bulk of the reduced protein. Furthermore, this early activity is sensitive to the reaction with N -ethyl-maleimide, showing that it is due to completely or partially reduced molecules. The amount of protein responsible for this early activity represents a small fraction of the total reduced RNAase A, and possesses binding properties similar to those of the native enzyme towards a substrate, 2′, 3′ CMP and an inhibitor, 2′ CMP. These results are interpreted as evidence for the existence of an equilibrium between native and unfolded conformations in reduced RNAase A, and are discussed with respect to the protein folding mechanism.

Detection of the homology among proteins by immunochemical cross-reactivity between denatured antigens: Application to the glyceraldehyde 3-phosphate dehydrogenases from different species

Abstract It was previously proposed that an immunological cross-reaction between two denatured proteins is evidence for an homology betweeen their amino sequence (Arnon & Maron, 1971; Arnheim et al ., 1971) and that detection of such a cross-reaction could then be a rapid method to detect sequence homologies (Zakin et al ., 1978). In order to test the possibilities of such a methodology, using proteins of known structure, glyceraldehyde 3-phosphate dehydrogenases from different sources are compared by immunochemical techniques. The antibodies raised against the native enzyme from E. coli K 12 can only recognize the homologous antigen, the glyceraldehyde 3-phosphate dehydrogenase from B. stearothermophilus and to a lesser extent that from halibut. In contrast, the antibodies raised against the denatured enzyme from E. coli K 12 can recognize the glyceraldehyde 3-phosphate dehydrogenases from man, ostrich, chicken, sturgeon, halibut, lobster and yeast, when in their denatured state. The present results show unambiguously that through exposure of buried sequences, the immunochemical detection of sequence homologies among proteins is more discriminating when unfolded proteins are used, rather than native ones. It is also proposed that the use of denatured proteins both as immunogens and antigens would be a useful tool in studying biochemical evolution.

Kinetics of reduction of the disulfide bonds in ribonuclease A.

Breaking the disulphide bonds is one of the steps involved in the complete denaturation of proteins which have such intramolecular crosslinks. This breaking is usually achieved by reduction with a thiol reagent, using a thiol concentration of 0.1 M or above, in the presence of a denaturing agent, at pH values above 8, and for a few hours at 25°C or room temperature [ 1,2] . These conditions have been shown to indeed lead to a completely reduced protein. We have attempted to follow the kinetics of reduction of the disulphide bonds of ribonuclease in these almost standard conditions, and we have found that this kinetics was too fast to be measured. We have therefore studied the range of conditions under which the kinetics of reduction of disulphide bonds could be observed, and this article reports some results about the kinetics of reduction of ribonuclease by P-mercaptoethanol. It is found that complete reduction of ribonuclease by /3-mercaptoethanol can be achieved in milder conditions than those generally used, conditions under which the kinetics of reduction can be observed.

Optical measurement of a solvent-induced isomerization in a proline-containing hexapeptide

Abstract The hexapeptide Gly-Gly-Pro-Tyr-Gly-Gly has been synthesized and its tyrosine residue converted to nitrotyrosine by reaction with tetranitromethane. When diluted from dimethylsulfoxide into aqueous solution, the nitrated hexapeptide undergoes a slow conformational change characterized by a change in the ionization state of the nitrotyrosine group. This slow reaction is not observed with peptides containing nitrotyrosine and no proline. Also, the rate and activation enthalpy of this slow conformational change suggest that it could be due to proline cis-trans isomerization. The possibility of measuring the rate of cis-trans isomerization of proline residues in a polypeptide chain is discussed.

Refolding kinetics of nitrated ribonuclease A

Summary The refolding from concentrated Gu-HCl of nitrated RNase A is measured by the changes in absorbance of the three exposed nitrotyrosines. The slow refolding kinetics are complex, with a major faster phase and a minor slower phase (amplitude ratio: 80%–20%). The major phase resembles the main folding reaction of unmodified RNase A in the dependence on Gu-HCl of its rate and activation enthalpy. The minor phase has both a rate and activation enthalpy independent of Gu-HCl. These biphasic kinetics are observed in conditions where the folding reaction is probably rate-limited by proline cis - trans isomerization (in 2 M final Gu-HCl), suggesting that different prolines of the polypeptide chain are the origin of these complex kinetics.