Proline peptide isomerization and protein folding

Transcription

1 J. Biosci., Vol. 6, Number 4, October 1984, pp Printed in India. Proline peptide isomerization and protein folding A. SALAHUDDIN Department of Biochemistry, J. Ν. Medical College, Aligarh Muslim University, Aligarh , India MS received 6 August 1984 Abstract. The unfolding-refolding of proteins is a cooperative process and, as judged by equilibrium properties, occurs in one step involving the native, N, and the unfolded U, conformational states. Kinetic studies have shown that the denatured protein exists as a mixture of slow-(u S ) and fast-(u F ) refolding forms produced by proline peptide cis-trans isomerization. Proline residues in U F are in the same configuration as in the native protein while they are in non-native configuration in U S. For protein folding to occur quickly U S must be converted into U F. The fact that the equilibrium and kinetic properties of U S U F are the same as those found for proline cis-trans isomerization taken together with the absence of slow phase in the kinetics of refolding of a protein devoid of proline, support this view. However, the absence of a linear correlation between half-time of reactivation of denatured enzymes and their proline-contents, as well as the dissimilarities in the kinetic properties of U S U F in unfolding and refolding experiments are not consistent with the model. Conformational energy calculation and experimental results on refolding of proteins suggest that some proline residues are non-essential. They will not block protein folding even in wrong isomeric form. The native-like folded structure with incorrect proline isomers will serve as intermediate state(s) in which these prolines will more readily isomerize to the correct isomeric form. The picture becomes more complex when one considers the consequence of cis-trans isomerism of non-proline residues on protein folding. Keywords. Protein folding; cis-trans proline isomerization. Introduction Protein folding is a complex process through which the protein polypeptide chain acquires its native conformation under physiological conditions. What type of native structure a protein molecule will form is specified in its amino acid sequence which in turn is dictated by the specific base sequence of DNA. Why should a protein polypeptide chain fold? An eucaryotic cell cannot accommodate 3 4 million different polypeptides if all of them decide to exist in an unfolded form. Besides, unfolded proteins with exposed peptide bonds will be subject to enormous proteolytic hazards. Further, formation of a crevice in a protein molecule which serves as an active centre recognizing specific substrates/ligands is inconceivable without protein folding. Experimental approach to the problem of protein folding includes studies on unfolding and refolding reaction which involves the interconversion of the native (N) and the unfolded (U) conformational states of a protein. The native conformation is often compact and globular in which the protein molecule exhibits its characteristic Abbreviations used: NMR, nuclear magnetic resonance; BPTI, bovine pancreatic trypsin inhibitor. 349

2 350 Salahuddin biological activity, whereas the unfolded state is devoid of any element of native structure and exists as a structureless random coil (Tanford, 1968). Thus the reversible unfolding of proteins can be expressed as N U (1) For a number of relatively low molecular weight globular proteins the transition from Ν to U approximates to a two-state mechanism (Tanford, 1968, 1970; Privalov and Khechinashvili, 1974). The latter implies that states other than Ν and U, if any, exist only transiently and accumulates in quantities too low to have any marked effect on the properties (observables) that are employed in following the process 1. It should, however, be emphasized that conformational transitions within microstates of a macrostate (N or U) are perfectly feasible. Equilibrium (Tanford, 1968) and calorimetric (Privalov and Khechinashvili, 1974) results support the two-state hypothesis of protein unfolding refolding reaction, in contrast, a large body of kinetic data (Baldwin, 1975) on protein denaturations are not consistent with this hypothesis. The time course of unfolding and refolding reactions for a protein would obey the first order kinetic law if the two-state model is valid. On the other hand the kinetics for the reversible denaturation of several globular proteins (Baldwin, 1975) was not simple first-order but more complex. In fact the unfolding and refolding reactions were shown to occur in two kinetic phases, slow and fast phases (Baldwin, 1975). Two factors, among others, may account for the observed complexity. First, the transition Ν U may involve a kinetically stable intermediate state which can be detected in kinetic experiments but not in equilibrium studies. Alternatively, one need not involve the existence of a stable intermediate since the non-first order kinetics for unfoldingrefolding reaction of a protein can be obtained if there are more than one unfolded states that differ in the rates of refolding. As noted above, a fully unfolded protein behaves as a random coil free from any residual native structure, so that the difference between the kinetic behaviour of two different unfolded states cannot be attributed to the presence of varying residual native structures. Do they (unfolded states) refold to give back the same native state? The answer for this question is likely to be in the affirmative for most, if not all, proteins. Why do different unfolded states differ in their rates of folding? This difference according to Brandts et al. (1975), arises from the cis-trans isomerization. of proline residues in the unfolded protein. Thus the biphasic kinetics of refolding observed with several proteins are consistent with the scheme, U S U F N (2) where U S and U F are slow- and fast-refolding unfolded states. The two differ in cistrans configuration about the peptide bonds NH 2 -terminal to proline residues but are indistinguishable with respect to their spectroscopic properties (Brandts et al., 1975). As U S and U F do not significantly differ in enthalpy the two forms cannot be distinguished by calorimetric method. Therefore, the process 2 will be judged as a two-state process both by direct [calorimetric (Privalov and Khechinashvili, 1974)] and indirect [spectroscopic (Tanford, 1968)] methods. The fast refolding species (U F ) have proline residues in the same configuration (cis or trans) as they do in the native state, N, whereas the proline residues in the slow-refolding forms (U s ) exist in non-native or wrong

3 Proline isomerism and protein folding 351 configuration. The proline residues in non-native forms in U S must isomerize to the correct native configuration before the latter can refold to give the state Ν according to eq. 2. Therefore, the conversion of U S into U F would involve cis-trans isomerization, which as we shall see below is a slow process. Kinetic experiments have shown that proteins unfolded by acid and/or denaturant exist as a mixture of fast- and slowrefolding forms (Baldwin, 1975, 1978; Schmid and Baldwin, 1978; Kim and Baldwin, 1982). Structural constraints would greatly reduce the possibility of such cis-trans isomerism in the native state, N. Proline isomerization What are the lines of evidence that support cis-trans isomerization of an isolated proline in an unfolded protein? As proton or carbon nuclear magnetic resonance gives separate signals (resonances) for the cis and trans isomer this technique is ideally suited to investigate this issue. Based on nuclear magnetic resonance (NMR) data, it has been shown that proline-containing peptides exist as a mixture of cis and trans isomers (Brandts et al., 1975). Although the fraction of cis isomer appears to depend on nearby isonizable groups, bulkiness of the neighbouring residue and on the nature of solvent, available data suggest that an isolated proline in an unfolded polypeptide chain might possess about 10 30% cis character. Thus using [ 13 C]-NMR it was found that cis isomer in Ala-Pro is 11 % for the cationic form of the dipeptide and 35 % for the zwitterionic form. The ratio, cis/trans, is independent of temperature and the two isomers appears to have the same enthalpy. The kinetics of cis-trans isomerization of proline containing dipeptides in aqueous solution was first studied by Brandts et al. (1975) using ph jump technique in which the ph of an acidified (ph 1 8) dipeptide solution was raised suddenly (i.e. ph jump) by quickly adding a requisite volume of 1 Μ KOH; the transient ph can be followed by a ph meter equipped with a stripchart recorder. The dipeptides used were Gly-Pro, Ala- Pro, and Val-Pro; in which the bulkiness of the side chain of the N-terminal residue increases from Gly-Pro to Val-Pro. For a given peptide, the relaxation time for the cistrans isomerization decreased substantially upon lowering the ph between ph 5 and 3. The relaxation time for zwitter-ionic Ala-Pro at 22 5 C was 300 s while that for the cationic dipeptide was about 75 s. The isomerization process was expectedly slowest with Val-Pro followed by Ala-Pro and Gly-Pro due to the increase in the bulkiness of the adjacent residue. From the dependence of the relaxation time on temperature an activation energy, E a of 19 8 K cal was computed for Ala-Pro (Brandts et al., 1975). Similar E a values have been found for polyproline. However, it would not be safe to extrapolate quantitatively the conclusions based on studies on model proline peptides to isolated prolines in an unfolded proteins. But the conclusion that the proline isomerization is a slow process at room temperature would be true for proteins also. Interconversion of slow (U S ) and fast (U F ) refolding forms of proteins and proline cis trans transition There are several features of the slow phase. U S U F, of the protein refolding process which are shared by proline isomerization. For several proteins the activation energies

4 352 Salahuddin for the slow phase lie in the range Kcal/mol while the corresponding values for model proline containing peptides range from 16 to 23 K cal /mol. The relaxation time at 25 C for cis-trans proline isomerization is s (Schmid and Baldwin, 1978), whereas that for the process U S U F is 8 55 s for six small proteins (Brandts et al., 1975). Other similarities between proline isomerization and U S U F reaction for the best studied protein, ribonuclease A are summarized in table 1. Thus both equilibrium and kinetic properties showed high degree of similarity between the two processes. Table 1. Properties of proline isomerization and U S ribonuclease A*. U F reaction of * Taken from Schmid and Baldwin (1978). The experimental evidence for the involvement of cis-trans isomerization of proline residues in U S U F process has been provided, among others, by the kinetics of denaturation of parvalbumin (band 5) which is devoid of any proline residues. The characteristic slow-phase with relaxation time of s seen with the refolding of other globular protein (see table 1) could not be found in the denaturation of parvalbumin (Brandts et al., 1977; Lin and Brandts, 1978). Although, two fast kinetic phases were detected in parvalbumin denaturation the slowest one was about times faster than that found for the denaturation of proline-containing globular protein (see table 1). Further support to the cis-trans proline isomerism model comes from the results on the kinetics of slow phase reactivation of denaturated enzymes (Stellwagen, 1979). The half-times for the reactivation process was found between 9 s and 726 s. The activation energy for the reactivation of denaturated adenosine deaminase was determined to be 19 K cal /mol which is similar to that found for the process, U S U F (see table 1). Some of the basic assumptions of Brandts et al. (1975) proline isomerism model that adequately explain the formation of slow refolding species (U S ) according to eq. 2 above are as follows: (i) each proline residue will have an unique cis or trans configuration in the native protein where cis-trans isomerism would be negligible due to structural constraints; (ii) in the denatured protein, isomerization would give a mixture of cis and trans forms; (iii) each proline residue in the unfolded protein must be in the same configuration as in the native protein before refolding occur. Once the proline peptides in the denatural proteins are in proper isomeric forms protein

5 Proline isomerism and protein folding 353 molecules would quickly acquire their native conformation. It should, however, be emphasized that the model predicts that the kinetic properties of U S U F would be the same under native and denaturing conditions. But this prediction was found by actual experimentation to be not true (Schmid and Baldwin, 1978; Kim and Baldwin, 1982; Cook et al., 1979). While in unfolding experiments the kinetics features of U S U F were similar to those of proline cis-trans isomerization the kinetic properties in the refolding experiments were complex and showed dependence on ph, temperature and denaturant concentration. Further, the model predicts that greater the number of proline residues in a denatured protein/enzyme slower will be the rate of protein unfolding or reactivation. However, this prediction was not supported by actual experiments (Stellwagen, 1979). These results can be reconciled with the Brandts proposal if the third assumption is modified to read that "each essential proline residue in the unfolded protein must be in the same configuration as in the native protein before refolding occurs". This would mean that certain proline residues in a protein are essential and others are non-essential for the formation of folded protein conformation (Kim and Baldwin, 1982; Baldwin and Creighton, 1980). For protein folding to occur nonessential proline residues in a denatured protein need not isomerize to their native form. Even the wrong isomers can be accommodated in the native structure; they will not block the rapid protein folding. Refined X-ray data along with results on refolding of denatured protein (Baldwin, 1975; Kim and Baldwin, 1982; Baldwin and Creighton, 1980; Levitt, 1981) have demonstrated the existence of such non-essential prolines. Thus proline residues in the terminal sequence, interdomain connectors, and lobes can be tolerated in either configuration (cis or trans). In view of these considerations, it seems quite possible that segments containing non-essential proline residues in a denatured protein would rapidly fold to give rise to native-like intermediate state I, containing non-essential proline in wrong configuration. Results on unfolding of ribonuclease (Cook et al., 1979) taken together with the fact that many large proteins containing proline residues fold much faster (Creighton, 1980) than would be expected from Brandts model are consistent with the view. The eq. 2 for refolding of denatured proteins should, therefore, be revised as follows: (3) where I represents one, or a sequence of intermediate states containing proline peptides in wrong configuration. In the state I, the rate of proline isomerization can be enhanced as much as 50-fold. It is closer to the native state in conventional properties that are used in following the denaturation transition but may differ from Ν in other properties (Nall, 1983; Zuniga and Nall, 1983; Muller and Garel, 1984; Schmid and Blaschek, 1984). Both the pathways i. e. (a) U S I N and (b) U S U F N lead to the formation of the native protein. Which one of the two pathways will prevail? According to Levitt (1981) the refolding route will be determined by the difference in the conformational energies of states I and N. If the difference is large the pathway (b) will be more important and the refolding process will approximate to Brandts model. But if the difference is small the first pathway (a) will predominate and significant Β 2

6 354 Salahuddin amounts of intermediate state (b), I, will be formed under native conditions. In bovine pancreatic trypsin inhibitor (BPTI) the four proline residues are in trans configuration in the native state, N. Proline in cis forms can be incorporated in the folded protein with small conformational change to give rise to a native-like state, I. Incorporation of a proline residue in wrong isomeric form will destabilize the folded conformation. The strain (or destabilization) energy can be calculated (Levitt, 1981). It turns out, that there are three types of proline residues in BPTI and probably in other proteins. Type I includes those residues which can be freely accommodated in either configuration in the native protein e.g. Pro-13 in BPTI in which case the strain energy for the incorporation of its cis form was calculated to be only 1 K cal /mol. Type II proline residues significantly destabilize native protein conformation when incorporated in wrong isomeric forms but the strain or destabilization energy is not so large as to prevent protein folding. Pro-2 and Pro-9 of BPTI for which the strain (or destabilization) energy is II K cal /mol belong to this category. Type III proline residues if forced into the native protein as incorrect isomers will destabilize the native protein to such an extent that the protein folding is blocked and can occur only upon isomerization of the proline residues to the correct isomeric forms. For example, Pro-8 in BPTI if inserted in the protein structure in cis form will destabilize the protein by 33 K cal /mol. It must isomerize to its trans form before refolding can take place. In such situations the refolding of proteins will occur through the pathway (b) involving the slow process, U S U F as envisaged in Brandts proposal. The molecules of BPTI having types I and II proline residues will refold much more rapidly than those with type III residues. The proportion of fast refolding BPTI molecules was calculated to be 77 % (Levitt, 1981). This is in excellent agreement with the results on refolding on slightly modified BPTI (Jullien and Baldwin, 1981) where the relative amplitude of the fast refolding U F Ν reaction was found to be 75 %. Type III behaviour could not be seen with BPTI. It would, therefore, seem that an unfolded polypeptide chain rapidly folds to a compact folded conformation in which proline residues can find their correct native configuration at leisure. However, any generalization in this rapidly changing area is likely to be refuted in future, since we have considered here only the consequences of proline isomerism on protein folding. Isomerization of non-proline peptide bonds, although sterically and energetically less probable (Levitt, 1981; Ramachandran and Mitra, 1976), cannot be ruled out. That such bonds exist in actual proteins is evident from the recent X-ray data on carboxypeptidase A showing three non-proline peptide bonds in cis configuration (Rees et al., 1981). Acknowledgements Financial support from the Council of Scientific and Industrial Research, New Delhi, and facilities from the Aligarh Muslim University, Aligarh are gratefully acknowledged. References Baldwin, R. L. (1975) Ann. Rev. Biochem., 44, 453. Baldwin, R. L. (1978) Trends Biochem. Sci., 3, 66.

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