Stress and strain in staphylococcal nuclease.

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease.

The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His124, is 1.9 kcal.mol-1 less stable (at pH 5.5 and 20 degrees C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains Leu124. In addition, the population of the trans conformer at the Lys116-Pro117 peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease H124L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and H124I variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys116-Pro117 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys116-Pro117. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro117 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H124L, and compared it with the published high-resolution X-ray structure of the cis form of the most stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the trans/cis equilibrium of the Lys116-Pro117 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination technique. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the His46-Pro47 peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L + P117G and H124L + P47G + P117G and the kinetic parameters of H124L, H124L + P47G, H124L + P117G, and H124L + P47G + P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kcat affected more than Km, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conformational rearrangement described for the P117G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 33:5021-5030). In both P117G mutants, the loop formed by residues 112-117 is located closer to the adjacent loop formed by residues 77-85, and residues 115-118 adopt a type I' beta-turn conformation with the Lys116-Gly117 peptide bond in the trans configuration, as compared with the parent protein in which these residues have a typeVIa beta-turn conformation with the Lys116-Pro117 peptide bond in the cis configuration. Addition of the P47G mutation appears not to cause any additional structural changes. However, the electron density for part of the loop containing this peptide bond was not strong enough to be interpreted.     

The importance of anchorage in determining a strained protein loop conformation.

We examine the role of the conformational restriction imposed by constrained ends of a protein loop on the determination of a strained loop conformation. The Lys 116-Pro 117 peptide bond of staphylococcal nuclease A exists in equilibrium between the cis and trans isomers. The folded protein favors the strained cis isomer with an occupancy of 90%. This peptide bond is contained in a solvent-exposed, flexible loop of residues 112-117 whose ends are anchored by Val 111 and Asn 118. Asn 118 is constrained by 2 side-chain hydrogen bonds. We investigate the importance of this constraint by replacing Asn 118 with aspartate, alanine, and glycine. We found that removing 1 or more of the hydrogen bonds observed in Asn 118 stabilizes the trans configuration over the cis configuration. By protonating the Asp 118 side chain of N118D through decreased pH, the hydrogen bonding character of Asp 118 approached that of Asn 118 in nuclease A, and the cis configuration was stabilized relative to the trans configuration. These data suggest that the rigid anchoring of the loop end is important in establishing the strained cis conformation. The segment of residues 112-117 in nuclease A provides a promising model system for study of the basic principles that determine polypeptide conformations. Such studies could be useful in the rational design or redesign of protein molecules.     

NMR analysis of staphylococcal nuclease thermal quench refolding kinetics.

Thermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 degree C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116-Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis-trans equilibrium. The rate of trans-->cis isomerization in the native-like nuclease intermediate is approximately 100-fold faster than that observed in a Lys-Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116-Pro 117 peptide bond is comparable to that observed for other X-Pro isomerizations.     

A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease.

Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.     

Coupling between local structure and global stability of a protein: mutants of staphylococcal nuclease

Staphylococcal nuclease exists in solution as a mixture of two folded (N and N') and two unfolded (U and U*) forms. Earlier workers [Evans et al. (1989) Biochemistry 28, 362] have proposed that the N'/N and U/U* structural differences involve cis/trans isomerization about the Lys116-Pro117 peptide bond with N and U cis and N' and U* trans. The present results show that residue changes throughout the nuclease structure have large effects on the distribution of the N and N'forms. The N'/N ratios at 313 K for nuclease H124L (N'/N = 0.07) and nuclease G79S (N'/N = 12) differ by 2 orders of magnitude. Thermodynamic parameters for equilibria linking the two folded and two unfolded substates were evaluated for seven mutants of nuclease which were found by kinetic assays to have similar enzymatic activities but by NMR spectroscopy to have a wide dispersion of thermal stabilities. Our results indicate that mutational perturbations of the N'/N equilibrium in folded nuclease (delta G for the N in equilibrium N' reaction) are strongly coupled to changes in the stability of the N form (delta G for the N in equilibrium U reaction), but much less so to the stability of the N' form (delta G for the N' in equilibrium U* reaction).     

Inhibition of beta-lactamase by clavulanate. Trapped intermediates in cryocrystallographic studies.

Crystallographic studies of the complex between beta-lactamase and clavulanate reveal a structure of two acyl-enzymes with covalent bonds at the active site Ser70, representing two different stages of inhibitor degradation alternately occupying the active site. Models that are consistent with biochemical data are derived from the electron density map and refined at 2.2 A resolution: cis enamine, in which the carboxylate group of the clavulanate molecule makes a salt bridge with Lys234 of beta-lactamase; decarboxylated trans enamine, which is oriented away from Lys234. For both acyl-enzymes, the carbonyl oxygen atom of the ester group occupies the oxyanion hole in a manner similar to that found in inhibitor binding to serine proteases. Whereas the oxygen atom in the trans product is optimally positioned in the oxyanion hole, that of the cis product clashes with the main-chain nitrogen atom of Ser70 and the beta-carbon atom of the adjacent Ala69. In contrast to cis to trans isomerization in solution that relieves the steric strain inherent in a cis double bond, at the enzyme-inhibitor interface two additional factors play an important role. The salt bridge enhances the stability of the cis product, while the steric strain introduced by the short contacts with the protein reduces its stability.     

Native-like partially folded conformations and folding process revealed in the N-terminal large fragments of staphylococcal nuclease: a study by NMR spectroscopy

The N-terminal large fragments of staphylococcal nuclease (SNase), SNase110 (1-110 residues), SNase121 (1-121 residues), and SNase135 (1-135 residues), and the fragment mutants G88W110, G88W121, V66W110 and V66W121 were studied by heteronuclear multidimensional NMR spectroscopy. Ensembles of co-existent native-like partially folded and unfolded states were observed for fragments. The persistent native-like tertiary interaction drives fragments to be in partially folded states, which reveal native-like beta-barrel conformations. G88W and V66W mutations modulate the extent of inherent native-like tertiary interaction in fragment molecules, and in consequence, fragment mutants fold into native-like beta-subdomain conformations. In cooperation with the inherent tertiary interaction, 2 M TMAO (trimethylamine N-oxide) can promote the folding reaction of fragments through the changes of unfolding free energy, and a native-like beta-subdomain conformation is observed when the chain length contains 135 residues. Heterogeneous partially folded conformations of 1-121 and 1-135 fragments due to cis and trans X-prolyl bond of Lys116-Pro117 make a non-unique folding pathway of fragments. The folding reaction of fragments can be characterized as a hierarchical process.     

Restricted backbone conformational and motional flexibilities of loops containing peptidyl-proline bonds dominate the enzyme activity of staphylococcal nuclease

The role of cis-trans isomerizations of peptidyl-proline bonds in the enzyme activity of staphylococcal nuclease (SNase) was examined by mutation of proline residues. The proline-free SNase ([Pro-]SNase), namely, P11A/P31A/P42A/P47T/P56A/P117G-mutant SNase, was adopted for elucidating the correlation between the nuclease activity and the backbone conformational and dynamic states of SNase. The 3D solution structure of [Pro-]SNase has been determined by heteronuclear NMR experiments. Comparing the structure of [Pro-]SNase with the structure of SNase revealed the conformational differences between the two proteins. In the structure of [Pro-]SNase, conformational rearrangements were observed for the loop of residues Ala112-His121 containing a trans Lys116-Gly117 peptide bond and for the C-terminal alpha-helical loop of residues Leu137-Glu142. Mutation of proline at position 117 also caused the conformational rearrangement of the p-loop (Asp77-Leu89), which is remote from the Ala112-His121 loop. The Ala112-His121 loop and p-loop are placed closer to each other in [Pro-]SNase than in SNase. The backbone dynamic features of the omega-loop (Pro42-Pro56) of SNase are different from those of [Pro-]SNase. The backbone of the omega-loop exhibits restricted flexibility with slow conformational exchange motions in SNase, but is highly flexible in [Pro-]SNase. The analysis indicates that the restrained backbone conformation of the Ala112-His121 loop and restricted flexibility of the omega-loop are two dominant factors determining the enzyme activity of SNase. Of the two factors, the former is correlated with the strained cis Lys116-Pro117 peptide bond and the latter is correlated with the cis-trans isomerizations of the His46-Pro47 peptide bond.     

Structural mechanism governing cis and trans isomeric states and an intramolecular switch for cis/trans isomerization of a non-proline peptide bond observed in crystal structures of scorpion toxins

Non-proline cis peptide bonds have been observed in numerous protein crystal structures even though the energetic barrier to this conformation is significant and no non-prolyl-cis/trans-isomerase has been identified to date. While some external factors, such as metal binding or co-factor interaction, have been identified that appear to induce cis/trans isomerization of non-proline peptide bonds, the intrinsic structural basis for their existence and the mechanism governing cis/trans isomerization in proteins remains poorly understood. Here, we report the crystal structure of a newly isolated neurotoxin, the scorpion alpha-like toxin Buthus martensii Karsch (BmK) M7, at 1.4A resolution. BmK M7 crystallizes as a dimer in which the identical non-proline peptide bond between residues 9 and 10 exists either in the cis conformation or as a mixture of cis and trans conformations in either monomer. We also determined the crystal structures of several mutants of BmK M1, a representative scorpion alpha-like toxin that contains an identical non-proline cis peptide bond as that observed in BmK M7, in which residues within or neighboring the cis peptide bond were altered. Substitution of an aspartic acid residue for lysine at residue 8 in the BmK M1 (K8D) mutant converted the cis form of the non-proline peptide bond 9-10 into the trans form, revealing an intramolecular switch for cis-to-trans isomerization. Cis/trans interconversion of the switch residue at position 8 appears to be sequence-dependent as the peptide bond between residues 9 and 10 retains its wild-type cis conformation in the BmK M1 (K8Q) mutant structure. The structural interconversion of the isomeric states of the BmK M1 non-proline cis peptide bond may relate to the conversion of the scorpion alpha-toxins subgroups.     

A kinetic study of the folding of staphylococcal nuclease using size-exclusion chromatography.

The kinetics of the hydrodynamic volume change accompanying the reversible unfolding of staphylococcal nuclease have been observed by size-exclusion chromatography at 4 degrees C and pH 7.0 using the denaturant guanidine hydrochloride. The observed chromatographic profiles have been simulated by a six-component unfolding/refolding mechanism using a consistent set of equilibrium and kinetic parameters. The native protein is an equilibrium mixture of the cis and trans isomers of the peptide bond preceding proline-117. The native conformation containing the cis isomer dominates the equilibrium mixture, is more stable, and unfolds more slowly at its transition midpoint. The denatured protein is an equilibrium mixture of at least four components, the cis/trans isomers of proline-117 and one of the five remaining prolines. The dominant refolding pathway is initiated from the denatured component containing the trans isomer of proline-117. The six-component mechanism is consistent with tryptophan fluorescence kinetic measurements of the wild-type protein and with chromatographic measurements of a mutant P117G protein.     

Proline cis-trans isomerization controls autoinhibition of a signaling protein

Autoinhibition is being widely used in nature to repress otherwise constitutive protein activities and is typically regulated by extrinsic factors. Here we show that autoinhibition can be controlled by an intrinsic intramolecular switch afforded by prolyl cis-trans isomerization. We find that a proline on the linker tethering the two SH3 domains of the Crk adaptor protein interconverts between the cis and trans conformation. In the cis conformation, the two SH3 domains interact intramolecularly, thereby forming the basis of an autoinhibitory mechanism. Conversely, in the trans conformation Crk exists in an extended, uninhibited conformation that is marginally populated but serves to activate the protein upon ligand binding. Interconversion between the cis and trans, and, hence, of the autoinhibited and activated conformations, is accelerated by the action of peptidyl-prolyl isomerases. Proline isomerization appears to make an ideal switch that can regulate the kinetics of activation, thereby modulating the dynamics of signal response.     

Crystallographic studies on the role of the C-terminal segment of human angiogenin in defining enzymatic potency

Human angiogenin (Ang) is an RNase in the pancreatic RNase superfamily that induces angiogenesis. Its catalytic activity is comparatively weak, but nonetheless critical for biological activity. The crystal structure of Ang has shown that enzymatic potency is attenuated in part by the obstructive positioning of Gln117 within the B(1) pyrimidine binding pocket, and that the C-terminal segment of residues 117-123 must reorient for Ang to bind and cleave RNA. The native closed conformation appears to be stabilized by Gln117-Thr44 and Asp116-Ser118 hydrogen bonds, as well as hydrophobic packing of Ile119 and Phe120. Consistent with this view, Q117G, D116H, and I119A/F120A variants are 4-30-fold more active than Ang. Here we have determined crystal structures for these variants to examine the structural basis for the activity increases. In all three cases, the C-terminal segment remains obstructive, demonstrating that none of the residues that has been replaced is essential for maintaining the closed conformation. The Q117G structure shows no changes other than the loss of the side chain of residue 117, whereas those of D116H and I119A/F120A reveal C-terminal perturbations beyond the replacement site, suggesting that the native closed conformation has been destabilized. Thus, the interactions of Gln117 seem to be less important than those of residues 116, 119, and 120 for stabilization. In D116H, His116 does not replicate either of the hydrogen bonds of Asp116 with Ser118 and instead forms a water-mediated interaction with catalytic residue His114; residues 117-121 deviate significantly from their positions in Ang. In I119A/F120A, the segment of residues 117-123 has become highly mobile and all of the interactions thought to position Gln117 have been weakened or lost; the space occupied by Phe120 in Ang is partially filled by Arg101, which has moved several angstroms. A crystal structure was also determined for the deletion mutant des(121-123), which has 10-fold reduced activity toward large substrates. The structure is consistent with the earlier proposal that residues 121-123 form part of a peripheral substrate binding subsite, but also raises the possibility that changes in the position of another residue, Lys82, might be responsible for the decreased activity of this variant.     

Lipids as cofactors in protein folding: stereo-specific lipid-protein interactions are required to form HAMLET (human alpha-lactalbumin made lethal to tumor cells)

Proteins can adjust their structure and function in response to shifting environments. Functional diversity is created not only by the sequence but by changes in tertiary structure. Here we present evidence that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes. We have identified unsaturated C18 fatty acids in the cis conformation as the cofactors that bind apo alpha-lactalbumin and form HAMLET (human alpha-lactalbumin made lethal to tumor cells). The complexes were formed on an ion exchange column, were stable in a molten globule-like conformation, and had attained the novel biological activity. The protein-fatty acid interaction was specific, as saturated C18 fatty acids, or unsaturated C18:1trans conformers were unable to form complexes with apo alpha-lactalbumin, as were fatty acids with shorter or longer carbon chains. Unsaturated cis fatty acids other than C18:1:9cis were able to form stable complexes, but these were not active in the apoptosis assay. The results demonstrate that stereo-specific lipid-protein interactions can stabilize partially unfolded conformations and form molecular complexes with novel biological activity. The results offer a new mechanism for the functional diversity of proteins, by exploiting lipids as essential, tissue-specific cofactors in this process.     

Hydrogen-1 NMR evidence for three interconverting forms of staphylococcal nuclease: effects of mutations and solution conditions on their distribution

It has been known for several years that 1H NMR spectra of the enzyme staphylococcal nuclease contain resonances due to conformational heterogeneity [Markley, J. L., Williams, M. N., & Jardetzky, O. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 645-651]. One source of conformational heterogeneity has been attributed recently to cis/trans isomeriation of the Lys116-Pro117 peptide bond [Evans, P. A., Dobson, C. M., Kautz, R. A., Hatfull, G., & Fox, R. O. (1987) Nature (London) 329, 266-268]. In this paper we present evidence for three interconverting folded forms of nuclease. Forms N and N' are monomeric; form N" appears at higher nuclease concentrations and probably corresponds to dimerized enzyme. Saturation transfer was used to demonstrate that exchange occurs between the denatured state and N". The effects of temperature, pH, and Ca2+ and nucleotide binding on NMR spectra of nuclease were examined. When the temperature is increased or the pH is lowered, form N' is favored relative to N. Binding of a competitive inhibitor (thymidine 3',5'-bisphosphate plus calcium ion) strongly favors one form of nuclease. 1H NMR spectra of wild-type nuclease, the single-mutant nucleases L89F and H124L, and the double-mutant nuclease F76V+H124L were compared. In the unligated proteins, the equilibrium constant for the conformational equilibrium N in equilibrium with N' is approximately 0.1 in wild-type nuclease and nuclease H124L; by contrast, this equilibrium constant is about 0.7 in nuclease L89F and 1.2 in nuclease F76V+H124L under similar conditions.     

Investigating the structural basis of purine specificity in the structures of MS2 coat protein RNA translational operator hairpins

We have determined the structures of complexes between the phage MS2 coat protein and variants of the replicase translational operator in order to explore the sequence specificity of the RNA–protein interaction. The 19-nt RNA hairpins studied have substitutions at two positions that have been shown to be important for specific binding. At one of these positions, –10, which is a bulged adenosine (A) in the stem of the wild-type operator hairpin, substitutions were made with guanosine (G), cytidine (C) and two non-native bases, 2-aminopurine (2AP) and inosine (I). At the other position, –7 in the hairpin loop, the native adenine was substituted with a cytidine. Of these, only the G-10, C-10 and C-7 variants showed interpretable density for the RNA hairpin. In spite of large differences in binding affinities, the structures of the variant complexes are very similar to the wild-type operator complex. For G-10 substitutions in hairpin variants that can form bulges at alternative places in the stem, the binding affinity is low and a partly disordered conformation is seen in the electron density maps. The affinity is similar to that of wild-type when the base pairs adjacent to the bulged nucleotide are selected to avoid alternative conformations. Both purines bind in a very similar way in a pocket in the protein. In the C-10 variant, which has very low affinity, the cytidine is partly inserted in the protein pocket rather than intercalated in the RNA stem. Substitution of the wild-type adenosine at position –7 by pyrimidines gives strongly reduced affinities, but the structure of the C-7 complex shows that the base occupies the same position as the A-7 in the wild-type RNA. It is stacked in the RNA and makes no direct contact with the protein.     

A conserved cis peptide bond is necessary for the activity of Bowman-Birk inhibitor protein

The Bowman-Birk inhibitor (BBI) family of protease inhibitors has an inhibitory region comprising a disulfide-linked nine-residue loop that adopts the characteristic canonical motif found in many serine protease inhibitors. A unique feature of the BBI loop is the presence of a cis peptide bond at the edge of the inhibitory loop. BBI-related protein fragments that encapsulate this loop retain the structure and inhibitory activity of the parent protein. The most common BBI loop sequence has a proline-proline element with a cis-trans geometry at P3'-P4'. We have examined this element by analysis of the inhibitory activity and structure for a series of synthetic fragments where each of these proline residues has been systematically replaced with alanine. The results show that only when a proline is present at P3' are potent inhibition and a cis peptide bond at that position in the solution structure observed, suggesting that this conformation is required for biological activity. Though a P4' proline is not essential for activity, it effectively stabilizes the cis conformation at P3' by suppressing alternative conformations. This is most evident from the Pro-Ala variant, which comprises a 1:1 mixture of slowly exchanging and structurally different cis and trans isomers. Monitoring the action of trypsin on this mixture by NMR shows that this protease interacts selectively with the cis P3' structure, providing direct evidence for the link between activity and the nativelike structure of the cis isomer. This is, to the best of our knowledge, the first example where cis isomer selectivity can be demonstrated for a proteinase.     

Catalysis of proline-directed protein phosphorylation by peptidyl-prolyl cis/trans isomerases

Proline-directed protein phosphorylation was shown to depend on the capacity of the targeted Ser(Thr)-Pro bond to exhibit conformational polymorphism. The cis/trans isomer specificity underlying ERK2-catalyzed phosphate transfer leads to a complete discrimination of the cis Ser(Thr)-Pro conformer of oligopeptide substrates. We investigated in vitro the ERK2-catalyzed phosphorylation of Aspergillus oryzae RNase T1 containing two Ser-Pro bonds both of which share high stabilization energy in their respective native state conformation, the cis Ser54-Pro and the trans Ser72-Pro moiety. Despite trans isomer specificity of ERK2, a doubly phosphorylated RNase T1 was found as the final reaction product. Similarly, the RNase T1 S54G/P55N and RNase T1 P73V variants, which retain the prolyl bond conformations of the RNase T1-wt, were both monophosphorylated with a catalytic efficiency kcat/KM of 425 M(-1) s(-1) and 1228 M(-1) s(-1), respectively. However, initial phosphorylation rates did not depend linearly on the ERK2 concentration. The phosphorylation rate of the resulting plateau region at high ERK2 concentrations can be increased up to threefold for the RNase T1 P73V variant in the presence of the peptidyl-prolyl cis/trans isomerase Cyclophilin 18, indicating a conformational interconversion as the rate limiting step in the catalyzed phosphate group transfer. Using peptidyl-prolyl cis/trans isomerases with different substrate specificity, we identified a native state conformational equilibrium of the Ser54-Pro bond with the minor trans Ser54-Pro bond as the phosphorylation-sensitive moiety. This technique can therefore be used for a determination of the ratio and the interconversion rates of prolyl bond isomers in the native state of proteins.