11810 Biochemistry zyxwvu 1993,32, 11810-1 1818 NMR Strategy for Determining Xaa-Pro Peptide Bond Configurations in Proteins: Mutants of Staphylococcal Nuclease with Altered Configuration at Proline- 1 177 Andrew P. Hinck,* Eric S. Eberhardt, and John L. Markley' Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, 420 Henry Mall, Madison, Wisconsin 53706 Received May zyxwvuts 5, 1993; Revised Manuscript Received August 20, 1993' ABSTRACT: A general approach has been developed for configurational analysis (cis or trans) of Xaa-Pro peptide bonds in proteins. This approach, which entails selective 13C labeling of Xaa and Pro residues in the protein and isotope-edited NMR, has been applied to mutants of staphylococcal nuclease with suspected altered configurations of the Lys116-Pro'17 peptide bond. The technique for monitoring proline configurations is based on differences in interproton distances between the Ha of residue Xaa and the proline H6 or Ha protons. Short (C2.5 zyxwvutsr A) Xaa Ha-Pro H6 interproton distances are diagnostic for the trans configuration, whereas short (C2.5 A) Xaa Ha-Pro Ha interproton distances are diagnostic for the cis configuration. Biosynthetic incorporation of [cr-13C]Xaaand [6-13C]proline facilitates detection of trans Xaa-Pro peptide bonds, whereas incorporation of [a-13C]Xaa and [cr-13C]proline facilitates detection of cis Xaa-Pro peptide bonds. Provided that the Xaa-Pro peptide bond is unique within the protein sequence, symmetric off- diagonal NOE cross peaks in the isotope-edited NOE spectrum allow for simultaneous chemical shift assignment and determination of the prolyl peptide bond geometry. We have used this technique to determine the predominant configuration of the L y ~ l ~ ~ - P r o l l7 peptide bond in recombinant V8 staphylococcal nuclease A (H124L) and two of its single amino acid mutants (D77A+H124L and G79S+H124L). The results are consistent with conclusions reached on the basis of indirect arguments concerning changes in the chemical shifts of histidine lHfl N M R signals. The Lys116-Pro117 peptide bond was found by this direct isotope-edited NOE method to be predominantly cis in H124L but predominantly trans in D77A+H124L and G79S+H124L. However, when a saturating amount of an inhibitor (pdTp plus Ca2+)was added to either D77A+H124L or G79S+H124L, the peptide bond became predominantly cis. The propensity of proline residues to form cis peptide bonds in folded proteins is unique among the twenty common amino acids. Structurally, cis Xaa-Pro peptide bonds, which are essential to the formation of type VI reverse turns, provide an important means of reversing the chain direction and allowing the backbone to packonto itself. zyxwvutsr TransXaa-pro peptide bonds introduce steric interactions between the pyrrolidine ring 8-position and the preceding residue. This raises the free energy of the trans form and effectively diminishes the net free energy difference between the cis and trans configurations relative to non-proline-containing peptide bonds, and it provides one explanation for the common Occurrence of cis prolines among protein structures (Wiithrich & Grathwohl, 1974). The relatively low activation barrier for isomerization, - 13 kcal/mol (MacArthur & Thornton, 1991), combined with the small energy difference between cis and trans Xaa-Pro peptide bond configurations provides a mechanism for slow interconversion of alternative forms of proteins in the folded and unfolded states. The role of prolyl trans to cis isomer- ization in the kinetics of protein folding reactions has been thoroughly investigated, and it is now widely accepted that peptide bond isomerization provides a mechanism for slow steps in protein folding reactions (Kim & Baldwin, 1982). The structural and functional consequences of peptide bond A.P.H. was supported by Molecular Biophysics Training Grant GM08293 (NIH), and E.S.E. is a Wharton Predoctoral Fellow. This work was supported by NIH Grant GM35976. * Author to whom correspondence should be addressed. t Present address: Department of Chemistry, University of Wisconsin, e Abstract published in Advance ACS Abstracts, October 15, 1993. 1101 University Ave., Madison, WI 53706. 0006-2960/93/0432-118 10$04.00/0 isomerization in folded proteins has been characterized less thoroughly (Chazin et al., 1989; Alexandrescu et al., 1989; Evans et al., 1987; Ludwig & Luschinsky, 1992), but the strict evolutionary conservation of some proline residues (Barlow & Thorton, 1988; Langsetmo et al., 1989) and the recent discovery of widely abundant proteins with prolyl peptide bond isomerase activity suggest that peptide bond isomerization may play an important role in protein structure and function. Suggestive examples include the sodium pump of Escherichia coli and the protein disulfide isomerase/ thioredoxin class of enzymes. Several lines of evidence now suggest that isomerization of a proline residue, which is centrally located in a transmembrane a-helix of the sodium pump, is synchronous with ion translocation (Brand1 & Deber, 1986). In E. coli thioredoxin, it has been suggested that proline isomerization may be linked thermodynamically with the cleavage and reformation of the active site disulfide (Lang- setmo et al., 1989). Staphylococcalnuclease (nuclease) is the best characterized protein in which isomerization of a prolyl peptide bond in the folded state has been studied. The existence of conformational substates in staphylococcal nuclease was first observed in the splitting of the well-resolved histidine lHfl resonanca (Mar- kley et al., 1970). Mechanisms for conformational hetero- geneity in staphylococcal nuclease have been attributed to isomerization about the Lys116-Pro117 (Fox et al., 1986; Alexandrescuetal., 1989) and H is'Wr~~~ peptide bonds (Loh et al., 1991). Changes in the signals from Hiss, HidZ1, and His124 are thought to report on isomerization of the Lys116- peptide bond, whereas changes in the signals from His46 report on isomerization of the peptide bond zy 0 1993 American Chemical Society