Evidence for a salt bridge interaction between the amino terminus and the carboxyl terminus in the basic pancreatic trypsin inhibitor was obtained from the pH titration curves of the terminal amino acid residues determined by 'H and I3C nuclear magnetic resonance (NMR). This interpretation of the titration behavior was confirmed by studies of an inhibitor modified by transamination of the a-amino group of Arg-1. The NMR techniques thus revealed a local difference between the protein surface structures in the crystal and in solution. Furthermore, the influence of the salt bridge between the end groups on the protein conformation was investigated by comparative studies of the native and the transaminated inhibitor. In all, over 200 corresponding NMR spectral and structural parameters in the two species were considered. The molecular conformations of the native and the transaminated inhibitor were found to be nearly identical, with small differences strictly localized in the immediate spatial environment of the chain termini. However, the absence of the salt bridge in the transaminated inhibitor affected the overall stability of the globular protein conformation, as evidenced in a lower denaturation temperature and an increased rate of exchange of interior amide protons with the solvent. The contribution of the single salt bridge between the chain termini to the stability of the globular protein conformation was estimated to be approximately 4.2 kJ mol-'.While it is generally acknowledged that under physiological conditions the polypeptide chains in globular proteins adopt an apparently unique 'native' conformation which is largely responsible for the protein function, the forces responsible for the outstanding stability of the native conformation are not fully understood [I]. It would appear that model considerations [2,3] and the rapidly increasing amount of structural data from single-crystal X-ray studies [4] clearly indicate the importance of short-range interactions between neighboring residues in the amino acid sequence. The fair degree of success recently achieved by prediction algorithms for regular secondary structures and bends [3] also seems to support Ahhrrviutions. NMR, nuclear magnetic resonance; 6 , chemical shift; ppm, parts per million; the inhibitor, basic pancreatic trypsjn inhibitor; the transaminated inhibitor, basic pancreatic trypsin inhibitor modified by transamiuation of the N-terminus. the idea that certain aspects of short-range interactions can by now be handled with some confidence. Shortrange interactions alone, however, could not possibly be the sole cause for the large variety of spatial arrangements of polypeptide chains. Inclusion of long-range interactions, i.e. interactions between polypeptide segments widely separated in the primary structure, into model considerations on polypeptide folding and stability of globular protein conformations has on the other hand so far been faced with serious difficulties [3]. The situation might possibly be improved considerably if more experi...

1H NMR spectra of the porcine pancreatic secretory trypsin inhibitor (PSTI) have been recorded vs. pH and temperature. Of the two tyrosines, one titrates with a pK of 11.25, while the resonances from the other are pH insensitive in the investigated range 4.8 less than or equal to pH less than or equal to 12. This is consistent with PSTI having one Tyr solvent exposed (Tyr-20) and the other buried (Tyr-31). The resonances from the lysyl epsilon-CH2 protons titrate with a pK of 10.95. The titration is accompanied by a pronounced line broadening, which starts near pH 8.5. Between pH 11.5 and pH 12 the epsilon-CH2 resonances recover their low pH line width. Titration curves for the lysines and Tyr-20 reflect single proton ionization equilibria, suggesting that these residues do not interact among themselves. On the basis of double resonance experiments, combined with analysis of chemical shifts, spin-spin couplings, and line widths, all methyl resonances are identified and followed as functions of pH and temperature. The gamma-CH3 doublet from the N-terminal Thr-1 is assigned by comparison between spectra of forms I and II of the inhibitor, the latter lacking the first four residues of form I. The beta-CH3 resonance from Ala-7 is also assigned. Proton resonance parameters of methyl groups are shown to afford useful NMR probes for the characterization of local nonbonded interactions, microenvironments, and mobilities.