pH and temperature effects on the molecular conformation of the porcine pancreatic secretory trypsin inhibitor as detected by hydrogen-1 nuclear magnetic resonance.
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.[1]References
- pH and temperature effects on the molecular conformation of the porcine pancreatic secretory trypsin inhibitor as detected by hydrogen-1 nuclear magnetic resonance. De Marco, A., Menegatti, E., Guarneri, M. Biochemistry (1982) [Pubmed]
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