Analysis of electrostatic interactions and their relationship to conformation and stability of bovine pancreatic trypsin inhibitor

Abstract

The modified Tanford-Kirkwood electrostatic theory has been employed to evaluate pK values for all charge sites in the bovine pancreatic trypsin inhibitor (BPTI). 13C NMR titration data were obtained for all titrating groups except arginine residues in BPTI at nearly constant ionic strength in 0.1 M NaCl, at 41 degrees C. The chemical shifts of 46 resonances were found to be sensitive to pH. The pK values of these titrating resonances compared well with those computed by the modified Tanford-Kirkwood electrostatic theory. A conformational change involving the NH2- and COOH-terminal and nearby residues is shown to be partly electrostatically driven by the formation of a salt bridge between the alpha-amino and alpha-carboxyl groups at mid-pH values. The computed total electrostatic free energy of the molecule is found to be stabilizing at neutral pH despite the substantial net positive charge borne by the protein under such conditions.

Selectivity in the Post-Translational, Transglutaminase-Dependent Acylation of Lysine Residues

“…In the reactions of the proteins with TG2, the potential amine site concentrations are constant. For BPTI, there is also convincing evidence that the ε-ammonium ions of K15, K26, and K46 have identical pK a values (35,36), so that these Lys residues also represent the same effective nucleophilic concentrations. Thus, the finding that there is only a single TG2-reactive K site in BPTI would seem to indicate that it is the binding affinity of this specific Lys residue in being able to form a Michaelis complex with the acylenzyme intermediate that is mainly responsible for substrate selection.…”

“…To further investigate whether K15 is merely a favored substrate of TG2 which outcompetes the other lysines in BPTI (K26 and K46) with similar solvent accessibility and identical basicity/nucleophilicity (35,36) or whether TG2 exhibits true discrimination toward K15, we compared the reactivity of the K15A protein mutant with that of the wild type. As seen in Figure 5, the absence of a Lys residue at position 15 which was identified earlier by the direct labeling approach as the sole target for TG2 (Figure 4) eliminated the ability of the protein to react with the enzyme altogether.…”

Prediction of titration properties of structures of a protein derived from molecular dynamics trajectories

“…pKas of individual groups Table 1 contains the calculated apparent pK, and intrinsic pKint, values, averaged over the trajectories, for all ionizable groups of BPTI. Also included are the calculated values based on the fullgroup model for the crystal structure (Antosiewicz et al, 1996a) and the available experimental data (Brown et al, 1976(Brown et al, , 1978Richarz & Wuthrich, 1978;March et al, 1982). The data presented in Table 1 can be analyzed from several points of view.…”

Thermodynamics of bpti folding

“…The dependence of the transition temperature on pH can be used to determine the number of protons released upon protein unfolding, A t v (Privalov et al, 1969): Temperature ("C) gives a value of 1.01 f 0.05 for A, v. According to March et al (1982), the group titrating in this pH range is the terminal carboxyl group. The ionization effects of this group in the protein are compensated by ionization effects of the glycine and sodium acetate buffers used (Privalov & Potekhin, 1986;.…”