James B. Matthew

pH-dependent processes in proteins.

Recent improvements in the understanding of electrostatic interactions in proteins serve as a focus for the general topic of pH-dependent processes in proteins. The general importance of pH-dependent processes is first set out in terms of hydrogen ion equilibria, stability, ligand interactions, assembly, dynamics, and events in related molecular systems. The development of various theoretical treatments includes various formalisms in addition to the solvent interface model developed by Shire et al. as an extension of the Tanford-Kirkwood treatment. A number of detailed applications of the model are presented and future potentialities are sketched.

[17] Calculation of electrostatic interactions in proteins

Publisher Summary The chapter discusses methods for the calculation of electrostatic interactions in proteins. By use of the static accessibility modification of the Tanford–Kirkwood model, a simple and efficient computational procedure yields quantitative and qualitative results in agreement with experimental data. The assumption that the structure of the protein in solution throughout the pH range of interest is essentially that of the crystal seems to hold reasonably well. Minor perturbations occur in solution and alter charge configurations. The working electrostatic model for the protein molecule adapts easily to a variety of problems and enables one to evaluate electrostatic contributions to such diverse phenomena as the binding of charged ligands, amino acid substitutions of site-directed mutagens, and any known tertiary or quaternary structural change. The static-accessibility-modified theory has been successfully applied to a variety of proteins that include sperm whale myoglobin and 11 species variations, oxy- and to deoxyhemoglobins and their interactions involving hydrogen ions, chloride ions, carbamino adducts, and organic phosphate polyanions, Bovine pancreatic trypsin inhibitor (BPTI) and ribonuclease. The generality of this approach is illustrated by the fact that all computations are based on the same consistent set of intrinsic p K values with the appropriate solvent accessibility parameter obtained from the known atomic coordinates.

Discrete charge calculations of potentiometric titrations for globular proteins: sperm whale myoglobin, hemoglobin alpha chain, cytochrome c.

Abstract The modified Tanford-Kirkwood theory of Shire et al. for intramolecular electrostatic interactions has been applied to hydrogen ion equilibria of sperm whale ferrimyoglobin, human hemoglobin α-chain and horse cytochrome c. The model employs two sets of parameters derived from the crystalline protein structures, first, the atomic coordinates of charged amino acid residues and, second, static accessibility factors to reflect their solvent exposure. In addition, a consistent set of intrinsic pK values (pK int ) for the individual groups is employed. The theoretical pK values at half-titration for individual groups in each protein correspond to the available observed pK values, and the theoretical titration curves compare closely with experimental potentiometric curves.

[18] Stabilization and destabilization of protein structure by charge interactions

Publisher Summary Protein molecules are unique polyelectrolytes and carry their own partial complement of counterions that exhibit charge cancellations at their characteristic isoionic point, and tight binding in the form of intramolecular salt bridges. When the proteins own charge array is sufficiently asymmetric by either composition, geometry, or both, the surface electrostatic potential in the absence of steric hindrance will dictate specific ion binding sites. Any calculation of protein stabilization as a function of pH and ionic strength include the phenomenon of specific ion binding where applicable. Electrostatic stabilization accounts for approximately half of the energy of dimer-tetramer assembly in hemoglobin; its role is marginal in the combination of trypsin with bovine pancreatic trypsin inhibitor (BPTI). Each protein studied shows a specific pattern of stabilization or destabilization resulting from a unique set of long-range, overlapping coulombic fields associated with each charge site. The individual contributions, and hence, the overall stabilization are affected as the array is modified by changes in pH, ionic strength, binding of charge components, or alteration of solvent exposure.

Solvent accessibility calculations for sperm whale ferrimyoglobin based on refined crystallographic data.

Abstract The calculation of solvent accessibility parameters from protein crystallographic data, by the method of Lee and Richards has been simplified to allow treatment of single atoms and their immediate environment. New accessibility values for all titratable amino acid residues in myoglobin have been computed from recent X-ray structure data of Takano. A number of prominent differences appear between these values and those from the older structure data of Watson. Differences are interpreted in terms of the proximity of neighboring residues.

[29] Measurement of CO2 binding: The 13C NMR method

Publisher Summary This chapter describes the 13C nuclear magnetic resonance (NMR) method. The carbamino adduct, R—NH—COO−, is a unique chemical species within the realm of proteins. This fact, together with the development over the past decade of highly sensitive Fourier transform 13C NMR spectrometers, has allowed 13C NMR to emerge as a powerful method for the detection of bound CO2 in protein samples. By virtue of the unique nature of the carbamino adduct, its 13C resonances arise in a region of the NMR spectrum unobscured by the protein itself. Hence, in a single 13C NMR experiment, both the nature and abundance at equilibrium of the interconverting forms of CO2 can be simultaneously determined. The sensitivity of NMR to exchange processes also allows, under favorable conditions, information to be obtained concerning the average lifetime of the carbamino adduct. To realize these objectives, several theoretical and practical considerations need to be satisfied. The typical 13C-Fourier transform instrument operating at 67 MHz is able to resolve clearly 13C natural abundance single-carbon resonances at 10 mM after the accumulation of approximately 10,000 free induction decay signals.