Biomedical subjects

R C Willson

Publications and source records attributed to R C Willson.

27 records · Page 2Linked to original sources

PubMed1995

Free energy simulations of the HyHEL-10/HEL antibody-antigen complex.

Free energy simulations are reported for the N31LD mutation, both in the HyHEL-10-HEL antibody-lysozyme complex and in the unliganded antibody, using the thermodynamic-cycle perturbation method. The present study suggests that the mutation would change the free energy of binding of the complex by -5.6 kcal/mol (unrestrained free energy simulations), by -0.5 kcal/mol (free energy simulations with a restrained backbone) and by 1.8 kcal/mol (Poisson-Boltzmann calculations, which also use a restrained geometry model). A detailed structural analysis helps in estimating the contributions from various residues and regions of the system. Enhanced recognition of HEL by the mutant HyHEL-10 would arise from the combination of thermodynamically more favorable conformational changes of the CDR loops upon association and subsequent charge pairing with Lys96 in the antigen.

Antigen-Antibody Complex↗

PubMed1995

Determination of the pKa values of titratable groups of an antigen-antibody complex, HyHEL-5-hen egg lysozyme.

The titration behavior of the ionizable residues of the HyHEL-5-hen egg lysozyme complex and its individual components has been studied using continuum electrostatic calculations. Several residues of HyHEL-5 had pKa values shifted away from model values for isolated residues by more than three pH units. Shifts away from the model values were smaller for the residues of hen egg lysozyme. A moderate variation in the pKa values of the titratable groups was observed upon increase of the ionic strength from 0 to 100 mM, amounting to 1-2 pH units in most cases. Under physiological conditions, the net charge of HyHEL-5 was opposite that for hen egg lysozyme. Several residues, including those involved in the Arg-Glu salt bridges that have been proposed to be important in antibody-antigen binding, had pKa values that were changed significantly upon binding. The main titration event upon antibody-antigen binding appears to be loss of a proton from residue GluH50 of the Fv molecule. The limitations of our calculation methods and the role they might play in the design of antibodies for use in assays, sensors and separations are discussed.

Amino Acids↗

PubMed1994

Presence of a preferred anion-exchange binding site on cytochrome b5: structural and thermodynamic considerations.

A preferred chromatographic contact region for adsorption of recombinant soluble tryptic fragment of rat cytochrome b5 on the hydrophilic anion-exchanger Mono Q has been identified using conservative carboxylate-to-amide mutations of charged residues. Equilibrium adsorption isotherms were measured under conditions of full reversibility by high ionic strength, as confirmed by explicit mass balances performed for each experiment. Although cytochrome b5 displays several clusters of negative charge, mutations in one cluster consistently reduce binding affinity and the stoichiometric displacement parameter Z by much greater factors than do mutations in other areas of the molecule. Adsorption heterogeneity derived by fitting isotherms to the Hill equation is reduced by factors which reduce the overall affinity of adsorption. Van't Hoff analyses gave uniformly positive enthalpies of adsorption, and mutational changes in adsorption enthalpy were relatively independent of the site of mutation. These results suggest that enthalpy does not play a dominant role in either affinity or selectivity of anion-exchange adsorption in this system.

Adsorption↗

PubMed1994

Isothermal titration calorimetric study of the association of hen egg lysozyme and the anti-lysozyme antibody HyHEL-5.

The thermodynamics of association of hen egg lysozyme and the antibody HyHEL-5 was characterized by isothermal titration calorimetry. The structure of this complex has been determined to 2.8-A resolution by Sheriff et al. [Sheriff, S., Silverton, E. W., Padlan, E. A., Cohen, G. H., Smith-Gill, S. J., Finzel, B. C., & Davies, D. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8075-8079]. The calorimetric enthalpy of association is negative and declines linearly with temperature in the range 10-37 degrees C (delta Cp = -340 +/- 40 cal mol-1 K-1). Entropic contributions calculated using previously determined values of the affinity of association are negative (unfavorable) in this temperature range. This result is consistent with the loss of mobility upon association of the unusually mobile segments of HEL which form the HyHEL-5 epitope. As the affinity of association is approximately constant in this temperature range, an enthalpy-entropy compensation effect is implied. The hydrophobic and vibrational contributions to delta S and delta Cp are estimated using the method of Sturtevant [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2236-2240]. The experimental value of delta Cp is in rather close agreement with the delta Cp estimated from the polar and nonpolar surface areas buried upon association.

Animals↗

PubMed1994

Electrostatic potentials and electrostatic interaction energies of rat cytochrome b5 and a simulated anion-exchange adsorbent surface.

Electrostatic potentials were determined for the soluble tryptic core of rat cytochrome b5 (using a structure derived from homology modeling) and a simulated anion-exchange surface through application of the linearized finite-difference Poisson-Boltzmann equation with the simulation code UHBD. Objectives of this work included determination of the contributions of the various charged groups on the protein surface to electrostatic interactions with a simulated anion-exchange surface as a function of orientation, separation distance, and ionic strength, as well as examining the potential existence of a preferred contact orientation. Electrostatic interaction free energies for the complex of the model protein and the simulated surface were computed using the electrostatics section of UHBD employing a 110(3) grid. An initial coarse grid spacing of 2.0 A was required to obtain correct boundary conditions. The boundary conditions of the coarse grid were used in subsequent focusing steps until the electrostatic interaction free energies were relatively independent of grid spacing (at approximately 0.5 A). Explicit error analyses were performed to determine the effects of grid spacing and other model assumptions on the electrostatic interaction free energies. The computational results reveal the presence of a preferred interaction orientation; the interaction energy between these two entities, of opposite net charge, is repulsive over a range of orientations. The electrostatic interaction free energies appear to be the summation of multiple fractional interactions between the protein and the anion-exchange surface. The simulation results are compared with those of ion-exchange adsorption experiments with site-directed mutants of the recombinant protein. Comparisons of the results from the computational and experimental studies should lead to a better understanding of electrostatic interactions of proteins and charged surfaces.

Adsorption↗

PubMed1994

Bacteriophage surface display of an immunoglobulin-binding domain of Staphylococcus aureus protein A.

As a model system for the optimization of separation ligands by bacteriophage surface display, we have constructed a phage surface expression system for a single immunoglobulin-binding domain of Protein A of Staphylococcus aureus. Protein A domain B is genetically fused to the gpIII adsorption protein of the filamentous bacteriophage M13, and hence displayed on the phage surface. Phage displaying the Protein A domain are selectively retained on human IgG-sepharose. Retention is due to specific Protein A-IgG interactions, as demonstrated by competitive inhibition by soluble Protein A or polyclonal human IgG. Polyclonal goat IgG, which is known to bind less well to Protein A than does human IgG, inhibits phage adsorption less effectively. Phage expressing Protein A can be purified in a few rounds of selective adsorption from a vast excess of wild type phage. Diverse libraries constructed by mutagenesis of this construct will allow massive screening of mutant forms of Protein A for alterations in binding and elution properties. We anticipate that phage display will prove to be a widely-applicable method of identification and optimization of affinity ligands for separations.

Amino Acid Sequence↗

PubMed1994

Tertiary structure of the heme-binding domain of rat cytochrome b5 based on homology modeling.

The in vitro complexes formed between cytochrome b5 and other proteins (e.g. cytochrome c) have served as a useful means to probe electrostatic contributions to macromolecular recognition. Extensive experimentation has been carried out to test the specificity and stability of these complexes, including site-directed mutagenesis based on the heterologous expression of rat cytochrome b5 in E. coli. Despite this interest, there has not been a determination of the complete structure of cytochrome b5. Here we report coordinates for the complete tertiary structure of the heme-binding domain of rat cytochrome b5 based on homology modeling. Protein Data Bank (PDB) coordinates derived from the crystal structure of the highly homologous bovine cytochrome b5 were used for main chain scaffolding. Secondary structures for the termini missing in the bovine structure were generated using homologous sequences derived from an exhaustive search of the PDB database. The model structure was solvated and further refined using energy minimization techniques. The N-terminal residues of the model appear to be in a beta sheet conformation while the carboxy terminus is in a helical conformation. The rest of the rat model is folded virtually identically to the bovine x-ray crystal structure (r.m.s. deviation 1.28 A), despite six sequence differences between the two cores. This homology-based structure should be useful for structure-function analyses of molecular recognition involving cytochrome b5.

Amino Acid Sequence↗

PubMed1993

Anion-exchange chromatographic behavior of recombinant rat cytochrome b5. Thermodynamic driving forces and temperature dependence of the stoichiometric displacement parameter Z.

The HPLC anion-exchange isocratic retention behavior of the recombinant soluble core of wild type rat cytochrome b5 on Mono Q HR 5/5 was investigated as a function of temperature and sodium chloride concentration at fixed eluent flow-rates. Retention was measured over a range of eluent flow-rates at a specified temperature to determine if true adsorption equilibrium could be approximated by the HPLC method. Apparent Van 't Hoff enthalpies of adsorption obtained from the HPLC retention data were positive, indicating an entropically driven spontaneous adsorption process, and were found to decline with increasing ionic strength. The retention results were interpreted in terms of the stoichiometric displacement model to obtain the apparent number of binding sites in the contact region, Z, as a function of temperature and of protein concentration. Z was found to depend significantly on temperature, even under conditions of nearly complete protein recovery, but did not depend on protein concentration at the low loadings studied.

Animals↗

PubMed1991

Measurement of translational accuracy in vivo: missense reporting using inactive enzyme mutants.

The measurement and potential technological significance of in vivo missense errors are briefly reviewed. A recently developed approach is described in which reporter enzyme activity is generated by mistranslation of a gene coding for an inactive mutant form of the enzyme. Initial results obtained using the alpha subunit of E coli tryptophan synthetase and bacterial luciferase are discussed, as well as the prospects for further development of this method.

Escherichia coli↗