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R A Mariuzza

Publications and source records attributed to R A Mariuzza.

76 records · Page 5Linked to original sources
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The effect of water activity on the association constant and the enthalpy of reaction between lysozyme and the specific antibodies D1.3 and D44.1.

The reactions of lysozyme with the specific monoclonal antibody D1.3, its Fv fragment and a mutant of the Fv, were studied under conditions of reduced water activity through the addition of the cosolutes glycerol, ethanol, dioxane and methanol. Titration calorimetry, BIAcoreTM and ultracentrifugal analyses were used to determine enthalpy of reactions and affinity constants. There was a decrease in the values of the enthalpies of reactions as well as in the association constants which was proportional to the decrease in water activity. These results are consistent with a structural model in which water molecules bound to the antigen and the antibody are conserved upon complex formation and provide bonds which are important for the stability of the complex. In contrast, the reaction of lysozyme with the specific monoclonal antibody D44.1, or its Fab, showed the inverse effect: a small increase in the value of the association constant with decreasing water molarities. This is in agreement with a model in which binding of antigen to antibody D44.1 is accompanied by the release of a very small number of water molecules.

Alcohols↗
PubMedDate not supplied

X-ray diffraction studies of an anti-azophenylarsonate antibody and of an antigen-antibody complex.

X-ray crystallographic studies of the Fab fragments of two murine monoclonal antibodies of predefined specificity are under way. Diffracted X-ray intensities of the crystalline native Fab fragment of an anti-azophenylarsonate antibody and of three heavy atom derivatives have been measured to a resolution of 3.5 A. A preliminary 6-A resolution electron density map has been obtained. The 6-A resolution structure of an antigen-antibody (hen lysozyme-Fab) complex has been determined. There are close contacts between the antigen and the antibody over a large contact area, about 20 X 25 A. At least two segments of the polypeptide chain of lysozyme, of about 10 amino acids each (positions 19-27 and 116-129), are involved in the contacts, as well as all six complementarity-determining regions of the antibody. No gross conformational changes are observed in the antigen at this resolution, although there are some smaller local changes in areas in contact with the antibody and elsewhere. The effects of amino acid substitutions on antigen recognition by the monoclonal anti-hen lysozyme antibody were investigated using different, closely related lysozymes. These effects can be readily explained in terms of the three-dimensional model presented here. A 3.5-A resolution electron density map has been calculated and is currently under study.

Animals↗
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Three-dimensional structure of an antigen-antibody complex at 6 A resolution.

Present understanding of the three-dimensional structure of antibody combining sites is based on X-ray diffraction studies of myeloma immunoglobulins. The structures of the antigen-binding fragment (Fab) complexes of two of these immunoglobulins with small ligands have also been determined. However, there is no crystallographic information concerning the interactions of an antibody with an antigen, nor do we know the precise structure of antigenic determinants on protein molecules. We now report the first structure determination of an antigen-antibody complex at 6 A resolution. The structure of the complex between hen egg-white lysozyme and the Fab of a monoclonal anti-lysozyme antibody (D1.3) shows that the combining site of antibodies is not merely a cleft delineated by the complementarity-determining regions of the variable regions of the light and heavy chains, but is a larger area extending beyond it. A correspondingly large area of the antigen makes close contacts with the antibody, in agreement with the notion of a 'topographical' rather than 'sequential' antigenic determinant. The structural basis of cross-reactivities of an antibody with heterologous antigens and the effect of a single amino acid substitution on antigenic specificity can thus be visualized in the structural model presented here.

Amino Acid Sequence↗