Antibodies structural analysis

The molecular mechanism of the enantioselective protonation reaction by antibody 14D9 was revealed by a crystal structure analysis [19[. A catalytic carboxyl group AspH 101 was found at the bottom of the catalytic pocket and found to be necessary for catalysis by mutagenesis to Asn or Ala. The mechanism or protonation involves an overall syn addition of water to the enol ether in a chiral binding pocket ensuring complete enantioselectivity (Figure 3.4). 

The CFR 21 part 11 compliant software newly released by Convergent Bioscience makes imaged cIEF a viable technique for use in QC environments for release testing of therapeutic proteins or antibody molecules. i-cIEF has also been used heavily for characterization of protein modifications such as deamidation and isomerization, as well as oligosaccharide structure analysis. ... 

Even though these approaches are powerful methods for determining functional sites on proteins, they are limited if not coupled with some form of structural determination. As Figure 2 illustrates, molecular biology and synthetic peptide/antibody approaches are not only interdependent, they are tied in with structural determination. Structural determination methods can take many forms, from the classic x-ray crystallography and NMR for three-dimensional determination, to two-dimensional methods such as circular dichroism and Fourier Transformed Infrared Spectroscopy, to predictive methods and modeling. A structural analysis is crucial to the interpretation of experimental results obtained from mutational and synthetic peptide/antibody techniques. 

Mazzoni, M. R., Malinski, J. A, and Hamm, H E. (1991) Structural analysis of rod GTP-binding protein, Gt-limited proteolytic digestion pattern of Gt with four proteases defines monoclonal antibodies epitope J Biol. Chem 266,14,072—14,081... 

In this context, Greene and coworkers [159, 160] have reported the first low-molecular-mass immunoglobulin mimetic 207, Scheme 62, developed on the basis of an X-ray structure analysis of the antigen-antibody complex. Compound 207 is resistant toward proteases and imitates the binding and functional properties of the native antibody. 

F6. Foster, M. H., Kieber-Emmons, T., Ohliger, M., and Madaio, M. P., Molecular and structural analysis of nuclear localizing anti-DNA lupus antibodies. Immunol. Res. 13, 186-206 (1994). 

In the most comprehensive structural analysis to date, Lo Conte et al.11 studied 75 protein-protein complexes comprising 24 protease-inhibitor, 19 antibody-antigen, and 32 other complexes (including 9 further enzyme-inhibitor and 11 signal transduction complexes). The authors found that protein-protein interfaces typically have a size of 1600 400 A2 with a few complexes exhibiting very large (2000—4660 A2) or very small (less than 1000 A2) interfaces. With respect to their chemical nature, the interfaces were found to... 

STRUCTURAL ANALYSIS OF AFFINITY MATURED ANTIBODIES AND LABORATORY-EVOLVED ENZYMES... 

Over half of the structurally characterized catalytic antibodies are from the esterolytic class of reactivity. This discussion will focus on 48G7, the most exemplary antibody from this class. In this antibody, the bound and free forms of both germline and mature antibodies have been structurally characterized, allowing for the first structural analysis of the affinity maturation process in a catalytic antibody system (Patten et al., 1996 Wedemayer et al., 1997a Wedemayer et al., 1997b). 

Directed evolution and antibody affinity maturation offer efficient routes to redesigning proteins for new functional characteristics. Adaptive mutations and well-defined selection pressures allow structural analysis of the evolved products to provide insights into the molecular basis of protein structure and function. It is interesting to note that the majority of mutations that were obtained in the present maturation and directed evolution experiments were located at positions away from the enzymatic active sites. Perhaps this is due to the inherent difficulty in retaining catalytic activity with most active site amino acid substitutions. 

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