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A Tramontano

Publications and source records attributed to A Tramontano.

99 records · Page 6Linked to original sources
PubMedDate not supplied

Structural conservation in single-domain proteins: implications for homology modeling.

Large-scale sequencing projects are widening the gap between the known protein universe and the fraction for which structural information has been experimentally obtained. Through the application of homology (comparative) modeling and more general structure prediction techniques, this gap can, however, be narrowed, providing indirect structural information for a considerable number of proteins. Moreover, the estimated number of existing protein folds seems to be limited and many of these yet unknown folds should be discovered by dedicated large-scale structural genomics projects. Within this perspective, homology (comparative) modeling will gain in importance, as will the use of models derived by this technique. Here we discuss how well a sequence alignment, the most common starting point for generating a model, reflects the structural conservation between homologous proteins and we show that sequence information is able to direct construction of acceptable models as far as the structural core is concerned. We also show here that the regions surrounding insertions and deletions are much less conserved than the core and discuss the implications of this observation for loop modeling.

Amino Acid Sequence↗
PubMedDate not supplied

Immune recognition, antigen design, and catalytic antibody production.

Catalytic antibodies have been developed by experimental approaches exploiting the analogy between antibody-antigen and enzyme-substrate interaction. Haptens have been prepared to model the electrostatic or geometric attributes of a reaction's transition state and to induce combining sites having appropriate catalytic residues. The relative merits of these design strategies may be gleaned from the apparent activities and efficiencies of the respective catalysts. The implications of screening strategies on the kinetic characteristics of the resulting abzymes are also considered. Combining-site hypermutation provides the variation in the antibody repertoire from which high-affinity clones are selected. The same mechanism can also lead to a subset of antibodies with reduced hapten affinity, but improved catalytic activity. This possibility has not been adequately characterized, but is suggested by a number of considerations. These include the unexplained efficiency and diversity of mechanisms utilized by various antibody catalysts, and the observed catalytic activity of antibodies found in autoimmune serum. This article attempts to assess critically the evidence for rational design of catalytic activity in antibodies. Correlations among abzymes and their relevant models could lead to revised or novel strategies for producing better catalysts.

Antibodies, Catalytic↗
PubMedDate not supplied

[A structure-activity study of a catalytic antiidiotypic antibody to the human erythrocyte acetylcholinesterase].

The catalytic monoclonal antibody 9A8 (MA 9A8), antiidiotypic to the antibody AE-2 (MA AE2) produced to the active site of acetyl cholinesterase from human erythrocytes, was subjected to a structure-function study. The specific binding of MA 9A8 to MA AE2 (K 2.26 x 10(9) M-1) was shown by the method of surface plasmon resonance, and the functional activity of MA 9A8 was demonstrated. Unlike acetyl cholinesterase, this antibody specifically reacted with the irreversible phosphonate inhibitors of esterases. A peptide map of MA 9A8 was analyzed by MALDI mass spectrometry. The Ser99 residue of its heavy chain was shown to be within the active site of the catalytic antibody. A computer modeling of the MA 9A8 active site suggested the existence of a catalytic dyad formed by Ser99 and His35. A comparison of the tertiary structures of the MA 9A8 and the 17E8 monoclonal antibody, which also exhibited an esterase activity and was produced to the stable analogue of the reaction transition state, indicated a practically complete coincidence of the structures of their presumed active sites.

Acetylcholinesterase↗
PubMedDate not supplied

Conformations of immunoglobulin hypervariable regions.

On the basis of comparative studies of known antibody structures and sequences it has been argued that there is a small repertoire of main-chain conformations for at least five of the six hypervariable regions of antibodies, and that the particular conformation adopted is determined by a few key conserved residues. These hypotheses are now supported by reasonably successful predictions of the structures of most hypervariable regions of various antibodies, as revealed by comparison with their subsequently determined structures.

Amino Acid Sequence↗
PubMedDate not supplied

Nucleophilic proteolytic antibodies.

Proteolytic antibodies appear to utilize catalytic mechanisms akin to nonantibody serine proteases, assessed from mutagenesis and protease-inhibitor studies. The catalytic efficiency derives substantially from the ability to recognize the ground state with high affinity. Because the proteolytic activity is germline-encoded, catalysts with specificity for virtually any target polypeptide could potentially be developed by applying appropriate immunogens and selection strategies. Analysis of transition-state stabilizing interactions suggests that chemical reactivity of active-site serine residues is an important contributor to catalysis. A prototype antigen analog capable of reacting covalently with nucleophilic serine residues permitted enrichment of the catalysts from a phage-displayed lupus light-chain library. Further mechanistic developments in understanding proteolytic antibodies may lead to the isolation of catalysts suitable for passive immunotherapy of major diseases, and elicitation of catalytic immunity as a component of prophylactic vaccination.

Animals↗
PubMedDate not supplied

Inhibition and labeling of enzymes and abzymes by phosphonate diesters.

Reactive phosphonate diesters were designed and prepared as inhibitors of serine proteases and esterases. Inactivation of trypsin, chymotrypsin, and butyrylcholinesterase was determined by residual enzymatic activity as well as by the release of a chromogenic or fluorogenic product of the inhibition reaction. Second-order rate constants were determined from rates of nitrophenol formation. Application of the reaction for active-site titration of enzyme preparations is demonstrated. A basic functional group present in the nitrophenyl tropane phosphonate diester was shown to confer selectivity for inactivation of trypsin and chymotrypsin. Biotinylated derivatives of the phosphonate diesters were prepared to permit analysis of proteins modified in the inhibition reaction. Labeled polypeptides were resolved by SDS-PAGE, electroblotted, and detected by streptavidin-peroxidase staining. A detection limit of less than 4 ng, corresponding to 20 nM of trypsin, was demonstrated. Pretreatment of enzymes with DFP or nonbiotinylated phosphonates specifically blocks the labeling. This technique permits identification of serine proteases in complex mixtures with good sensitivity and specificity.

Animals↗