Biomedical subjects
Publications and source records attributed to C Mattos.
Catalysis in organic solvents and the mapping of protein surfaces using multiple solvent crystal structures are two rapidly developing areas of research. Recent advances include the study of protein folding and stability in different solvents, and the demonstration that it is possible to qualitatively rank the affinities of protein binding sites for a given organic solvent using the multiple solvent crystal structures method.
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We have developed an experimental approach to map the complete binding surface of any crystalline macromolecule that is fast and flexible. Crystals of the target protein are transferred into organic solvents and the crystal structures are determined at high (about 2A) resolution. The sites where the solvent molecules bind to the protein are thus identified directly. Different solvents serve as probes for different organic functional groups; thus, benzene is a probe for where aromatic groups like to bind, dimethyl formamide is a probe for peptide binding sites, and so forth. A series of about six such experiments suffices to locate the major binding regions on the protein surface unambiguously. These different sites can then be targeted with "Hydra-headed" inhibitors that interact simultaneously with more that one site, thereby providing specificity for the desired target. We have used this method to map the complete binding surface of elastase, and find that three regions, including the active site cleft, are generally "sticky" and can make interactions with almost any functional group. Analyses of these binding sites on elastase and other proteins suggests that what makes a binding site is amphipathicity and the ease with which water can be displaced.
OBJECTIVE - To analyze immediate and late results of percutaneous mitral valvotomy (PMV) in patients < or = 18 year. METHODS - Between August '87 and July '97, 48 procedures were performed on 40 patients. The mean age was 15.6 years; 68.7% were females four of whom were pregnant. RESULTS - Success was obtained in 91.7% of the procedures. Immediate complications were severe mitral regurgitation (6.3%) and cardiac tamponade (2.0%). Late follow-up was obtained in 88.8% of the patients (mean value=43.2+/-33.9 months). NYHA functional class (FC) I or II was observed in 96.2% of the patients and restenosis developed in five patients, at a mean follow-up of 29.7+/-11.9 months. Three patients presented with severe mitral insufficiency and underwent surgery. Two patients died. CONCLUSION - PMV represents a valid therapeutic option in young patients. In these patients, maybe because of subclinical rheumatic activity, restenosis may have a higher incidence and occur at an earlier stage than in others persons.
This review article begins with a discussion of fundamental differences between substrates and inhibitors, and some of the assumptions and goals underlying the design of a new ligand to a target protein. An overview is given of the methods currently used to locate and characterize ligand binding sites on protein surfaces, with focus on a novel approach: multiple solvent crystal structures (MSCS). In this method, the X-ray crystal structure of the target protein is solved in a variety of organic solvents. Each type of solvent molecule serves as a probe for complementary binding sites on the protein. The probe distribution on the protein surface allows the location of binding sites and the characterization of the potential ligand interactions within these sites. General aspects of the application of the MSCS method to porcine pancreatic elastase is discussed, and comparison of the results with those from X-ray crystal structures of elastase/inhibitor complexes is used to illustrate the potential of the method in aiding the process of rational drug design.
The X-ray crystal structures of two new (trifluoroacetyl)dipeptide p-(trifluoromethyl)anilide (TFA-dipeptide-TFM) inhibitors complexed to porcine pancreatic elastase are presented. TFA-Val-Ala-TFM and TFA-Phe-Ala-TFM both bind to elastase with the TFA group in the S1 subsite, Val or Phe in the S2 subsite, Ala in the S3 subsite, and the TFM group in the S4 subsite. Five other TFA-dipeptide-anilide/elastase crystal structures are available (two TFA-X-Ala-p-(trifluoromethyl)anilide, X = Lys, Leu, and three TFA-Lys-X-p-isopropylanilide, X = Pro, Leu, Phe). The four inhibitors with the trifluoromethyl substituent on the anilide ring bind in a single mode to elastase, whereas superposition of the three inhibitors with the isopropyl substituent on the anilide ring show three different modes of binding to the protein [Mattos, C., et al. (1994) Nature Struct. Biol. 1, 55-58]. The seven structures are taken together in a detailed analysis of the active site of porcine pancreatic elastase. The inhibition constants for the inhibitors are used in combination with the crystal structures to understand the specificity of the different elastase subsites.
The conformational properties of tight two-residue beta-turns in proteins are examined by empirical energy function calculations. Twenty-five tight turns are studied in isolation, in the presence of the protein, and in the presence of the protein and crystal water molecules. The conformational properties are subdivided into those that are intrinsic to the turn and those that depend on the protein and water environment. Two factors are shown to determine the conformation of a tight beta-turn. One is the twist of the beta-sheet (responsible for selecting either a type I' or II' conformation as opposed to the more common types I or II) and the other is a local electrostatic effect (responsible for distinguishing between the type I' and II' conformations). In the rare cases where a two-residue turn is found in a type I conformation, there exists a stabilizing feature (turn-protein interaction, a side-chain in a conformation that stabilizes type I, etc.) which compensates for the unfavorable twist of the turn relative to the beta-sheet.
It has been assumed that the structure of a single inhibitor complex is sufficient to define the available subsites of an enzyme that has a unique binding site and a uniquely defined mode for ligand binding--the specificity for these subsites can thus be probed by kinetic experiments. Elastase is an enzyme for which these traditional assumptions, which underlie such structural and kinetic studies, do not hold. Three new crystal structures of elastase complexed to chemically similar inhibitors with similar binding affinities reveal a diversity of binding modes as well as two new subsites on elastase. The existence of multiple binding sites and different binding modes for such similar inhibitors indicates that researchers must proceed with caution when using kinetics to map out protein subsites.