Dye-sensitized photooxidation as a tool for determining the degree of exposure of amino acid residues in proteins. The methionyl residues in ribonuclease A.

Abstract Optical rotation measurements and absorption difference spectroscopy of ribonuclease A in different water-acetic acid mixtures showed that at least three conformational situations exist for the protein molecule at different levels of acetic acid concentration. Hematoporphyrin-sensitized photooxidation of the native as well as of the denatured proteins allowed us to detect three discrete states of reactivity for the 4 methionyl residues of ribonuclease A. On the basis of the photooxidation kinetics and of the conformational analyses, we concluded that methionine-29 is partly exposed in the native protein and methionine-13 is partially buried, whereas methionine-30 and methionine-79 are deeply buried. The importance of the single methionyl residues for the catalytic activity of the enzyme has been also resolved. The described procedure appears to be a reliable tool for examining the state of the amino acid residues in proteins

Site-directed spin labeling of the mitochondrial membrane. Synthesis and utilization of the adenosine triphosphatase inhibitor (N-(2, 2, 6, 6-tetramethyl-piperidyl-1-oxyl)-N'-(cyclohexyl)-carbodiimide).

Abstract NCCD (N-(2,2,6,6-tetramethyl-piperidyl-1-oxyl)-N'-(cyclohexyl)carbodiimide) is a spin label inhibitor of ATPase of mitochondrial membrane fragments. Upon binding (∼0.5 nmole per mg of protein) its electron paramagnetic resonance spectrum becomes highly immobilized (τc = 2.10-8 s). The bound but not the free label is reduced by succinate, indicating that electrons can be transferred from the respiratory chain to the ATPase system. The Mn++ATP complex decreases the paramagnetic signal of NCCD bound to membrane fragments about 30%. Such an interaction can be the consequence of the vicinity of the binding sites of the two species

Polypeptide models of elastin: CD and NMR studies on synthetic poly(Ile-Gly-Gly)

Poly(X-Gly-Gly), simple structural models for the hydrophobic, proline-devoid, regions of elastin, have been synthesized and studied by circular dichroism and NMR spectroscopies. The results gave evidence of type II beta-turns as the only ordered structure present in the polymers. The stability of the turns has been shown to decrease on hydration and to increase in the series Leu less than Ala less than Val less than Ile

Etude de peptides amyloïdogéniques dérivés de la tropoélastine humaine par simulations numériques

Ce travail de thèse porte sur l étude de peptides dérivés de la tropoélastine par simulations de dynamique moléculaire en solvant explicite. Ces peptides sont issus des exons 7, 28 et 30 et possèdent une séquence consensus XGGZG (X, Z = Valine, Leucine) identifiée expérimentalement comme responsable de la formation d assemblages supramoléculaire ayant des propriétés amyloïdes. Nous montrons tout d abord que le motif minimal XGGZG et (XGGZG) adoptent un grand nombre de coudes quel que soit la permutation entre valine et leucine et que localement les résidus non glycine présentent une conformation de type polyproline II dans des proportions significatives. L étude des répétitions (XGGZG)3 fait apparaître des structures allant du feuillet b antiparallèle pour (VGGVG)3 à de l hélice a et 310 pour (LGGLG)3. Les répétitions (VGGLG) 3 et (LGGVG)3 donnent pour leur part à la fois des structures comportant de faibles proportions d hélices ou de feuillets. Des résultats équivalents sont obtenus sur les exons 28 et 30 entiers, ainsi que sur le peptide comprenant les 17 premier résidus de l exon 30. La création d un plancher poly(VGGVG) de brins b virtuellement infini nous permet de montrer qu en sa présence, des peptides VGGVG peuvent s organiser parallèlement ou perpendiculairement à ce dernier. Enfin, es simulations préliminaires distinctes ont été menées afin d évaluer le rôle du cholestérol dans les phénomènes d agrégation ou dynamiques des peptides dérivés de la tropélastine. Des études de l effet biologique des peptides ont été débutées et sont une perspectives intéressantes de ce travail.This work deals with the study of tropoelastin-derived peptides by molecular dynamics simulations in explicit solvent. These peptides are identified in exons 7, 28 and 30 possess a consensus sequence XGGZG (X, Z = Valine, Leucine) described experimentally as responsible for the formation of supramolecular assemblies with amyloid properties. We first show that the minimal motif XGGZG and (XGGZG) adopt a large number of turns, independently of the permutation of valine and leucine residues. Locally, non-glycine residues exhibit polyproline-II conformation in significant proportions. The study of (XGGZG)3 show that it gives rise to structure form antiparallel b sheets for (VGGVG)3 to a and 310 helices for (LGGLG)3. (VGGLG)3 and (LGGVG)3 sequences give rise to both structures in small proportions. Equivalent results are obtained on the entire exons 28 and 30, and the peptide including the first 17 residues of exon 30. The construction of a poly(VGGVG) plan with virtually infinite b strand allows us to show that in contact with it, VGGVG peptides can organize parallel or perpendicularly to the plan. Finally, separate preliminary simulations were conducted to evaluate the role of cholesterol in the phenomena of aggregation and dynamics to tropoelastin-derived peptides. Studies of the biological effect of these peptides were initiated and are an interesting perspective of this work.REIMS-BU Sciences (514542101) / SudocSudocFranceF