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A Amadei

Publications and source records attributed to A Amadei.

25 records · Page 2Linked to original sources
PubMed1995

Essential dynamics of the cellular retinol-binding protein--evidence for ligand-induced conformational changes.

The cellular retinol-binding protein (CRBP) is an intracellular retinol carrier protein belonging to a family of hydrophobic ligand-binding proteins. It transports retinol to specific locations in the cell where, for instance, it is esterified for storage. Recently solved crystallographic structures of CRBP homologues with and without bound ligand do not provide evidence for a ligand-induced conformational change. However, it has been shown that there is a difference in binding of holo-CRBP and apo-CRBP to lecithin-retinol acyltransferase. Moreover, proteolysis of holo-CRBP and apo-CRBP yields different products, indicating a difference in structure or dynamics between the two forms. Here, we present the results of molecular dynamics simulations of holo-CRBP and apo-CRBP. The simulations show a significant difference in conformation, in agreement with experimental results. The essential dynamics method was used to study differences in dynamics between the apo and holo forms of CRBP, and showed inhibition of essential motions upon ligand binding. It also revealed large correlated motions of retinol with regions of the protein, pointing to a possible retinol entry/exit site.

Apoproteins↗
PubMed1993

Essential dynamics of proteins.

Analysis of extended molecular dynamics (MD) simulations of lysozyme in vacuo and in aqueous solution reveals that it is possible to separate the configurational space into two subspaces: (1) an "essential" subspace containing only a few degrees of freedom in which anharmonic motion occurs that comprises most of the positional fluctuations; and (2) the remaining space in which the motion has a narrow Gaussian distribution and which can be considered as "physically constrained." If overall translation and rotation are eliminated, the two spaces can be constructed by a simple linear transformation in Cartesian coordinate space, which remains valid over several hundred picoseconds. The transformation follows from the covariance matrix of the positional deviations. The essential degrees of freedom seem to describe motions which are relevant for the function of the protein, while the physically constrained subspace merely describes irrelevant local fluctuations. The near-constraint behavior of the latter subspace allows the separation of equations of motion and promises the possibility of investigating independently the essential space and performing dynamic simulations only in this reduced space.

Animals↗
PubMed1991

The conformation of constitutive DNA interaction sites for eukaryotic DNA topoisomerase I on intrinsically curved DNAs.

The analysis of the sites which are cleaved constitutively and preferentially by eukaryotic DNA topoisomerase I on two intrinsically curved DNAs reveals the conformational features that provoke the cleavage reaction on the curve-inducing sequence elements in the absence of supercoiling. This analysis is based on the observation (Caserta et al. (1989) Nucleic Acids Res. 17, 8521-8532 and (1990) Biochemistry 29, 8152-8157) that the reaction of eukaryotic DNA topoisomerase I occurs on two types of DNA sites: sites S (Supercoiled induced) and sites C (Constitutive, whose presence is topology-independent). We report that sites C are abundant on the intrinsically curved DNAs analyzed. The DNAs studied were two intrinsically curved segments of different origin: the Crithidia fasciculata kinetoplast DNA and the bent-containing domain B of the Saccharomyces cerevisiae ARS1. On these DNA segments DNA topoisomerase I cleaves at the junctions between the poly(A) tracts and mixed-sequence DNA. Analysis of the conformation of the double helix around the cleavage sites has revealed that the reaction occurs in correspondence of a defined DNA conformational motif. This motif is described by the set of Eulerian angular values that define the axial path of DNA (helical twist, deflection angle, direction) and of the orthogonal components of wedge (roll and tilt).

Animals↗
PubMed1990

Regulation of the function of eukaryotic DNA topoisomerase I: analysis of the binding step and of the catalytic constants of topoisomerization as a function of DNA topology.

It was previously observed that two steps of the reaction of eukaryotic DNA topoisomerase I (topoisomerization and cleavage) depend upon the conformation of the DNA substrate: in both instances the supercoiled form is a more efficient substrate than the relaxed one. This paper reports the analysis of two other steps of the reaction: the binding of DNA topoisomerase I to DNA and the catalytic constants (Kcs) of topoisomerization as a function of the topology of the substrate. Binding. Competition assays show that supercoiled DNA binds the enzyme with even slower kinetics than the relaxed form. Therefore, the preferential topoisomerization of supercoiled DNA is not due to the binding step. Additional evidence that the rate-limiting step of the topoisomerization reaction is not the binding of the enzyme to DNA is provided by the fact that the kinetics of relaxation is first order. Catalysis. The Kcs of the topoisomerization reaction have been calculated and it was shown that they do not vary as a function of the topology of the substrate or of its size. Taken together, the data on binding, cleavage, topoisomerization, and Kcs suggest that the preferential topoisomerization of torsionally strained DNA is due to the higher availability, on this topological form, of DNA sites that allow the onset of the reaction.

Animals↗
PubMed1989

In vitro preferential topoisomerization of bent DNA.

The steps of the topoisomerization reaction by calf thymus DNA topoisomerase I on a DNA domain containing an intrinsically bent DNA sequence have been analyzed. High preferentiality of binding, cleavage and topoisomerization on the bent segment relative to the rest of the DNA domain was observed. These studies show the importance of the local DNA conformation in the reaction of eukaryotic DNA topoisomerase I and the interest of the use of this enzyme as a tool for the analysis of DNA conformation and DNA dynamics.

Animals↗
PubMed1989

[The anatomo-pathologist as a necroscopy physician in the field: a balance-sheet of 4 years' experience].

The quality of the certificates of death for 2429 cases, all the residents died at home in the USL 70 during the period 1.8.83-31.12.87, was evaluated thanks to the necroscopic activity of the Legnano's Hospital pathologist. All the information, oral or written, available about each case was used to check the family doctor's certificate. Any correction to the certificate was discussed with the certifier. During the period a gradual and progressive reduction of error in the statement of the underlying cause of death was shown. The improvement comes out using different quality indicators.

Autopsy↗