Biomedical subjects

S A Sanchez

Publications and source records attributed to S A Sanchez.

10 recordsLinked to original sources

PubMed2001

Solution and interface aggregation states of Crotalus atrox venom phospholipase A2 by two-photon excitation fluorescence correlation spectroscopy.

The dimeric Crotalus atrox venom PLA2 is part of the secreted phospholipase A2 (PLA2) enzyme family that interacts at the lipid-solution interface to hydrolyze the sn-2 acyl ester bond of phospholipids. We have employed fluorescence correlation spectroscopy (FCS) to study the monomer-dimer equilibrium of the C. atrox venom PLA2 in solution, in the presence of urea, and in the presence of monomeric and micellar n-dodecylphosphocholine (C12-PN), a phosphatidylcholine analogue. Dilution experiments show that PLA2 is an extremely tight dimer, Kd < or = 0.01 nM, in solution. Urea was introduced to weaken the subunit's association, and an estimate for the PLA(2) dimer dissociation constant in buffer was obtained by linear extrapolation. The derived dissociation constant was at least several orders of magnitude greater than that suggested from the dilution experiments, indicating a complex interaction between urea and the PLA2 dimer. FCS data indicate that the PLA2 dimer begins to dissociate at 10 mM C12-PN in 10 mM Ca2+ and at 5 mM C12-PN in 1 mM EDTA. The PLA2 tryptophan fluorescence displayed spectral shifts and intensity changes upon interacting with C12-PN. On the basis of the FCS and tryptophan fluorescence results, we postulate an intermediate state where the two monomers are in loose interaction within a protein-lipid comicelle. As the concentration of C12-PN was increased, complete dissociation of the dimer was observed, inferred from the doubling of the particle number, and the average diffusion constant decreased to approximately 60 microm2/s, consistent with PLA2 associated with a C12-PN micelle. The presence of Ca2+ makes the comicelle intermediate more stable, retarding the separation of the monomers in the micellar suspension. Our data clearly indicate that PLA2, though a strong dimer in the absence of lipids, is dissociated by micellar C12-PN and supports the monomer hypothesis for PLA2 action.

Animals↗

PubMed2001

Arrangement of apolipoprotein A-I in reconstituted high-density lipoprotein disks: an alternative model based on fluorescence resonance energy transfer experiments.

The folding and organization of apolipoprotein A-I (apoA-I) in discoidal, high-density lipoprotein (HDL) complexes with phospholipids are not yet completely resolved. For about 20 years, it was generally accepted that the amphipathic helices of apoA-I lie parallel to the acyl chains of the phospholipids ("picket fence" model). However, based on the X-ray crystal structure of a large, lipid-free fragment of apoA-I, a "belt model" was recently proposed. In this model, the helices of two antiparallel apoA-I molecules are extended in a circular arrangement and lie perpendicular to the phospholipid acyl chains. To obtain conclusive information on the spatial organization of apoA-I in discoidal HDL, we engineered three separate cysteine mutants of apoA-I (D9C, A124C, A232C) for specific labeling with the fluorescence probes ALEXA-488 or ALEXA-546 (fluorescein and rhodamine derivatives). The labeled apoA-I was reconstituted into well-defined HDL complexes containing two molecules of protein and dipalmitoylphosphatidylcholine, and the complexes were used in three quantitative fluorescence resonance energy transfer (FRET) experiments to determine the distances between two specific sites in an HDL particle. Comparison of the distances measured by FRET (4.7-7.8 nm) with those predicted from the existing models indicated that neither the picket fence nor the belt model can account for the experimental results; rather, a hairpin folding of each apoA-I monomer with most helices perpendicular to the phospholipid acyl chains and a random head-to-tail and head-to-head arrangement of the two apoA-I molecules in the HDL particles are strongly suggested by the distance and lifetime data.

Apolipoprotein A-I↗

PubMed2000

Characterization of apolipoprotein A-I structure using a cysteine-specific fluorescence probe.

Two new Cys mutants of proapolipoprotein A-I, D9C and A232C, were created and expressed in Escherichia coli systems. Specific labeling with the thiol-reactive fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), was used to study the structural organization and dynamic properties of the extreme regions of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound states. Spectroscopic approaches, including circular dichroism and various fluorescence methods, were used to examine the properties of the mutant proteins and of their covalent adducts with the fluorescence probe. The mutations themselves had no effect on the structure and stability of apoA-I in the lipid-free state and in reconstituted HDL (rHDL) complexes. Furthermore, covalent modification with acrylodan did not alter the properties of the apoA-I variants in the lipid-bound state nor in the lipid-free A232C mutant, but it affected the structure and local stability of the lipid-free protein in the D9C mutant. Fluorescence results using the acrylodan probe confirmed a well-organized structure in the N-terminal region of apoA-I. Also, they suggested a three-dimensional structure in the C-terminal region, stabilized by protein-protein contacts. When Trp residues and acrylodan were used as donor-acceptor pairs for fluorescence resonance energy transfer (FRET), average distances could be measured. Both intensity and lifetime changes of the Trp emission indicated a protein folding in solution that brings the C-terminus of the protein near the Trp residues in the N-terminal half of the sequence. Also, the N- and C-terminal domains of apoA-I appeared to be near each other in rHDL having two apoA-I per particle.

2-Naphthylamine↗

PubMed1974