0000000000012875
AUTHOR
M.c. Bañó
The Closed/Open Model for Lipase Activation. Addressing Intermediate Active Forms of Fungal Enzymes by Trapping of Conformers in Water-Restricted Environments
The behavior of prototypic fungal lipases in a water-restricted environment has been investigated by exploiting the reported experimental strategy that allows the trapping (freeze-drying) of the enzyme in the conformation present in aqueous solution and to subsequently assay it in nonaqueous media [Mingarro, I., Abad, C., and Braco, L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3308-3312]. We now report, using simple esterification as well as acidolysis (triglycerides as substrates) as nonaqueous model reactions, that the presence of a detergent (n-octyl-beta-glucopyranoside) in the freeze-drying buffer, at concentrations below the critical micellar concentration, generates different catalyti…
New high-performance liquid chromatography-based methodology for monitoring the conformational transitions of self-associating hydrophobic peptides, incorporated into liposomes.
A new high-performance size-exclusion chromatographic strategy is reported for the analysis of the hydrophobic self-associating peptide gramicidin A, incorporated into artificial phospholipid vesicles (liposomes). The method is based on the direct injection of a few microlitres of the gramicidin A-containing liposome suspension into the column, which is eluted with a non-polar solvent, such as tetrahydrofuran. The type and amount of information which can be derived from this methodology have been evaluated. Using this chromatographic approach, a correlation has been unambiguously shown to exist between the organization of the peptide in the vesicles and a number of variables involved in the…
Size-exclusion high-performance liquid chromatography in the study of the autoassociating antibiotic gramicidin A in micellar milieu.
Gramicidin A (gA) is a polypeptide antibiotic which forms dimeric channels specific for monovalent cations in biological membranes. It is a polymorphic molecule that adopts several different conformations, double-stranded (ds) helical dimers (pore conformation) and single-stranded beta-helical dimers (channel conformation). This study investigated the conformational adaptability of gramicidin A when incorporated into micelles as membrane-mimetic model system. Taking advantage of our reported, versatile, size-exclusion high-performance liquid chromatography (SE-HPLC) strategy that allows the separation of double-stranded dimers and monomers, we have quantitatively characterized the conformat…
Regulation of cell cycle transcription factor Swi5 by karyopherin Msn5
AbstractInactivation of S. cerevisiae β-karyopherin Msn5 causes hypersensitivity to the overexpression of mitotic cyclin Clb2 and aggravates growth defects of many mutant strains in mitotic exit, suggesting a connection between Msn5 and mitotic exit. We determined that Msn5 controlled subcellular localization of the mitotic exit transcription factor Swi5, since it was required for Swi5 nuclear export. Msn5 physically interacted with the N-terminal end of Swi5. Inactivation of Msn5 caused a severe reduction in cellular levels of Swi5 protein. This effect occurred by a post-transcriptional mechanism, since SWI5 mRNA levels were not affected. The reduced amount of Swi5 in msn5 mutant cells was…
HPLC study on the ‘history’ dependence of gramicidin A conformation in phospholipid model membranes
AbstractA novel HPLC methodology for the study of gramicidin A reconstituted in model membranes has been tested in comparison with circular dichroism data. It is shown that this chromatographic technique not only corroborates most of the recent spectroscopic results but allows one to explain them in terms of mass fractions of different actual conformational species of GA in the phospholipid assemblies. In particular, the dependence of the inserted peptide configuration on the organic solvent and other parameters involved in the ‘history’ of the sample preparation and handling has been analyzed by HPLC in two phospholipid model systems: small unilamellar vesicles and micelles. Moreover, a sl…
Targeting and membrane insertion into the endoplasmic reticulum membrane of Saccharomyces cerevisiae essential protein Rot1
Rot1 is an essential yeast protein that has been related to cell wall biosynthesis, actin cytoskeleton dynamics and protein folding. Rot1 is an N -glycosylated protein anchored to the nuclear envelope–endoplasmic reticulum (ER) membrane by a transmembrane domain at its C-terminal end. Rot1 is translocated to the ER by a post-translational mechanism. Here, we investigate the protein domain required to target and translocate Rot1 to the ER membrane. We found that several deletions of the N-terminal region of Rot1 prevented neither membrane targeting nor the insertion of this protein. Interestingly, we obtained the same results when different truncated forms in the C-terminal transmembrane dom…
A semi-empirical approach for the simulation of circular dichroism spectra of gramicidin A in a model membrane
In an extension of our previous work (Bañó, M. C., Braco, L., and Abad, C. 1991. Biochemistry. 30:886-94), the kinetics of dissociation of gramicidin A double-stranded dimers into beta 6.3-helical monomers in small unilamellar vesicles prepared following different protocols, were investigated using in combination circular dichroism (CD) and high-performance liquid chromatography (HPLC). The analysis of the data from both techniques according to a two-component model strongly supports that any given CD pattern of gramicidin incorporated in the phospholipid bilayer can be deconvoluted essentially as a linear combination of the reference subspectra calculated for the double-stranded dimer and …
Determination of the relative quantum yields of the conformational species of autoassociating polypeptide gramicidin A in organic solvent using combined fluorescence spectroscopy and high performance liquid chromatography
Abstract A simple, novel method is proposed for the accurate determination of the relative quantum yields of each of the interconverting conformational species of the autoassociating polypeptide gramicidin A in organic solution. The method is based on fitting the experimental results obtained independently from fluorescence emission spectroscopy and high performance liquid chromatography. The fluorescence parameters obtained are discussed in terms of the structural features of the individual conformational species. The advantages of this approach and its possible application to other different organic solvents or to other autoassociating polypeptides are also considered.
Conformational transitions of gramicidin A in phospholipid model membranes. A high-performance liquid chromatography assessment.
We have investigated the conformation of gramicidin A reconstituted in different phospholipid environments, small unilamellar vesicles, extensive bilayers, and micelles by exploiting a recently proposed experimental approach based on high-performance liquid chromatography [Bano et al. (1988) J. Chromatogr. 458, 105; Bano et al. (1989) FEBS Lett. 250, 67]. The method allows the separation of conformational species of the peptide namely, antiparallel double-stranded (APDS) dimers and β 6.3 -helical monomers, and quantitation of their proportions in the lipid environment. Various experimental parameters (e.g., nature of organic solvent, time of incubation in organic solvent, lipid-to-peptide m…
HPLC demonstration that an all Trp--Phe replacement in gramicidin A results in a conformational rearrangement from beta-helical monomer to double-stranded dimer in model membranes.
We have taken advantage of our previously reported high performance liquid chromatographic (HPLC) strategy to investigate the conformational behavior of the optically reversed gramicidin M (gM-), an analog of gramicidin A with all tryptophans replaced by phenylalanines, in different model membranes. It is quantitatively demonstrated for the first time that once inserted in the lipid environment, gM- (unlike the native peptide) undergoes a conformational transition from beta-helical monomers to thermodynamically stable double-stranded dimers. This transition is faster the higher the incubation temperature and can be neatly observed in both small unilamellar phospholipid vesicles and lysophos…