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Patrick Groves

Publications and source records attributed to Patrick Groves.

12 recordsLinked to original sources
PubMed2006

Structure and dynamics of the conserved protein GPI anchor core inserted into detergent micelles.

A suitable approach which combines nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have been used to study the structure and the dynamics of the glycosylphosphatidylinositol (GPI) anchor Manalphal-2Manalpha1-6Manalphal -4GlcNalpha1-6myo-inositol-1-OPO(3)-sn-1,2-dimyristoylglycerol (1) incorporated into dodecylphosphatidylcholine (DPC) micelles. The results have been compared to those previously obtained for the products obtainable from (1) after phospholipase cleavage, in aqueous solution. Relaxation and diffusion NMR experiments were used to establish the formation of stable aggregates and the insertion of (1) into the micelles. MD calculations were performed including explicit water, sodium and chloride ions and using the Particle Mesh Ewald approach for the evaluation of the electrostatic energy term. The MD predicted three dimensional structure and dynamics were substantiated by nuclear overhauser effect (NOE) measurements and relaxation data. The pseudopentasaccharide structure, which was not affected by incorporation of (1) into the micelle, showed a complex dynamic behaviour with a faster relative motion at the terminal mannopyranose unit and decreased mobility close to the micelle. This motion may be better described as an oscillation relative to the membrane rather than a folding event.

Computer Simulation↗
PubMed2006

Exploring the use of conformationally locked aminoglycosides as a new strategy to overcome bacterial resistance.

The emergence of bacterial resistance to the major classes of antibiotics has become a serious problem over recent years. For aminoglycosides, the major biochemical mechanism for bacterial resistance is the enzymatic modification of the drug. Interestingly, in several cases, the oligosaccharide conformation recognized by the ribosomic RNA and the enzymes responsible for the antibiotic inactivation is remarkably different. This observation suggests a possible structure-based chemical strategy to overcome bacterial resistance; in principle, it should be possible to design a conformationally locked oligosaccharide that still retains antibiotic activity but that is not susceptible to enzymatic inactivation. To explore the scope and limitations of this strategy, we have synthesized several aminoglycoside derivatives locked in the ribosome-bound "bioactive" conformation. The effect of the structural preorganization on RNA binding, together with its influence on the aminoglycoside inactivation by several enzymes involved in bacterial resistance, has been studied. Our results indicate that the conformational constraint has a modest effect on their interaction with ribosomal RNA. In contrast, it may display a large impact on their enzymatic inactivation. Thus, the work presented herein provides a key example of how the conformational differences exhibited by these ligands within the binding pockets of the ribosome and of those enzymes involved in bacterial resistance can, in favorable cases, be exploited for designing new antibiotic derivatives with improved activity in resistant strains.

Acetyltransferases↗
PubMed2005

Useful applications of DOSY experiments for the study of mushroom polysaccharides.

DOSY analysis has been performed on different mushroom extracts and fractions with the aim to describe a general method for the study of high molecular weight polysaccharides. These NMR experiments can be exploited to monitor the fractionation pathways performed on crude extracts in order to isolate polysaccharides. DOSY can also rapidly verify the purity of the isolated compounds, as well as evaluate their molecular size. In spite of the complexity of DOSY spectra of mixtures, this NMR technique seems to be a valid analytical tool that could be adopted as a routine method for the study of polysaccharides from different sources.

Agaricales↗
PubMed2005

On the importance of carbohydrate-aromatic interactions for the molecular recognition of oligosaccharides by proteins: NMR studies of the structure and binding affinity of AcAMP2-like peptides with non-natural naphthyl and fluoroaromatic residues.

The specific interaction of a variety of modified hevein domains to chitooligosaccharides has been studied by NMR spectroscopy in order to assess the importance of aromatic-carbohydrate interactions for the molecular recognition of neutral sugars. These mutant AcAMP2-like peptides, which have 4-fluoro-phenylalanine, tryptophan, or 2-naphthylalanine at the key interacting positions, have been prepared by solid-phase synthesis. Their three-dimensional structures, when bound to the chitin-derived trisaccharide, have been deduced by NMR spectroscopy. By using DYANA and restrained molecular dynamics simulations with the AMBER 5.0 force field, the three-dimensional structures of the protein-sugar complexes have been obtained. The thermodynamic analysis of the interactions that occur upon complex formation have also been carried out. Regarding binding affinity, the obtained data have permitted the deduction that the larger the aromatic group, the higher the association constant and the binding enthalpy. In all cases, entropy opposes binding. In contrast, deactivation of the aromatic rings by attaching fluorine atoms decreases the binding affinity, with a concomitant decrease in enthalpy. The role of the chemical nature of the aromatic ring for establishing sugar contacts has been thus evaluated.

Amino Acid Sequence↗
PubMed2005

Characterization of calretinin I-II as an EF-hand, Ca2+, H+-sensing domain.

Calretinin, a neuronal protein with well-defined calcium-binding properties, has a poorly defined function. The pH dependent properties of calretinin (CR), the N-terminal (CR I-II), and C-terminal (CR III-VI) domains were investigated. A drop in pH within the intracellular range (from pH 7.5 to pH 6.5) leads to an increased hydrophobicity of calcium-bound CR and its domains as reported by fluorescence spectroscopy with the hydrophobic probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). The TNS data for the N- and C-terminal domains of CR are additive, providing further support for their independence within the full-length protein. Our work concentrated on CR I-II, which was found to have hydrophobic properties similar to calmodulin at lower pH. The elution of CR I-II from a phenyl-Sepharose column was consistent with the TNS data. The pH-dependent structural changes were further localized to residues 13-28 and 44-51 using nuclear magnetic resonance spectroscopy chemical shift analysis, and there appear to be no large changes in secondary structure. Protonation of His 12 and/or His 27 side chains, coupled with calcium chelation, appears to lead to the organization of a hydrophobic pocket in the N-terminal domain. CR may sense and respond to calcium, proton, and other signals, contributing to conflicting data on the proteins role as a calcium sensor or calcium buffer.

Amino Acid Sequence↗
PubMed2005

The relative orientation of the lipid and carbohydrate moieties of lipochitooligosaccharides related to nodulation factors depends on lipid chain saturation.

Lipochitooligosaccharides (LCOs) signal the symbiosis of rhizobia with legumes and the formation of nitrogen-fixing root nodules. LCOs 1 and 2 share identical tetrasaccharide scaffolds but different lipid moieties (1, LCO-IV(C16:1[9Z], SNa) and , LCO-IV(C16:2[2E,9Z], SNa)). The conformational behaviors of both LCOs were studied by molecular modeling and NMR. Modeling predicts that a small lipid modification would result in a different relative orientation of the lipid and tetrasaccharide moieties. Diffusion ordered spectroscopy reports that both LCOs form small aggregates above 1 mM. Nuclear Overhauser spectroscopy (NOESY) data, collected under monomeric conditions, reveals lipid-carbohydrate contacts only for 1, in agreement with the modeling data. The distinct molecular structures of 1 and 2 have the potential to contribute to their selective binding by legume proteins.

Carbohydrate Conformation↗
PubMed2004

NMR and modeling studies of protein-carbohydrate interactions: synthesis, three-dimensional structure, and recognition properties of a minimum hevein domain with binding affinity for chitooligosaccharides.

HEV32, a 32-residue, truncated hevein lacking eleven C-terminal amino acids, was synthesized by solid-phase methodology and correctly folded with three cysteine bridge pairs. The affinities of HEV32 for small chitin fragments--in the forms of N,N',N"-triacetylchitotriose ((GlcNAc)3) (millimolar) and N,N',N",N"',N"",N""'-hexaacetylchitohexaose ((GlcNAc)6) (micromolar)--as measured by NMR and fluorescence methods, are comparable with those of native hevein. The HEV32 ligand-binding process is enthalpy driven, while entropy opposes binding. The NMR structure of ligand-bound HEV32 in aqueous solution was determined to be highly similar to the NMR structure of ligand-bound hevein. Solvated molecular-dynamics simulations were performed in order to monitor the changes in side-chain conformation of the binding site of HEV32 and hevein upon interaction with ligands. The calculations suggest that the Trp21 side-chain orientation of HEV32 in the free form differs from that in the bound state; this agrees with fluorescence and thermodynamic data. HEV32 provides a simple molecular model for studying protein-carbohydrate interactions and for understanding the physiological relevance of small native hevein domains lacking C-terminal residues.

Allergens↗
PubMed2003

Diffusion ordered spectroscopy as a complement to size exclusion chromatography in oligosaccharide analysis.

A series of N-acetyl-chitooligosaccharides (GlcNAc)(1-6) have been studied by a nuclear magnetic resonance (NMR) method, diffusion ordered spectroscopy (DOSY). DOSY has also been applied to two additional synthetic related oligosaccharides [GlcNH(2)-(GlcNAc)(4) and GlcNH(2)-(GlcNAc)(2)-GlcNAcSO(3)Na]. A plot of the log of the determined diffusion coefficients (logD) of (GlcNAc)(n) versus the log of molecular weight was linear (6 points, R(2) = 0.995). The molecular weights of the two synthetic chitin derivatives could be estimated to within 10% error. The processed NMR data of all the chitooligosaccharides was also plotted in a polyacrylamide gel-like format to aid visual interpretation. Moreover, the logD value of the NMR signal resonances of a chitin-binding protein (hevein) changed as a function of a given titrated ligand, (GlcNAc)(6). Evidence for a 2:1 hevein:(GlcNAc)(6) complex is detected by DOSY at high hevein:(GlcNAc)(6) ratios. This data is consistent with published analytical ultracentrifugation and isothermal titration calorimetry data. A 1:1 complex is preferred at higher ligand concentrations. DOSY can complement size exclusion chromatography in carbohydrate research with the advantage that oligosaccharides are more readily detected by NMR.

Animals↗
PubMed2003

Calretinin and calbindin D28k have different domain organizations.

The domain organization of calretinin (CR) was predicted to involve all six EF-hand motifs (labeled I to VI) condensed into a single domain, as characterized for calbindin D28k (Calb), the closest homolog of calretinin. Unperturbed (1)H,(15)N HSQC NMR spectra of a (15)N-labeled calretinin fragment (CR III-VI, residues 100-271) in the presence of the unlabeled complimentary fragment (CR I-II, residues 1-100) show that these fragments do not interact. Size exclusion chromatography and affinity chromatography data support this conclusion. The HSQC spectrum of (15)N-labeled CR is similar to the overlaid spectra of individual (15)N-labeled CR fragments (CR I-II and CR III-VI), also suggesting that these regions do not interact within intact CR. In contrast to these observations, but in accordance with the Calb studies, we observed interactions between other CR fragments: CR I (1-60) with CR II-VI (61-271), and CR I-III (1-142) with CR IV-VI (145-271). We conclude that CR is formed from at least two independent domains consisting of CR I-II and CR III-VI. The differences in domain organization of Calb and CR may explain the specific target interaction of Calb with caspase-3. Most importantly, the comparison of CR and Calb domain organizations questions the value of homologous modeling of EF-hand proteins, and perhaps of other protein families.

Animals↗
PubMed2003

Concanavalin A-agarose removes mannan impurities from an extracellularly expressed Pichia pastoris recombinant protein.

Pichia pastoris secretes few native proteins. However, the more than 1 g l(-1)of extracellularly expressed mannan interfered with the purification of our extracellularly expressed, non-glycosylated recombinant protein. Concanavalin A-agarose removed more than 95% of the unwanted mannan as monitored by phenol reaction. A(13)C-based NMR assay confirmed this improvement. Concanavalin A-agarose can assist the purification of extracellular expressed, non-glycosylated proteins from yeasts.

Carbon Isotopes↗