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R Grandori

Publications and source records attributed to R Grandori.

18 recordsLinked to original sources
PubMed2001

Probing protein stabilization by glycerol using electrospray mass spectrometry.

This study shows that electrospray ionization mass spectrometry (ESI-MS), combined with a heated turbo ion-spray interface, allows monitoring protein stabilization by glycerol in solution. Measurements obtained with the two proteins lysozyme and cytochrome c are presented. The observed mass-to-charge (m/z) distributions reveal the stabilizing effect of the additive on the protein conformations against temperature and acid-induced unfolding, as well as against denaturation by acetonitrile. The data obtained with lysozyme allow detection of minor conformational changes upon glycerol addition to the native protein, and suggest that the protein structure in the presence of the additive is slightly compressed compared with its state in water. This result corroborates previous evidence obtained by nuclear magnetic resonance. It is also shown that analysis of the m/z distributions obtained by ESI-MS can lead to detection of partially folded and partially populated states in protein samples.

Animals↗
PubMed2001

Geometry dependent two-dimensional heteronuclear multiplet effects in paramagnetic proteins.

We report experimental observation and numerical simulation of a two-dimensional multiplet effect in the heteronuclear correlation spectrum of a paramagnetic protein that depends on molecular geometry. This effect arises as a consequence of cross-correlated relaxation involving the Curie spin relaxation and internuclear dipolar relaxation mechanisms. It also manifests itself in resolution and sensitivity improvement in transverse relaxation optimised spectroscopy (TROSY) kind of experiments. Characteristic multiplet patterns in heteronuclear coupled two-dimensional NMR spectra encode directional information for the heteronuclear bond with respect to the paramagnetic center. These patterns, which are simulated here using Redfield's relaxation theory, can be used to obtain a new type of geometry restriction for structure determination and refinement of paramagnetic macromolecular systems.

Animals↗
PubMed2000

Cloning, overexpression and characterization of micro-myoglobin, a minimal heme-binding fragment.

We report the cloning and expression of micro-myoglobin, a 78-amino-acid fragment containing residues 29-105 of sperm whale myoglobin, and spanning the region from mid-helix B to mid-helix G of the globin fold. In contrast to full-length myoglobin and to mini-myoglobin (residues 32-129), the micro-myoglobin apoprotein is almost unfolded. However, circular dichroism and absorption spectroscopy data indicate that this fragment is capable of folding into a functional heme-binding unit forming a complex with the prosthetic group with characteristics similar to native myoglobin. Therefore, this case represents a new example of cofactor-assisted folding. The experimental data suggest independence between myoglobin subdomains.

Amino Acid Sequence↗
PubMed1998

Biochemical characterization of WrbA, founding member of a new family of multimeric flavodoxin-like proteins.

The protein WrbA had been identified as an Escherichia coli stationary-phase protein that copurified and coimmunoprecipitated with the tryptophan repressor. Sequences homologous to WrbA have been reported in several species of yeast and plants. We previously showed that this new family of proteins displays low but structurally significant sequence similarity with flavodoxins and that its members are predicted to share the alpha/beta core of the flavodoxin fold but with a short conserved insertion unique to the new family, which could account for reports that some family members may be dimeric in solution. The general sequence similarity to flavodoxins suggests that the members of the new family might bind FMN, but their wide evolutionary distribution indicates that, unlike the flavodoxins, these proteins may be ubiquitous. In this paper, we report the purification and biochemical characterization of WrbA, demonstrating that the protein binds FMN specifically and is a multimer in solution. The FMN binding constant is weaker than for many flavodoxins, being approximately 2 microM at 25 degreesC in 0. 1 mM sodium phosphate, pH 7.2. The protein participates in a dimer-tetramer equilibrium over a wide range of solution conditions, with a midpoint at approximately 1.4 microM. One FMN binds per monomer and has no apparent effect on the multimerization equilibrium. WrbA has no effect on the affinity or mode of DNA binding by the tryptophan repressor; thus, its physiological role remains unclear. Although many proteins with flavodoxin-like domains are known to be multimers, WrbA is apparently the first characterized case in which multimerization is associated directly with the flavodoxin-like domain itself.

Bacterial Proteins↗
PubMed1998

Systematic fold recognition analysis of the sequences encoded by the genome of Mycoplasma pneumoniae.

A robust tool for fold recognition was applied to the systematic analysis of the sequences below 200 residues encoded by the genome of Mycoplasma pneumoniae. The goal was to determine the additional information gain achievable in genome analysis by fold recognition, beyond the intrinsic limits of homology studies. A list of 124 sequences encoding for soluble proteins or domains not homologous to each other, or to proteins with known three-dimensional structure, was analyzed, resulting in significant Z scores for the energy of the structural models in 12 of these cases. This result indicates that systematic application of fold recognition techniques to the analysis of structurally unassigned soluble proteins can lead to high-confidence structural predictions with an efficiency of about 10%, a relevant contribution besides the complementary approach of homology analysis. Four of the predictions presented include mapping of the putative active site of the target sequence and lead to the detection of probable catalytic and binding residues. The data are discussed with reference to the functional implications of the structural models and to the results reported for the homologous genome of Mycoplasma genitalium.

Adenine Phosphoribosyltransferase↗
PubMed1997

A three-step PCR protocol for construction of chimeric proteins.

A general method is described for creating chimeric proteins by transposition of subdomain-sized gene fragments. The method uses three sequential PCR steps to circumvent several limitations inherent in transposition of small insertions and has been optimized to avoid purification of intermediate products. The sequence-independence of the method permits flexibility in the choice of host molecule and insertion site.

Bacterial Proteins↗
PubMed1995

The DNA-binding domain of the hexameric arginine repressor.

The arginine repressor of Escherichia coli is a classical feedback regulator, signalling the availability of L-arginine inside the cell. It differs from most other bacterial repressors in functioning as a hexamer, but structural details have been lacking and its shares no clear sequence homologies with other transcriptional regulators. Analysis of the amino acid residue sequence and proteolytic cleavage pattern of the repressor was used to identify a region predicted to house the DNA-binding function. When this protein fragment is overexpressed from a clone of the corresponding gene fragment, it represses ornithine transcarbamylase levels in vivo, and binds to the operator DNA in vitro, both in an arginine-independent manner. Sedimentation equilibrium and gel filtration indicate that the purified protein fragment is a monomer in solution. The results thus define the domain organization of the repressor at low resolution, suggesting that the N and C-terminal portions of the polypeptide chain are separated by a structural and functional border that decouples hexamerization and arginine binding from DNA binding.

Allosteric Regulation↗
PubMed1995

Localization of lipase genes on Candida rugosa chromosomes.

In the yeast Candida rugosa the lipase isozymes are encoded by a family of genes, five of which have been cloned and sequenced in our laboratory. In this paper we report on the identification and preliminary characterization of two new related sequences, thus extending this multigene family to seven members. The total DNA content of Candida cells was estimated by laser flow-cytometry at about 20 Mb. Eight chromosomes with sizes ranging between 100 kb and 2.1 Mb, as determined by comparison with S. cerevisiae chromosomal bands, were resolved by pulsed-field gel electrophoresis. The lipase-encoding genes were localized on chromosome I, therefore suggesting that they have originated through multiple duplication events of an ancestral gene.

Base Sequence↗
PubMed1994

Autonomous subdomains in protein folding.

Proteolytic dissection of native trp repressor and horse heart cytochrome c has been used to infer some of the steps in the folding pathways of the intact proteins. For both proteins, small fragments are capable of undergoing spontaneous noncovalent association to form subdomains with native-like secondary and/or tertiary structural features, suggesting that dissection/reassembly may be a general method to gain insight into the structures of folding intermediates. The importance of this approach is its simplicity and potential applicability to studying the folding pathways of a wide range of proteins. The proteases report on the structure and dynamics of the native state, circumventing the need for prior knowledge of the structures of folding intermediates. The observation that small fragments of proteins can associated noncovalently suggests that protein folding can be viewed as an intramolecular "recognition" process. The results imply that substantial information about protein structure and folding is encoded at the level of subdomains, and that chain connectivity has only a minor role in determining the fold.

Animals↗
PubMed1994

Six new candidate members of the alpha/beta twisted open-sheet family detected by sequence similarity to flavodoxin.

Strong sequence similarity has been reported among WrbA (the Trp repressor-binding protein of Escherichia coli); Ycp4, a protein of unknown function from the budding yeast Saccharomyces cerevisiae; P25, the pap1-dependent protein of the fission yeast Schizosaccharomyces pombe; and the translation product of a partial cDNA sequence from rice seedling root (Oryza sativa, locus Ricr02421a; here referred to as RicR). Further homology search with the profile method indicates that all the above sequences are related to the flavodoxin family and, in turn, allows detection of the recently proposed flavodoxin-like proteins from E. coli, MioC and the hypothetical protein YihB. We discuss sequence conservation with reference to the known 3-dimensional structures of flavodoxins. Conserved sequence and hydrophobicity patterns, as well as residue-pair interaction potentials, strongly support the hypothesis that these proteins share the alpha/beta twisted open-sheet fold typical of flavodoxins, with an additional alpha/beta unit in the WrbA family. On the basis of the proposed structural homology, we discuss the details of the putative FMN-binding sites. Our analysis also suggests that the helix-turn-helix motif we identified previously in the C-terminal region of the WrbA family is unlikely to reflect a DNA-binding function of this new protein family.

Amino Acid Sequence↗
PubMed1993

Cloning and analysis of Candida cylindracea lipase sequences.

Lipases (Lip) hydrolyze triglycerides into fatty acids and glycerol. Lip produced by the yeast Candida cylindracea are encoded by multiple genomic sequences. We report the molecular cloning and characterization of three genes from this family. They encode putative mature 57-kDa proteins of 534 amino acids (aa). To date, five Lip-encoding genomic sequences from C. cylindracea have been characterized in our laboratory. The five deduced aa sequences share an overall homology of 80%. These sequences have been aligned with each other and with those of homologous enzymes, the Lip from the mould Geotrichum candidum and the acetylcholinesterase from Torpedo californica, whose three-dimensional structures have been solved by X-ray analysis. The C. cylindracea Lip appear to have a structural organization similar to that described for both enzymes.

Amino Acid Sequence↗
PubMed1992

Cloning and nucleotide sequences of two lipase genes from Candida cylindracea.

Two lipase-encoding genes (LIP1 and LIP2) have been isolated from a SacI genomic library of the yeast Candida cylindracea and their nucleotide sequences have been determined. Comparison with the sequence of a cDNA ruled out the presence of introns in the two genes. Both ORFs encode for mature proteins of 534 residues with putative signal peptides of 15 and 14 amino acids, respectively. When compared with other lipase sequences, the two C. cylindracea lipases showed homology only with the Geotrichum candidum lipase, whereas they shared a significant similarity with several esterases.

Amino Acid Sequence↗
PubMed1991

Identification by computer sequence analysis of transcriptional regulator proteins in Dictyostelium discoideum and Serratia marcescens.

We have performed computer searches in the database of known protein sequences for proteins similar in sequence to bacteriophage regulatory proteins of known 3-D structure. The searches are more selective than other methods due to the use of a length-dependent threshold in sequence similarity, above which structural homology is implied with high certainty. Two probable DNA binding proteins were identified which are predicted to have a three-dimensional structure very similar to bacteriophage cro and repressor proteins. Approximate three-dimensional model coordinates are available from the authors. Both proteins contain the helix-turn-helix sequence motif typical of a wide class of DNA binding proteins and their function is deduced by analogy to sequence-similar proteins of known function. We predict that the Y.Smal protein in the restriction-modification enzyme gene locus of the enterobacterium serratia marcescens is a regulator of endonuclease expression; and, that the vegetative specific gene VSH7 of the slime mold dictyostelium discoideum codes for a regulator of gene expression specific for the slime mold growth phase before the onset of the developmental program. Point mutations that would have a strong effect on growth regulation phenotype are suggested. The VSH7 protein would be the first eukaryotic representative of the cro/phage repressor class.

Amino Acid Sequence↗
PubMed1990

cAMP promotes the synthesis in early G1 of gp115, a yeast glycoprotein containing glycosyl-phosphatidylinositol.

The glycoprotein gp115 (Mr = 115,000, pI 4.8-5) is localized in the plasma membrane of Saccharomyces cerevisiae cells and maximally expressed during G1 phase. To gain insight on the mechanism regulating its synthesis, we have examined various conditions of cell proliferation arrest. We used pulse-labeling experiments with [35S]methionine and two-dimensional gel electrophoresis analysis, which allow the detection of the well characterized 100-kDa precursor of gp115 (p100). In the cAMP-requiring mutant cyr1, p100 synthesis is active during exponential growth, shut off by cAMP removal, and induced when growth is restored by cAMP readdition. The inhibition of p100 synthesis also occurs in TS1 mutant cells (ras1ras2-ts1) shifted from 24 to 37 degrees C. During nitrogen starvation of rca1 cells, a mutant permeable to cAMP, p100 synthesis is also inhibited. cAMP complements the effect of ammonium deprivation, promoting p100 synthesis, even when added to cells which have already entered G0. Experiments with the bcy1 and cyr1bcy1 mutants have indicated the involvement of the cAMP-dependent protein kinases in the control of p100 synthesis. Moreover, the synthesis of p100 was unaffected in A364A cells, terminally arrested at START B by alpha-factor. These results indicate that the switch operating on p100 synthesis is localized in early G1 (START A) and is one of the multiple events controlled by the cAMP pathway.

Cell Cycle↗
PubMed1990

The cell cycle modulated glycoprotein GP115 is one of the major yeast proteins containing glycosylphosphatidylinositol.

The cell cycle modulated protein gp115 (115 kDa, isoelectric point about 4.8-5) of Saccharomyces cerevisiae undergoes various post-translational modifications. It is N-glycosylated during its maturation along the secretory pathway where an intermediary precursor of 100 kDa (p100), dynamically related to the mature gp115 protein, is detected at the level of endoplasmic reticulum. Moreover, we have shown by the use of metabolic labeling with [35S]methionine, [3H]palmitic acid and myo-[3H]inositol combined with high resolution two-dimensional gel electrophoresis and immunoprecipitation with a specific antiserum, that gp115 is one of the major palmitate- and inositol-containing proteins in yeast. These results, and the susceptibility of gp115 to phosphatidylinositol-specific phospholipase C treatment strongly indicate that gp115 contains the glycosylphosphatidylinositol (GPI) structure as membrane anchor domain. The two-dimensional analysis of the palmitate- and inositol-labeled proteins has also allowed the characterization of other polypeptides which possibly contain a GPI structure.

Acetylglucosaminidase↗
PubMed1989

Identification of a protein cross-reacting with anti-phosphotyrosine antibodies in yeast insoluble cytoplasmic matrices.

Immunoblot analysis with anti-phosphotyrosine antibodies of total extracts from exponentially growing yeast cells reveals a unique cross-reactive polypeptide of about 75 Kd (p75). The specificity of the immunodecorations has been checked by experiments of competition with phosphoaminoacids. A common behaviour has been observed for the 75 kd band and the 170 kd band corresponding to the platelet-derived growth factor receptor from Swiss 3T3 cells, which it has been known to be autophosphorylated on tyrosine upon ligand binding and used as a control throughout this work. We have found that p75 is associated to detergent insoluble cytoplasmic matrices. The stability of p75 detection by antibodies following treatments that specifically hydrolyze phosphohistidine and its susceptibility to potato acid phosphatase treatment provide further evidences that the epitope recognized by these antibodies in the yeast p75 polypeptide is indeed phosphotyrosine.

Antibodies, Fungal↗
PubMed1988

Immunochemical characterization of gp115, a yeast glycoprotein modulated by the cell cycle.

A cell cycle-modulated glycoprotein (gp115, 115 kDa, isoelectric point 4.8-5) of Saccharomyces cerevisiae has been purified by Concanavalin A-affinity chromatography, followed by preparative two-dimensional gel electrophoresis, from yeast membrane proteins solubilized in Triton X-100. Antisera have been generated against the electrophoretically purified protein. Their specificity has been established by immunoblot analysis and by comparison of the partial proteolytic map obtained for the immunoprecipitated 35S-labeled 115 kDa polypeptide with that of the in vivo [35S]methionine-labeled gp115 isolated from two-dimensional gels. In tunicamycin-treated cells the immunoblot analysis identifies an unglycosylated precursor (86-88 kDa) and in sec18 mutant cells at the restrictive temperature an intermediary precursor of about 100 kDa. Six to seven carbohydrate chains have been estimated to be present on the gp115 protein, accounting for an electrophoretic shift corresponding to about 27 to 29 kDa of its relative molecular mass. Affinity-purified antibodies against the unglycosylated precursor (86-88 kDa) of gp115 were prepared and used to localize gp115 by indirect immunofluorescence microscopy. The similarity between the pattern of fluorescence obtained with these antibodies and that obtained using anti-plasma membrane H+-ATPase antibodies suggests an association of gp115 with the plasma membrane.

Antibody Specificity↗