RESUMEN
Erythrocyte-specific bisphosphoglycerate mutase is a trifunctional enzyme which modulates the levels of 2,3-bisphosphoglycerate (2,3-BPG) in red blood cells by virtue of its synthase and phosphatase activities. Low levels of erythrocyte 2,3-BPG increase the affinity of haemoglobin for oxygen, thus limiting the release of oxygen into tissues. 2,3-BPG levels in stored blood decline rapidly owing to the phosphatase activity of bisphosphoglycerate mutase, which is enhanced by a fall in pH. Here, the 1.94â Å resolution X-ray structure of bisphosphoglycerate mutase is presented, focusing on the dynamic nature of key ligand-binding residues and their interaction with the inhibitor citrate. Residues at the binding pocket are complete. In addition, the movement of key residues in the presence and absence of ligand is described and alternative conformations are explored. The conformation in which the ligand citrate would bind at the substrate-binding pocket is proposed, with discussion and representations of its orientation. The characterization of bisphosphoglycerate mutase-citrate interactions will provide a framework for the design of specific inhibitors of the phosphatase activity of this enzyme, which may limit the decline of 2,3-BPG in stored blood.
Asunto(s)
Bisfosfoglicerato Mutasa/química , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Estructura Terciaria de Proteína , Homología Estructural de ProteínaRESUMEN
In order to increase understanding of the basis of the stability of the native conformational state of porcine pepsin A, a strategy based on induction and monitoring of protein denaturation was developed. Structural perturbation was achieved by adding acetonitrile (MeCN) to the protein-solvent system. MeCN was found to induce non-coincident disruption of the secondary and tertiary structural features of pepsin A. It is proposed that gross unfolding is prompted by disruption of the protein hydration pattern induced by the organic co-solvent. It should be noted that the functional properties and thermal stability of the protein were already impaired before the onset of global unfolding. Low and intermediate contents of MeCN in the protein-solvent system affected the sharpness of the thermal transition and the degree of residual structure of the heat-denatured state. The importance of hydration to the conformational stability of pepsin A in its biologically active state is discussed.
Asunto(s)
Acetonitrilos/farmacología , Pepsina A/química , Pepsina A/metabolismo , Pliegue de Proteína/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Agua/química , Animales , Cromatografía en Gel , Hidrólisis/efectos de los fármacos , Péptidos/metabolismo , Conformación Proteica , Porcinos , Temperatura de Transición/efectos de los fármacosRESUMEN
AIM: The primary aim of this large pilot survey was to demonstrate the use and benefits of electronic data collection with respect to rapidly monitoring the access, delivery, and outcome of cataract surgery in the NHS and to update benchmark standards for these parameters of care. METHOD: Eight NHS departments that currently use specialty-specific electronic clinical systems or Electronic Patient Records (EPR) to collect a minimum preoperative, operative, and anaesthetic data set for cataract surgery agreed to pool their data. RESULTS: A total of 162 surgeons from 50 consultant teams and eight NHS Trusts agreed to submit their data on a total of 16,541 operations for age-related cataract. This report describes the age, sex, and ethnic profiles of the patients, waiting time for surgery, ocular copathology causing a reason for a guarded visual prognosis, visual impairment on admission, visual acuity in the operated eye, and the characteristics of the anaesthetic and surgical procedures. CONCLUSIONS: This survey has raised the benchmark standards established by the last National Survey in 1997. There has been a near universal switch to day case, phacosurgery under local anaesthesia (all used in > or =99.1% of cases compared with 70, 77, and 86%, respectively in 1997). The visual impairment in the operated eye is lower with 45% having 6 / 12 or better compared with 27% in 1997. Waiting times and visual impairment in the fellow eye have probably improved although data collection for these variables was incomplete. All departments require specialty-specific clinical systems to efficiently collect and analyse these data and this survey proves their potential to form the basis for national electronic surveys in the future.
Asunto(s)
Extracción de Catarata/estadística & datos numéricos , Atención a la Salud/estadística & datos numéricos , Sistemas de Registros Médicos Computarizados/organización & administración , Anciano , Anciano de 80 o más Años , Catarata/etnología , Extracción de Catarata/métodos , Extracción de Catarata/normas , Competencia Clínica , Bases de Datos como Asunto/organización & administración , Atención a la Salud/normas , Femenino , Accesibilidad a los Servicios de Salud/normas , Accesibilidad a los Servicios de Salud/estadística & datos numéricos , Investigación sobre Servicios de Salud/métodos , Humanos , Masculino , Auditoría Médica/métodos , Evaluación de Resultado en la Atención de Salud , Proyectos Piloto , Pronóstico , Medicina Estatal/organización & administración , Reino Unido/epidemiología , Agudeza VisualRESUMEN
The polyprotein allergens/antigens of nematodes (NPAs) are the only lipid binding proteins known to be produced as polyproteins. Cleavage of the large polyprotein precursors at regularly spaced proteinase cleavage sites produces 10 or 11 individual protein units of approximately 15 kDa. The sequences of these units are highly diverse within and between species, but there are five absolutely or strongly conserved amino acid positions (Trp15, Gln20, Leu42, Cys64, and Cys120). We have tested the role of these signature amino acids by mutational or chemical alteration of the ABA-1 protein of Ascaris, and examined the resulting modified proteins for perturbations of their lipid binding activities and structural integrity. Substitution of Trp15 and Gln20 both affect the stability of the protein in terms of resistance to thermal or chemical denaturation, but the ligand binding function is unaffected. Mutation of Leu42, however, disrupts both the protein's structural stability and functional integrity, as does chemical disruption of the disulfide bridge formed between Cys64 and Cys120. We also find that the C-terminal, but not the N-terminal, half of the protein binds fatty acids, indicating that the binding site may be confined to this part of the protein. This also supports the idea that the NPA units are themselves derived from an ancient duplication event, and that they may comprise two functionally distinct domains.
Asunto(s)
Alérgenos , Proteínas del Helminto/química , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Antígenos de Plantas , Ascaris , Sitios de Unión , Dicroismo Circular , Cisteína/química , Glutamina/química , Glutatión Transferasa/metabolismo , Metabolismo de los Lípidos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Triptófano/química , Rayos UltravioletaRESUMEN
Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.
Asunto(s)
Lisina/química , Lisina/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacología , Adenosina Trifosfato/metabolismo , Arginina/química , Ácido Aspártico/química , Sitios de Unión , Rastreo Diferencial de Calorimetría , Catálisis , Dicroismo Circular , Cristalografía por Rayos X , Dickeya chrysanthemi/química , Disulfuros/farmacología , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Oxidantes/farmacología , Unión Proteica , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Reactivos de Sulfhidrilo/farmacología , Temperatura , Ácido Trinitrobencenosulfónico/farmacología , Rayos UltravioletaRESUMEN
The molybdate-dependent transcriptional regulator ModE of Escherichia coli functions as a sensor of intracellular molybdate concentration and a regulator for the transcription of several operons that control the uptake and utilization of molybdenum. We present two high-resolution crystal structures of the C-terminal oxyanion-binding domain in complex with molybdate and tungstate. The ligands bind between subunits at the dimerization interface, and analysis reveals that oxyanion selectivity is determined primarily by size. The relevance of the structures is indicated by fluorescence measurements, which show that the oxyanion binding properties of the C-terminal domain of ModE are similar to those of the full-length protein. Comparisons with the apoprotein structure have identified structural rearrangements that occur on binding oxyanion. This molybdate-dependent conformational switch promotes a change in shape and alterations to the surface of the protein and may provide the signal for recruitment of other proteins to construct the machinery for transcription. Sequence and structure-based comparisons lead to a classification of molybdate-binding proteins.
Asunto(s)
Proteínas Bacterianas , Proteínas de Escherichia coli , Molibdeno/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Compuestos de Tungsteno/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Factores de Transcripción/químicaRESUMEN
The structure and backbone dynamics of a double labelled (15N,13C) monomeric, 23.7 kD phosphoglycerate mutase (PGAM) from Schizosaccharomyces pombe have been investigated in solution using NMR spectroscopy. A set of 3125 NOE-derived distance restraints, 148 restraints representing inferred hydrogen bonds and 149 values of (3)J(HNHalpha) were used in the structure calculation. The mean rmsd from the average structure for all backbone atoms from residues 6-205 in the best 21 calculated structures was 0.59 A. The core of the enzyme includes an open, twisted, six-stranded beta-sheet flanked by four alpha-helices and a short 3(10)-helix. An additional smaller domain contains two short antiparallel beta-strands and a further pair of alpha-helices. The C(alpha) atoms of the S. pombe PGAM may be superimposed on their equivalents in one of the four identical subunits of Saccharomyces cerevisiae PGAM with an rmsd of 1.34 A (0.92 A if only the beta-sheet is considered). Small differences between the two structures are attributable partly to the deletion in the S. pombe sequence of a 25 residue loop involved in stabilising the S. cerevisiae tetramer. Analysis of 15N relaxation parameters indicates that PGAM tumbles isotropically with a rotational correlation time of 8.7 ns and displays a range of dynamic features. Of 178 residues analysed, only 77 could be fitted without invoking terms for fast internal motion or chemical exchange, and out of the remainder, 77 required a chemical exchange term. Significantly, 46 of the slowly exchanging (milli- to microsecond) residues lie in helices, and these account for two-thirds of all analysed helix residues. On the contrary, only one beta-sheet residue required an exchange term. In contrast to other analyses of backbone dynamics reported previously, residues in slow exchange appeared to correlate with architectural features of the enzyme rather than congregating close to ligand binding sites.
Asunto(s)
Fosfoglicerato Mutasa/química , Fosfoglicerato Mutasa/metabolismo , Schizosaccharomyces/enzimología , Sustitución de Aminoácidos/genética , Cristalografía por Rayos X , Modelos Moleculares , Peso Molecular , Resonancia Magnética Nuclear Biomolecular , Fosfoglicerato Mutasa/genética , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/genética , SolucionesRESUMEN
The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis. The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site-directed mutagenesis. All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm:HIS) in good yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. cerevisiae. The S62A mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate (3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques.
Asunto(s)
Aminoácidos/metabolismo , Fosfoglicerato Mutasa/metabolismo , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Cartilla de ADN , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfoglicerato Mutasa/química , Fosfoglicerato Mutasa/genética , Pliegue de Proteína , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The structure of the Escherichia coli flavodoxin NADP(+) oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6 min(-1); R174A, 132.1 min(-1); R184A, 305.5 min(-1) versus wild type, 338.9 min(-1)) and increased K(m) for NADPH (R144A, 5.3 microM; R174A, 20.2 microM; R184A, 54.4 microM versus wild type, 3.9 microM). The k(cat) value for NADH-dependent cytochrome c reduction was increased for R174A (42.3 min(-1)) and R184A (50.4 min(-1)) compared with the wild type (33.0 min(-1)), consistent with roles for R174 and R184 in discriminating between NADPH/NADH by interaction with the adenosine ribose 2'-phosphate. Stopped-flow studies indicated that affinity (K(d)) for NADPH was markedly reduced in mutants R144A (635 microM) and R184A (2.3 mM) compared with the wild type (<5 microM). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH K(d) ratio >175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (k(red)=8.82 s(-1) versus 22.63 s(-1) for the wild type), which also exhibited tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C-terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosphates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2'-phosphate group, and R144 likely to interact with the nicotinamide ribose 5'-phosphate group.
Asunto(s)
Escherichia coli/enzimología , NADH NADPH Oxidorreductasas/metabolismo , NADP/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Cinética , Modelos Moleculares , Sondas Moleculares , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/aislamiento & purificación , Conformación Proteica , Homología de Secuencia de Aminoácido , Análisis EspectralRESUMEN
Dihydrolipoamide dehydrogenase (E3) from Escherichia coli, an FAD-linked homodimer, can be fully reconstituted in vitro following denaturation in 6 m guanidinium chloride. Complete restoration of activity occurs within 1-2 h in the presence of FAD, dithiothreitol, and bovine serum albumin. In the absence of FAD, the dihydrolipoamide dehydrogenase monomer forms a stable folding intermediate, which is incapable of dimerization. This intermediate displays a similar tryptic resistance to the native enzyme but is less heat-stable, because its ability to form native E3 is lost after incubation at 65 degrees C for 15 min. The presence of FAD promotes slow, additional conformational rearrangements of the E3 subunit as observed by cofactor-dependent decreases in intrinsic tryptophan fluorescence. However, after 2 h, the tryptophan fluorescence spectrum and far UV CD spectrum of E3, refolded in the absence of FAD, are similar to that of the native enzyme, and full activity can still be recovered on addition of FAD. Cross-linking studies show that FAD insertion is necessary for the monomeric folding intermediate to attain an assembly competent state leading to dimerization. Thus cofactor insertion represents a key step in the assembly of this enzyme, although its initial presence appears not to be required to promote the correct folding pathway.
Asunto(s)
Dihidrolipoamida Deshidrogenasa/metabolismo , Escherichia coli/enzimología , Flavina-Adenina Dinucleótido/metabolismo , Animales , Catálisis , Bovinos , Dihidrolipoamida Deshidrogenasa/química , Dimerización , Ditiotreitol/farmacología , Electroforesis en Gel de Poliacrilamida , Calor , Pliegue de Proteína , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Tripsina/metabolismoRESUMEN
The acidic isoform of phospholipase A(2) from Naja mossambica mossambica was activated by treatment with a molar equivalent of oleoyl imidazolide. Modification of the protein was accompanied by 50% quenching of tryptophan fluorescence and a significant red shift. The (3)H(9,10) labeled oleoyl residue was co-eluted with the enzyme during gel filtration in the presence of 20% 1-propanol or excess albumin, both of which remove free oleic acid from the enzyme. In contrast, the adduct was labile as to electrophoresis on SDS-PAGE and acid or alkali urea PAGE. The formation of a covalently linked adduct was demonstrated by electrospray mass spectrometry in the presence of 2% formic acid. No such adduct was formed by the phospholipase A(2) isoform from Naja naja atra, which differs in sequence from the N. mossambica mossambica isoform by seven residues including 2 histidine residues and 1 lysine residue. We conclude that oleoyl imidazolide activates the N. mossambica mossambica enzyme by forming an acyl adduct which is unstable as to protein denaturation. The magnitude of tryptophan fluorescence quenching indicates that the site of acylation lies in the sequence WWHF.
Asunto(s)
Venenos Elapídicos/enzimología , Ácidos Grasos/metabolismo , Fosfolipasas A/metabolismo , Procesamiento Proteico-Postraduccional , Acilación , Animales , Cromatografía en Gel , Elapidae , Electroforesis , Activación Enzimática , Espectrometría de Masas , Espectrometría de FluorescenciaRESUMEN
The conformation of a protein refers to the three-dimensional arrangement of its constituent atoms. Since expression of the biological activity of a protein depends on its conformation, it is clear that full characterization of a protein involves an understanding of its three-dimensional structure. This review outlines the principal techniques for determining the conformation of a protein, describes the crucial role played by the flexibility of proteins, and gives an account of current theories of the mechanisms by which proteins fold. A final section deals with strategies that can be adopted to preserve the conformational integrity of proteins; an aspect that is of increasing importance as a greater number of proteins are finding applications in industrial processes and as therapeutic and diagnostic agents.
Asunto(s)
Pliegue de Proteína , Proteínas/química , Dicroismo Circular , Cristalografía por Rayos X , Fluorescencia , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Proteica , Desnaturalización Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Two 3D experiments, (H)CCH(3)-TOCSY and H(C)CH(3)-TOCSY, are proposed for resonance assignment of methyl-containing amino acid side chains. After the initial proton-carbon INEPT step, during which either carbon or proton chemical shift labeling is achieved (t(1)), the magnetization is spread along the amino acid side chains by a carbon spin lock. The chemical shifts of methyl carbons are labeled (t(2)) during the following constant time interval. Finally the magnetization is transferred, in a reversed INEPT step, to methyl protons for detection (t(3)). The proposed experiments are characterized by high digital resolution in the methyl carbon dimension (t(2max) = 28.6 ms), optimum sensitivity due to the use of proton decoupling during the long constant time interval, and an optional removal of CH(2), or CH(2) and CH, resonances from the F(2)F(3) planes. The building blocks used in these experiments can be implemented in a range of heteronuclear experiments focusing on methyl resonances in proteins. The techniques are illustrated using a (15)N, (13)C-labeled E93D mutant of Schizosacharomyces pombe phosphoglycerate mutase (23.7 kDa).
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Proteínas/químicaRESUMEN
Circular Dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as alpha-helix and beta-sheet. The CD spectrum in the near UV region (320-260 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein. Other non-protein chromophores such as flavin and haem moieties can give rise to CD signals which depend on the precise environment of the chromophore concerned. Because of its relatively modest resource demands, CD has been used extensively to give useful information about protein structure, the extent and rate of structural changes and ligand binding. In the protein design field, CD is used to assess the structure and stability of the designed protein fragments. Studies of protein folding make extensive use of CD to examine the folding pathway; the technique has been especially important in characterising molten globule intermediates which may be involved in the folding process. CD is an extremely useful technique for assessing the structural integrity of membrane proteins during extraction and characterisation procedures. The interactions between chromophores can give rise to characteristic CD signals. This is well illustrated by the case of the light harvesting complex from photosynthetic bacteria, where the CD spectra can be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll molecules. It is therefore evident that CD is a versatile technique in structural biology, with an increasingly wide range of applications.
Asunto(s)
Dicroismo Circular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Aminoácidos/química , Disulfuros/química , Diseño de Fármacos , Ligandos , Modelos Moleculares , Pliegue de ProteínaRESUMEN
The 28.6 kDa vaccinia virus complement control protein (VCP) is an inhibitor of the complement system and has therapeutic potential. It is composed of four domains or modules and is a homologue of complement receptor 1 (CR1) and other mammalian regulators of complement activation. A key aspect to structure-function relationships in these proteins is the extent of intramolecular module-module interactions, since these dictate the overall shape and flexibility of the molecules. A protein fragment (VCP approximately 2,3) encompassing modules 2 and 3 of VCP was over-expressed in Pichia pastoris. Ultracentrifugation showed that VCP approximately 2,3 is highly asymmetric with an axial ratio of 5.3:1, which is consistent with an end-to-end arrangement of the two modules. NMR spectroscopy, differential scanning calorimetry, CD and intrinsic tryptophan fluorescence were used to monitor unfolding of VCP approximately 2,3. Experiments performed over a range of temperatures and concentrations of guanidinium chloride revealed that module 2 unfolds under milder conditions than, and independently of, module 3. Unfolding of module 2 is not associated with extensive changes in amide (15)N and (1)H chemical shifts of module 3, implying that the modules do not form an extensive intermodular interface. Results obtained in this work for VCP approximately 2,3 are compared with those obtained in a study of CR1 modules 15-17 [Kirkitadze, Krych, Uhrin, Dryden, Smith, Cooper, Wang, Hauhart, Atkinson and Barlow (1999) Biochemistry 38, 7019-7031].
Asunto(s)
Proteínas Inactivadoras de Complemento/química , Virus Vaccinia/química , Proteínas Virales/química , Secuencia de Aminoácidos , Animales , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Proteínas Inactivadoras de Complemento/genética , Proteínas Inactivadoras de Complemento/inmunología , Secuencia Conservada , Guanidina , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Desnaturalización Proteica , Estructura Terciaria de Proteína , Receptores de Complemento/química , Receptores de Complemento/genética , Espectrometría de Fluorescencia , Ultracentrifugación , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Complement receptor type 1 (CR1) has 30 modules in its extracellular portion. An understanding of structure-function relationships within CR1 is being assembled gradually from studies of overlapping protein fragments. A CR1 fragment corresponding to modules 16 and 17 was expressed recombinantly as a non-glycosylated protein and its stability and unfolding characteristics studied using biophysical techniques. The results were compared with data collected previously on a CR1 fragment encompassing modules 15, 16 and 17 which together constitute a C3b-binding site (Kirkitadze, M.D., Krych, M., Uhrin, D. , Dryden, D.T.F., Smith, B.O., Wang, X., Hauhart, R., Atkinson, J.P. and Barlow, P.N. (1999) Biochemistry 38, 7019-7031). Modules within CR1 were found to co-operate during unfolding. The folding, stability and flexibility of this protein is therefore likely to be a complex function, and not just the sum, of contributions from individual modules.
Asunto(s)
Complemento C3b/metabolismo , Receptores de Complemento/metabolismo , Sitios de Unión , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Complemento C3b/química , Guanidina/farmacología , Espectroscopía de Resonancia Magnética , Pichia , Conformación Proteica , Pliegue de Proteína , Receptores de Complemento/química , Receptores de Complemento/genética , Espectrometría de FluorescenciaRESUMEN
Polyproteins comprise long polypeptides that are post-translationally cleaved into proteins of different function, or tandemly repetitive polypeptides which are processed into multiple versions of proteins which are presumed to have the same function. In the latter case the individual units of the polyprotein can differ substantially in sequence. Identity of function between the different units therefore cannot be assumed. Here we have examined the ABA-1 polyprotein allergen of the parasitic nematode Ascaris suum and found it to contain units which show a 50% difference in amino acid sequence. The parasite therefore produces at least two radically different forms of the allergen encoded within the polyprotein array. In fluorescence-based ligand-binding assays, recombinant polypeptides representing the two forms (designated ABA-1A1 and ABA-1B1) showed similar binding affinities for a range of fluorescent active-site probes [retinol, dansylundecanoic acid, dansyl-DL-alpha-amino-octanoic acid, cis-parinaric acid (cPnA)] and for the non-specific hydrophobic surface probe 8-anilinonaphthalene-1-sulphonic acid. However, the molecular environments in the active sites are markedly different, as indicated by disparate fluorescence emission peaks and intensities of bound probes. CD showed that the proteins have similar secondary structures but differ in susceptibility to chemical denaturation/unfolding by guanidinium chloride. Both retain a single conserved tryptophan residue in a characteristic non-polar environment, as revealed by extreme fluorescence blue shift. Thus the gross differences in sequence of the two proteins are not reflected in their ligand-binding specificities but in their binding-site environments.
Asunto(s)
Alérgenos , Ascaris suum/química , Ácidos Grasos/metabolismo , Proteínas del Helminto/química , Vitamina A/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/química , Antígenos Helmínticos/genética , Antígenos Helmínticos/metabolismo , Antígenos de Plantas , Ascaris suum/genética , Sitios de Unión , Dicroismo Circular , Clonación Molecular , Biblioteca de Genes , Variación Genética/genética , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Ligandos , Datos de Secuencia Molecular , Unión Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Volumetría , Triptófano/genéticaRESUMEN
Site-specific mutants have been produced in order to investigate the role of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis-proline in the high resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T. maritima PGK, and may therefore have a role in the independent folding of the two domains or in the 'hinge' bending of the molecule during catalysis. The residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe), and the resulting proteins characterised by differential scanning calorimetry (DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic analysis. Although the secondary and tertiary structure of the Pro204His protein is generally similar to that of the wild-type enzyme as assessed by CD, the enzyme is less stable to heat and guanidinium chloride denaturation than the wild-type. In the denaturation experiments two transitions were observed for both the wild-type and the Pro204His mutant, as have been previously reported for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990) Biochemistry 29, 8683-8689]. The first transition is accompanied by an increase in fluorescence intensity leading to a hyperfluorescent state, followed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild-type protein, suggesting that sequence-distant interactions are more rapidly disrupted in this mutant enzyme than in the wild-type enzyme, while sequence-local interactions are disrupted in a similar way. The Michaelis constants (K(m)) for both 3-phospho-D-glycerate and ATP are increased only by three or fourfold, which confirms that, as expected, the substrate binding sites are largely unaffected by the mutation. However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of 'hinge' bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the wild-type and Pro204His enzymes, with considerable loss of both secondary and tertiary structure. It is proposed that the proline residue at 204 in the 'hinge' region of PGK plays a role in the stability and catalytic mechanism of the enzyme.