RESUMEN
The yeast prolyl isomerase, Ess1, has recently been shown to interact via its WW domain with the hyperphosphorylated form of the RNA polymerase II C-terminal domain (CTD). We have investigated folding of the Ess1 WW domain and its binding to peptides representing the CTD by circular dichroism and fluorescence. Ess1 WW folds and unfolds reversibly, but in the absence of ligand is only marginally stable with a melting temperature of 19 degrees C. The WW domain is stabilized by the addition of anionic ligands, namely, chloride, inorganic phosphate, phosphoserine, and phosphorylated CTD peptides. Dissociation constants were measured to be 70--100 microM for CTD peptides phosphorylated at one serine, and 16--21 microM for peptides with two or more phosphorylated serines. Weaker or no affinity was observed for nonphosphorylated CTD peptides. There is surprisingly little difference in the affinity for peptides phosphorylated at Ser 2 or Ser 5 of the consensus repeat, or for peptides with different patterns of multiple phosphorylation. The binding of Ess1 to phosphorylated CTD peptides is consistent with a model wherein the WW domain positions Ess1 to catalyze isomerization of the many pSer--Pro peptide bonds in the phosphorylated CTD. We suggest that cis/trans isomerization of prolyl peptide bonds plays a crucial role in CTD function during eukaryotic transcription.
Asunto(s)
Fragmentos de Péptidos/metabolismo , Isomerasa de Peptidilprolil/metabolismo , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Triptófano/metabolismo , Dicroismo Circular , Estabilidad de Enzimas , Ligandos , Peptidilprolil Isomerasa de Interacción con NIMA , Fosfatos/metabolismo , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae , Cloruro de Sodio/metabolismo , Espectrometría de Fluorescencia , Temperatura , VolumetríaRESUMEN
The folding and unfolding kinetics of the B-domain of staphylococcal protein A, a small three-helix bundle protein, were probed by NMR. The lineshape of a single histidine resonance was fit as a function of denaturant to give folding and unfolding rate constants. The B-domain folds extremely rapidly in a two-state manner, with a folding rate constant of 120,000 s-1, making it one of the fastest-folding proteins known. Diffusion-collision theory predicts folding and unfolding rate constants that are in good agreement with the experimental values. The apparent rate constant as a function of denaturant ('chevron plot') is predicted within an order of magnitude. Our results are consistent with a model whereby fast-folding proteins utilize a diffusion-collision mechanism, with the preorganization of one or more elements of secondary structure in the unfolded protein.
Asunto(s)
Pliegue de Proteína , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo , Dicroismo Circular , Difusión , Guanidina/farmacología , Isomerismo , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Prolina/química , Prolina/metabolismo , Desnaturalización Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Apolipoprotein E plays an important role in recovery from acute brain injury and risk of developing Alzheimer's disease. We demonstrate that biologically relevant concentrations of apoE suppress microglial activation and release of TNFalpha and NO in a dose-dependent fashion. Peptides derived from the apoE receptor-binding region mimic the effects of the intact protein, whereas deletion of apoE residues 146-149 abolishes peptide bioactivity. These results are consistent with the hypothesis that apoE modulates microglial function by binding specific cell surface receptors and that the immunomodulatory effects of apoE in the central nervous system may account for its role in acute and chronic neurological disease.
Asunto(s)
Apolipoproteínas E/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Neuroglía/metabolismo , Fragmentos de Péptidos/farmacología , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/farmacología , Sitios de Unión/genética , Sitios de Unión/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/citología , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuroglía/citología , Neuroglía/efectos de los fármacos , Óxido Nítrico/metabolismo , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Estructura Secundaria de Proteína , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Eliminación de Secuencia , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Two high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported. The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively. The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R-values of 19.0 and 17.5%, respectively. Refinement of the AP-inhibitor complexes was carried out using X-PLOR. The final round of refinement was done using SHELXL-97. Crystallographic analyses of the inhibitor complexes reveal different binding modes for the two phosphonate compounds. The significant difference in binding constants can be attributed to these alternative binding modes observed in the high resolution X-ray structures. The phosphinyl group of PAA coordinates to the active site zinc ions in a manner similar to the competitive inhibitor and product inorganic phosphate. In contrast, MMP binds with its phosphonate moiety directed toward solvent. Both enzyme-inhibitor complexes exhibit close contacts, one of which has the chemical and geometrical potential to be considered an unconventional hydrogen bond of the type C-H...X.
Asunto(s)
Fosfatasa Alcalina/antagonistas & inhibidores , Organofosfonatos/química , Ácido Fosfonoacético/química , Compuestos de Sulfhidrilo/química , Fosfatasa Alcalina/química , Sitios de Unión , Carbono/química , Cristalografía por Rayos X , Escherichia coli/enzimología , Hidrocarburos , Enlace de Hidrógeno , Cinética , Metano/análogos & derivados , Metano/química , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Organofosfonatos/metabolismo , Ácido Fosfonoacético/metabolismo , Unión Proteica , Electricidad Estática , Compuestos de Sulfhidrilo/metabolismo , Termodinámica , Zinc/químicaRESUMEN
The folding of coiled coil peptides has traditionally been interpreted in terms of native dimer and unfolded monomers. Calculations using AGADIR and experimental studies of fragments suggest that the monomers of the coiled coil peptide, GCN4-p1, contain significant residual helical structure. A simple model based on diffusion-collision theory predicts not only the measured folding rate within an order of magnitude, but also predicts remarkably well the effect of alanine to glyXcine mutations. We suggest that intrinsic helix stability is a major determinant of the folding rate of the GCN4 coiled coil.
Asunto(s)
Proteínas Fúngicas/química , Pliegue de Proteína , Proteínas Quinasas/química , Proteínas de Saccharomyces cerevisiae , Alanina , Algoritmos , Sustitución de Aminoácidos , Proteínas de Unión al ADN/química , Dimerización , Proteínas Fúngicas/metabolismo , Glicina , Cinética , Mutagénesis Sitio-Dirigida , Proteínas Quinasas/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
A hydrogen bond between the buried residues Asp 14 and Ser 77 in monomeric lambda repressor has been removed by mutation of these residues to alanine. Double mutant cycles show that the interaction stabilizes the native state of the protein by 1.5 kcal/mol. Removal of the interaction affects mainly the unfolding rates and not the folding rates, suggesting that this hydrogen bond is not substantially formed in the rate-limiting steps in the folding pathways of the protein. Mutations in two versions of lambda6-85, wild type and the faster folding G46A/G48A (WT), show similar effects. Diffusion-collision correctly predicts the behavior of WT but not of wild type. Our analysis suggests that folding of helix 3 is a crucial slow step along the various folding pathways and generally occurs before the formation of the 14-77 hydrogen bond. Experiments removing tertiary interactions, combined with experiments altering helical stability and diffusion-collision calculations, provide a strategy to unravel the folding mechanisms of small helical proteins.
Asunto(s)
Bacteriófago lambda/química , Proteínas de Unión al ADN , Pliegue de Proteína , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Bacteriófago lambda/genética , Enlace de Hidrógeno , Cinética , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Desnaturalización Proteica , Proteínas Represoras/genética , Temperatura , Proteínas Virales/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The development of a quantitative kinetic scheme is a central goal in mechanistic studies of biological phenomena. For fast-folding proteins, which lack experimentally observable kinetic intermediates, a quantitative kinetic scheme describing the order and rate of events during folding has yet to be developed. In the present study, the folding mechanism of monomeric lambda repressor is described using the diffusion-collision model and estimates of intrinsic alpha-helix propensities. The model accurately predicts the folding rates of the wild-type protein and five of eight previously studied Ala left and right arrow Gly variants and suggests that the folding mechanism is distributed among multiple pathways that are highly sensitive to the amino acid sequence. For example, the model predicts that the wild-type protein folds through a small number of pathways with a folding time of 260 micros. However, the folding of a variant (G46A/G48A) is predicted to fold through a large number of pathways with a folding time of 12 micros. Both folding times quantitatively agree with the experimental values at 37 degrees C extrapolated to 0 M denaturant. The quantitative nature of the diffusion-collision model allows for rigorous experimental tests of the theory.
Asunto(s)
Proteínas de Unión al ADN , Modelos Químicos , Modelos Moleculares , Pliegue de Proteína , Proteínas Represoras/química , Bacteriófago lambda , Difusión , Cinética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Solventes , Proteínas Virales , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Trifluoroethanol (TFE) is often used to increase the helicity of peptides to make them usable as models of helices in proteins. We have measured helix propensities for all 20 amino acids in water and two concentrations of trifluoroethanol, 15 and 40% (v/v) using, as a model system, a peptide derived from the sequence of the alpha-helix of ribonuclease T1. There are three main conclusions from our studies. (1) TFE alters electrostatic interactions in the ribonuclease T1 helical peptide such that the dependence of the helical content on pH is lost in 40% TFE. (2) Helix propensities measured in 15% TFE correlate well with propensities measured in water, however, the correlation with propensities measured in 40% TFE is significantly worse. (3) Propensities measured in alanine-based peptides and the ribonuclease T1 peptide in TFE show very poor agreement, revealing that TFE greatly increases the effect of sequence context.
Asunto(s)
Fragmentos de Péptidos/química , Ribonucleasa T1/química , Trifluoroetanol/química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Electricidad Estática , TermodinámicaRESUMEN
Our understanding of the factors stabilizing alpha-helical structure has been greatly enhanced by the study of model alpha-helical peptides. However, the relationship of these results to the folding of helices in intact proteins is not well characterized. Helix propensities measured in model peptides are not in good agreement with those from proteins. In order to address these questions, we have measured helix propensities in the alpha-helix of ribonuclease T1 and a helical peptide of identical sequence. We have previously demonstrated excellent agreement between peptide and protein for the nonpolar amino acids [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2833-2837]. Most other amino acids also show good agreement, although certain polar amino acids are exceptions. Helix propensities measured in the ribonuclease T1 peptide/protein are compared with those measured in other systems. Reasonable agreement is found between most systems; however, our propensities differ substantially from those measured in several model peptide systems. Alanine-based peptides overestimate the propensity differences by a factor of 2, and host/guest experiments underestimate them by a factor of 2-3.
Asunto(s)
Péptidos/química , Pliegue de Proteína , Proteínas/química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Ribonucleasa T1/químicaRESUMEN
Phosphatases are important in signal transduction, bacterial pathogenesis and several human diseases. So far, however, it is their opposite numbers, the kinases, that have received more attention from chemists. Recent progress in inhibitor development offers hope that new probes of cellular processes, and perhaps novel therapeutic agents, may soon become available.
Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Marcadores de Afinidad , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Transducción de SeñalRESUMEN
alpha-Helical secondary structure occurs widely in globular proteins and its formation is a key step in their folding. As a consequence, understanding the energetics of helix formation is crucial to understanding protein folding and stability. We have measured the helix propensities of the nonpolar amino acids for an alpha-helix in an intact protein, ribonuclease T1, and for a 17-residue peptide with a sequence identical to that of the alpha-helix in the protein. The helix propensities are in excellent agreement. This shows that when compared in the same sequence context, the helix propensities of the nonpolar amino acids are identical in helical peptides and intact proteins, and that conclusions based on studies of the helix-to-coil transitions of peptides may, in favorable cases, be directly applicable to proteins. Our helix propensities based on ribonuclease T1 are in good agreement with those from similar studies of barnase and T4 lysozyme. In contrast, our helix propensities differ substantially from those derived from studies of alanine-stabilized or salt bridge-stabilized model alpha-helical peptides.
Asunto(s)
Péptidos/química , Proteínas/química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Mutagénesis , Péptidos/genética , Proteínas/genética , TermodinámicaRESUMEN
Measurements of the change in conformational stability, delta(delta G), upon mutation of two acidic residues at the C terminus of the helix of ribonuclease T1 have recently been reported. Here, we investigate peptides based on the sequence of the helix with the same mutations: Glu28 replaced with Gln, Asp29 replaced with Asn, and the double mutant. In addition, the mutant Lys25 to Gln was studied. Changes in helix content of the peptides with pH confirm the conclusion found in the intact protein, that the charged forms of the acidic residues destabilize the protein by destabilizing the helix. The pH-dependence of the change in conformational free energy for the peptides and mutant proteins show fair correspondence for D29N and the double mutant. The mutants E28Q and K25Q, on the other hand, give striking agreement between the protein and peptide systems. This agreement suggests that the helix of ribonuclease T1 behaves as an independently stabilized structural unit of the intact protein and that stabilization of the helical form of the peptide is mirrored in the protein.
Asunto(s)
Estructura Secundaria de Proteína , Ribonucleasa T1/química , Secuencia de Aminoácidos , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación , Péptidos/síntesis química , Péptidos/química , Conformación Proteica , Ribonucleasa T1/genéticaRESUMEN
It is clear that intramolecular hydrogen bonds are essential to the structure and stability of globular proteins. It is not clear, however, whether they make a net favorable contribution to this stability. Experimental and theoretical studies are at odds over this important question. Measurements of the change in conformational stability, delta (delta G), for the mutation of a hydrogen bonded residue to one incapable of hydrogen bonding suggest a stabilization of 1.0 kcal/mol per hydrogen bond. If the delta (delta G) values are corrected for differences in side-chain hydrophobicity and conformational entropy, then the estimated stabilization becomes 2.2 kcal/mol per hydrogen bond. These and other experimental studies discussed here are consistent and compelling: hydrogen bonding stabilizes globular proteins.
Asunto(s)
Conformación Proteica , Proteínas/química , Bacteriófago T4/enzimología , Calorimetría , Enlace de Hidrógeno , Nucleasa Microcócica/química , Modelos Teóricos , Muramidasa/química , Mutagénesis Sitio-Dirigida , Mutación Puntual , Desnaturalización Proteica , Proteínas Recombinantes/química , TermodinámicaRESUMEN
The compound 4-(fluoromethyl)phenyl phosphate (FMPP), recently shown to be a mechanism-based inhibitor of prostatic acid phosphatase (Myers, J.K., and Widlanski, T.S. (1993) Science 262, 1451-1453), was examined for its effect on calcineurin. This compound inhibits calcineurin in a time-dependent, first order manner. Inactivation with [3H]FMPP led to a specific labeling of the catalytic subunit with a stoichiometry of 0.75 mol of label/mol of protein. A related substrate, 4-methylphenyl phosphate, is able to protect calcineurin from FMPP-mediated inhibition. Scavenging nucleophiles, such as cysteine, do not affect the rate of inhibition when included in the reaction. In addition, extensive dialysis indicates that inhibition is essentially irreversible. These results demonstrate that FMPP inactivates calcineurin in a mechanism-based fashion by forming a covalent adduct with calcineurin A, the catalytic subunit.
Asunto(s)
Proteínas de Unión a Calmodulina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Compuestos Organofosforados/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Sitios de Unión , Calcineurina , Cinética , Especificidad por SustratoRESUMEN
Denaturant m values, the dependence of the free energy of unfolding on denaturant concentration, have been collected for a large set of proteins. The m value correlates very strongly with the amount of protein surface exposed to solvent upon unfolding, with linear correlation coefficients of R = 0.84 for urea and R = 0.87 for guanidine hydrochloride. These correlations improve to R = 0.90 when the effect of disulfide bonds on the accessible area of the unfolded protein is included. A similar dependence on accessible surface area has been found previously for the heat capacity change (delta Cp), which is confirmed here for our set of proteins. Denaturant m values and heat capacity changes also correlate well with each other. For proteins that undergo a simple two-state unfolding mechanism, the amount of surface exposed to solvent upon unfolding is a main structural determinant for both m values and delta Cp.
Asunto(s)
Enzimas/química , Desnaturalización Proteica , Proteínas/química , Animales , Calorimetría , Guanidina , Guanidinas , Calor , Humanos , Cinética , Matemática , Muramidasa/química , Pliegue de Proteína , Análisis de Regresión , Ribonucleasa T1/química , Ribonucleasas/química , TermodinámicaRESUMEN
Protein phosphatases play important roles in the regulation of cell growth and metabolism, yet little is known about their enzymatic mechanism. By extrapolation from data on inhibitors of other types of hydrolases, an inhibitor of prostatic acid phosphatase was designed that is likely to function as a mechanism-based phosphotyrosine phosphatase inactivator. This molecule, 4-(fluoromethyl)phenyl phosphate, represents a useful paradigm for the design of potent and specific phosphatase inhibitors.
Asunto(s)
Fosfatasa Ácida/antagonistas & inhibidores , Compuestos Organofosforados/farmacología , Próstata/enzimología , Fosfatasa Ácida/metabolismo , Alquilación , Sitios de Unión , Diseño de Fármacos , Humanos , Hidrólisis , Cinética , Masculino , Compuestos Organofosforados/metabolismoRESUMEN
The associations between the one-month prevalence rates of mental disorders and sociodemographic characteristics were investigated for 18,571 people interviewed in the first-wave community samples of all 5 sites in the US National Institute of Mental Health (NIMH) Epidemiologic Catchment Area program. Men were found to have a significantly higher rate of cognitive impairment than women after controlling for the effects of age, race or ethnicity, marital status and socioeconomic status. Marital status was one of the most powerful correlates of mental disorder risk: the odds of separated or divorced people having any NIMH Diagnostic Interview Schedule disorder were twice that of married people after controlling for age, gender, race or ethnicity and socioeconomic status. The odds of those in the lowest socioeconomic status group having any Diagnostic Interview Schedule disorder was about 2.5 times that of those in the highest socioeconomic status group, controlling for age, gender, race or ethnicity and marital status. For all disorders except cognitive impairment, race or ethnicity did not remain statistically significant after controlling for age, gender, marital status and socioeconomic status.
Asunto(s)
Trastornos Mentales/epidemiología , Adolescente , Adulto , Anciano , Trastornos del Conocimiento/complicaciones , Etnicidad , Femenino , Humanos , Masculino , Estado Civil , Trastornos Mentales/complicaciones , Trastornos Mentales/diagnóstico , Persona de Mediana Edad , Prevalencia , Escalas de Valoración Psiquiátrica , Factores Sexuales , Factores Socioeconómicos , Estados Unidos/epidemiologíaRESUMEN
That a specified number of vascular surgery procedures must be performed annually to maintain competence and that these procedures require a specialized center have been suggested repeatedly. Recent national guidelines admonish us to check our own experience with carotid surgery. In our 10-year review of 215 operations performed for carotid stenosis and/or ulceration by two surgeons in a relatively small community, the combined major morbidity/mortality rate of 2.8% compares favorably to many other series. Our experience suggests that if done judiciously, gently, and with universal shunting, the operation can be accomplished safely in the community hospital setting.
Asunto(s)
Endarterectomía Carotidea , Adulto , Anciano , Anciano de 80 o más Años , Endarterectomía Carotidea/efectos adversos , Femenino , Hospitales Comunitarios , Humanos , Masculino , Persona de Mediana Edad , Complicaciones PosoperatoriasRESUMEN
One-month prevalence results were determined from 18,571 persons interviewed in the first-wave community samples of all five sites that constituted the National Institute of Mental Health Epidemiologic Catchment Area Program. US population estimates, based on combined site data, were that 15.4% of the population 18 years of age and over fulfilled criteria for at least one alcohol, drug abuse, or other mental disorder during the period one month before interview. Higher prevalence rates of most mental disorders were found among younger people (less than age 45 years), with the exception of severe cognitive impairments. Men had higher rates of substance abuse and antisocial personality, whereas women had higher rates of affective, anxiety, and somatization disorders. When restricted to the diagnostic categories covered in international studies based on the Present State Examination, results fell within the range reported for European and Australian studies.