RESUMEN
BACKGROUND AND PURPOSE: Few studies have investigated the differences in cognitive skills between the three subtypes of multiple sclerosis (MS) and they confounded the course of the disease with the duration of the disease and the physical disability. Moreover, they were not population based. METHODS: This was a retrospective analysis of cognitive testing from the database of a French programme for MS care. The pattern and the frequency of cognitive impairment in secondary progressive (SP), primary progressive (PP) and late relapsing-remitting (LRR, disease duration of more than 10 years) MS were compared. RESULTS: A total of 101 patients with MS (41 LRRMS, 37 SPMS, 23 PPMS) were included. 63.0% had a significant cognitive impairment. After controlling for age, sex, Expanded Disability Status Scale, disease duration and education level, patients with SPMS were at least 2-fold more frequently impaired than patients with LLRMS in information processing speed (P = 0.005), executive functions (P = 0.04), verbal fluency (P = 0.02), verbal episodic memory (P = 0.04), working memory (P = 0.02) and visuospatial construction (P = 0.01). The number of patients with at least one or two deficient cognitive domain(s) was higher in the SPMS group than in the LRRMS group (P = 0.002 and P < 0.001). Patients with PPMS were more frequently impaired in verbal fluency (P = 0.046) than patients with LRRMS and they more often presented at least one impaired cognitive domain (P = 0.03). SPMS and PPMS groups differed only for visuospatial construction (P = 0.02). CONCLUSION: In this population-based study, patients with a progressive subtype of MS were more frequently and more severely impaired than patients with RRMS, even after more than 10 years of disease.
Asunto(s)
Trastornos del Conocimiento/fisiopatología , Esclerosis Múltiple Crónica Progresiva/fisiopatología , Esclerosis Múltiple Recurrente-Remitente/fisiopatología , Adulto , Trastornos del Conocimiento/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Crónica Progresiva/complicaciones , Esclerosis Múltiple Recurrente-Remitente/complicaciones , Estudios RetrospectivosRESUMEN
In this work, we have analyzed the relative importance of secondary versus tertiary interactions in stabilizing and guiding protein folding. For this purpose, we have designed four different mutants to replace the alpha-helix of the GB1 domain by a sequence with strong beta-hairpin propensity in isolation. In particular, we have chosen the sequence of the second beta-hairpin of the GB1 domain, which populates the native conformation in aqueous solution to a significant extent. The resulting protein has roughly 30 % of its sequence duplicated and maintains the 3D-structure of the wild-type protein, but with lower stability (up to -5 kcal/mol). The loss of intrinsic helix stability accounts for about 80 % of the decrease in free energy, illustrating the importance of local interactions in protein stability. Interestingly enough, all the mutant proteins, included the one with the duplicated beta-hairpin sequence, fold with similar rates as the GB1 domain. Essentially, it is the nature of the rate-limiting step in the folding reaction that determines whether a particular interaction will speed up, or not, the folding rates. While local contacts are important in determining protein stability, residues involved in tertiary contacts in combination with the topology of the native fold, seem to be responsible for the specificity of protein structures. Proteins with non-native secondary structure tendencies can adopt stable folds and be as efficient in folding as those proteins with native-like propensities.
Asunto(s)
Proteínas Bacterianas/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/química , Desnaturalización Proteica , Termodinámica , Urea/farmacologíaRESUMEN
The usefulness of molecular dynamics to assess the structural integrity of mutants containing several mutations has been investigated. Our goal was to determine whether molecular dynamics would be able to discriminate mutants of a protein having a close-to-wild-type fold, from those that are not folded under the same conditions. We used as a model the B1 domain of protein G in which we replaced the unique central alpha-helix by the sequence of the second beta-hairpin, which has a strong intrinsic propensity to form this secondary structure in solution. In the resulting protein, one-third of the secondary structure has been replaced by a non-native one. Models of the mutants were built based on the three-dimensional structure of the wild-type GB1 domain. During 2 ns of molecular dynamics simulations on these models, mutants containing up to 10 mutations in the helix retained the native fold, while another mutant with an additional mutation unfolded. This result is in agreement with our circular dichroism and NMR experiments, which indicated that the former mutants fold into a structure similar to the wild-type, as opposed to the latter mutant which is partly unfolded. Additionally, a mutant containing six mutations scattered through the surface of the domain, and which is unfolded, was also detected by the simulation. This study suggests that molecular dynamics calculations could be performed on molecular models of mutants of a protein to evaluate their foldability, prior to a mutagenesis experiment.
Asunto(s)
Simulación por Computador , Proteínas de Unión al GTP/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Aminoácidos/química , Dicroismo Circular , Cinética , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Factores de TiempoRESUMEN
Recent studies have pointed out the important role of local water structures in protein conformational stability. Here, we present an accurate and computationally effective way to estimate the free energy contribution of the simplest water structure motif--the water bridge. Based on the combination of empirical parameters for accessible protein surface area and the explicit consideration of all possible water bridges with the protein, we introduce an improved protein solvation model. We find that accounting for water bridge formation in our model is essential to understand the conformational behavior of polypeptides in water. The model formulation, in fact, does not depend on the polypeptide nature of the solute and is therefore applicable to other flexible biomolecules (i.e., DNAs, RNAs, polysaccharides, etc.).
Asunto(s)
Conformación Proteica , Agua/química , Enlace de Hidrógeno , TermodinámicaRESUMEN
The design of a transmembrane four-helix bundle is described. We start with an idealized four-helix bundle geometry, then use statistical information to build a plausible transmembrane bundle. Appropriate residues are chosen using database knowledge on the sequences of membrane helices and loops, then the packing of the bundle core is optimized, and favorable side chain rotamers from rotamer libraries are selected. Next, we use explicit physical knowledge from biomolecular simulation force fields and molecular dynamics simulations to test whether the designed structure is physically possible. These procedures test whether the designed protein will indeed be alpha-helical, well packed and stable over a time scale of several nanoseconds in a realistic lipid bilayer environment. We then test a modeling approach that does not include sophisticated database knowledge about proteins, but rather relies on applying our knowledge of the physics that governs protein motions. This independent validation of the design is based on simulated annealing and restrained molecular dynamics simulation in vacuo, comparable to procedures used to refine NMR and X-ray structures.
Asunto(s)
Simulación por Computador , Proteínas de la Membrana/química , Modelos Moleculares , Péptidos/síntesis química , Péptidos/química , Estructura Terciaria de ProteínaRESUMEN
Annexin I homology models were built from the annexin V crystal structure. Three methods for side-chain prediction were tested based on molecular mechanics conformational search, the use of a rotamer database, or a combination of these two methods. We showed that rotamer-based methods were more efficient and that molecular mechanics energy minimizations, prior to rotamer selection, did not afford clearly improved predictions. Models built in vacuo and with an implicit solvation term were compared with the annexin I crystal structure which became available during the course of this study. The analysis of solvation energies, root mean square deviations, chi 1 angles and hydrogen bonds showed that models built with implicit solvation were of better quality. In annexin V, repeat III displays A-B and D-E loop conformations quite different from other repeats. Since the sequence differences suggest that repeat III in annexin I might present a conformation similar to other repeats, two annexin I models with different repeat III conformations were built and compared to determine whether the correct conformation could have been predicted. We show that using a combination of evaluation criteria, it is possible to discriminate unequivocally between the native and the incorrect fold, stressing that only one criterion should not be used to evaluate protein structures.
Asunto(s)
Anexina A1/química , Modelos Moleculares , Secuencia de Aminoácidos , Anexina A3/química , Anexina A5/química , Simulación por Computador , Cristalografía , Bases de Datos Factuales , Estudios de Evaluación como Asunto , Enlace de Hidrógeno , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Agua/químicaRESUMEN
In register and staggered models of tropomyosin coiled-coil were built from X-ray C alpha coordinates and refined via molecular dynamics. The two models show similar structural features with the X-ray structure of GCN4 leucine zipper. Empirical energetic methods used to compare the in register and staggered models indicate that both are equally probable. The two models have similar profiles of solvation free energy of folding for residues at positions a and d of the repeating heptad, indicating that residues at these positions are as well buried in an in register structure as in a staggered one. Neither the in register nor the 14 residues staggered structure can be ruled out based on hydrophobic or e-g' (g-e') electrostatic interactions which are not able to distinguish between the two models and are therefore not selective. However, the eg-b'c' electrostatic interactions, although smaller in magnitude, are in favor of the in register model. Furthermore, analysis of hydrophobic and electrostatic interactions along the tropomyosin sequence shows that bulky residues in positions a and d prevent the formation of inter-chain salt bridges.
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Proteínas de Unión al ADN , Modelos Moleculares , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae , Tropomiosina/química , Simulación por Computador , Proteínas Fúngicas/química , Pliegue de Proteína , Proteínas Quinasas/química , Soluciones/química , Factores de Transcripción/química , Difracción de Rayos XRESUMEN
Annexins are homologous proteins that bind to membranes in a calcium dependent manner, but for which precise physiological roles have yet to be defined. Most annexins are composed of a planar array of four homologous repeats, each containing five alpha-helices and associated into two modules. Annexin V forms a voltage-gated calcium channel in phospholipid bilayers. It has been proposed that the hydrophilic pore in the centre of the molecule may represent the ion conduction pathway and that a hinge movement in annexin V causes a variation of the inter-module angle and opens the calcium ion path. Here we present the results of molecular dynamics simulations of apo-annexin V and of calcium-bound annexin V and annexin I. The three simulations show significant differences in conformation and dynamics. The essential dynamics method was used to study the essential subspace of annexin V and showed that one of the essential motions corresponds to the postulated hinge motion. The hinge residues were located between repeats but belong to helices rather than to the links between helices. Calcium binding to annexin V led to a limitation of this hinge motion with more open conformations being favoured.
Asunto(s)
Anexina A1/química , Anexina A5/química , Apoproteínas/química , Calcio/metabolismo , Aminoácidos/metabolismo , Anexina A1/metabolismo , Anexina A5/metabolismo , Apoproteínas/metabolismo , Canales de Calcio/metabolismo , Simulación por Computador , Cristalización , Activación del Canal Iónico , Modelos Moleculares , Conformación Proteica , Factores de TiempoRESUMEN
In this work we describe the rational design of two helix coiled coil peptide mimetics of interleukin-4 (IL-4) which are able to recognize and bind its high affinity receptor (IL-4R alpha). We have used the leucine-zipper domain of the yeast transcription factor GCN4 as a scaffold into which the putative binding epitope of IL-4 for IL-4R alpha was transferred in a stepwise manner, using computer-aided molecular modeling. The resulting molecules bind IL-4R alpha with affinities ranging from 2 mM to 5 microM, depending on the fraction of the IL-4 binding site incorporated and on their stability. To our knowledge this is the first time a molecule capable of binding a cytokine receptor has been successfully designed in a rational manner.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Fúngicas/química , Interleucina-4/química , Proteínas Quinasas/química , Receptores de Interleucina-4/química , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Diseño de Fármacos , Proteínas Fúngicas/síntesis química , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Proteínas Quinasas/síntesis química , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Temperatura , TermodinámicaRESUMEN
The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third alpha-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.
Asunto(s)
Proteínas de la Membrana , Virus Vaccinia/química , Virus Vaccinia/fisiología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/fisiología , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/fisiología , Proteínas Virales/química , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Chlorocebus aethiops , Dicroismo Circular , Cartilla de ADN/genética , Escherichia coli/genética , Células HeLa , Humanos , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Puntual , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Virus Vaccinia/genética , Proteínas del Envoltorio Viral/genética , Proteínas Virales de Fusión/genética , Proteínas Virales/genéticaRESUMEN
All the functions of annexins in vitro as well as in vivo are mediated and probably regulated by calcium. We have used recombinant annexin I, synthesized by Escherichia coli, and we have performed site-directed mutagenesis. We have mutated the endonexin fold of domain 2 that binds calcium. Mutations were performed in this domain of the molecule because it perfectly matches the calcium binding consensus sequence. The two glycines of this fold were mutated into glutamic acid. The helix content and the stability of the mutants are identical to those of the wild-type, suggesting that the mutations did not drastically affect the structure of the protein. The two mutants showed modified calcium binding affinities. However, the calcium binding affinity of the G131E mutant was far more altered than that of the G129E mutant. Furthermore, other biochemical properties of these mutants were modified to different extents. The binding to phospholipid was not seriously affected, whereas the self-association was lost by the G131E mutant. In the same way, liposome aggregation is conserved, but modified, while the calcium affinity measured by equilibrium dialysis is dramatically altered.