RESUMEN
Automated methods for NMR structure determination of proteins are continuously becoming more robust. However, current methods addressing larger, more complex targets rely on analyzing 6-10 complementary spectra, suggesting the need for alternative approaches. Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra recorded using a single, fully protonated protein sample in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the structures of proteins can be determined accurately and in an unsupervised manner in a matter of days.
Asunto(s)
Algoritmos , Proteínas Bacterianas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Modelos Moleculares , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Thermoanaerobacter/químicaRESUMEN
AIMS: Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases involved in oxidant defense and signal transduction. Despite much study, the precise roles of conserved residues remain poorly defined. In this study, we carried out extensive functional and structural characterization of 10 variants of such residues in a model decameric bacterial Prx. RESULTS: Three active site proximal mutations of Salmonella typhimurium AhpC, T43V, R119A, and E49Q, lowered catalytic efficiency with hydrogen peroxide by 4-5 orders of magnitude, but did not affect reactivity toward their reductant, AhpF. pKa values of the peroxidatic cysteine were also shifted up by 1-1.3 pH units for these and a decamer disruption mutant, T77I. Except for the decamer-stabilizing T77V, all mutations destabilized decamers in the reduced form. In the oxidized form, three mutants-T77V, T43A, and T43S-exhibited stabilized decamers and were more efficiently reduced by AhpF than wild-type AhpC. Crystal structures of most mutants were solved and many showed alterations in stability of the fully folded active site loop. INNOVATION: This is the first study of Prx mutants to comprehensively assess the effects of mutations on catalytic activities, the active site cysteine pKa, and the protein structure and oligomeric status. CONCLUSION: The Arg119 side chain must be properly situated for efficient catalysis, but for other debilitating variants, the functional defects could be explained by structural perturbations and/or associated decamer destabilization rather than direct effects. This underscores the importance of our comprehensive approach. A remarkable new finding was the preference of the reductant for decamers. Antioxid. Redox Signal. 28, 521-536.
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Catálisis , Peróxido de Hidrógeno/química , Peroxidasas/química , Peroxirredoxinas/química , Secuencia de Aminoácidos/genética , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/química , Peróxido de Hidrógeno/metabolismo , Cinética , Modelos Moleculares , Mutación , Oxidación-Reducción , Peroxidasas/genética , Peroxidasas/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Salmonella typhimurium/enzimología , Salmonella typhimurium/genéticaRESUMEN
Ensembles of protein structures are increasingly used to represent the conformational variation of a protein as determined by experiment and/or by molecular simulations, as well as uncertainties that may be associated with structure determinations or predictions. Making the best use of such information requires the ability to quantitatively compare entire ensembles. For this reason, we recently introduced the Ensemblator (Clark et al., Protein Sci 2015; 24:1528), a novel approach to compare user-defined groups of models, in residue level detail. Here we describe Ensemblator v3, an open-source program that employs the same basic ensemble comparison strategy but includes major advances that make it more robust, powerful, and user-friendly. Ensemblator v3 carries out multiple sequence alignments to facilitate the generation of ensembles from non-identical input structures, automatically optimizes the key global overlay parameter, optionally performs "ensemble clustering" to classify the models into subgroups, and calculates a novel "discrimination index" that quantifies similarities and differences, at residue or atom level, between each pair of subgroups. The clustering and automatic options mean that no pre-knowledge about an ensemble is required for its analysis. After describing the novel features of Ensemblator v3, we demonstrate its utility using three case studies that illustrate the ease with which complex analyses are accomplished, and the kinds of insights derived from clustering into subgroups and from the detailed information that locates significant differences. The Ensemblator v3 enhances the structural biology toolbox by greatly expanding the kinds of problems to which this ensemble comparison strategy can be applied.
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Bases de Datos de Proteínas , Proteínas/química , Proteínas/clasificación , Programas Informáticos , Conformación Proteica , Proteínas/genéticaRESUMEN
In the original publication of the article, the given name and family name of the author P. Andrew Karplus was published incorrectly. The name should read as "P. Andrew" - Given name and "Karplus" - Family name.
RESUMEN
The solution NMR structure of the isolated thumb subdomain of HIV-1 reverse transcriptase (RT) has been determined. A detailed comparison of the current structure with dozens of the highest resolution crystal structures of this domain in the context of the full-length enzyme reveals that the overall structures are very similar, with only two regions exhibiting local conformational differences. The C-terminal capping pattern of the αH helix is subtly different, and the loop connecting the αI and αJ helices in the p51 chain of the full-length p51/p66 heterodimeric RT differs from our NMR structure due to unique packing interactions in mature RT. Overall, our data show that the thumb subdomain folds independently and essentially the same in isolation as in its natural structural context.
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Transcriptasa Inversa del VIH/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Dominios Proteicos , Humanos , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Multimerización de Proteína , SolucionesRESUMEN
Ultra-high resolution protein crystal structures have been considered as relatively reliable sources for defining details of protein geometry, such as the extent to which the peptide unit deviates from planarity. Chellapa and Rose (Proteins 2015; 83:1687) recently called this into question, reporting that for a dozen representative protein structures determined at â¼ 1 Å resolution, the diffraction data could be equally well fit with models restrained to have highly planar peptides, i.e. having a standard deviation of the ω torsion angles of only â¼ 1° instead of the typically observed value of â¼ 6°. Here, we document both conceptual and practical shortcomings of that study and show that the more tightly restrained models are demonstrably incorrect and do not fit the diffraction data equally well. We emphasize the importance of inspecting electron density maps when investigating the agreement between a model and its experimental data. Overall, this report reinforces that modern standard refinement protocols have been well-conceived and that ultra-high resolution protein crystal structures, when evaluated carefully and used with an awareness of their levels of coordinate uncertainty, are powerful sources of information for providing reliable information about the details of protein geometry.
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Péptidos/química , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Reproducibilidad de los ResultadosRESUMEN
During protein folding and as part of some conformational changes that regulate protein function, the polypeptide chain must traverse high-energy barriers that separate the commonly adopted low-energy conformations. How distortions in peptide geometry allow these barrier-crossing transitions is a fundamental open question. One such important transition involves the movement of a non-glycine residue between the left side of the Ramachandran plot (that is, Ï < 0°) and the right side (that is, Ï > 0°). We report that high-energy conformations with Ï ~ 0°, normally expected to occur only as fleeting transition states, are stably trapped in certain highly resolved native protein structures and that an analysis of these residues provides a detailed, experimentally derived map of the bond angle distortions taking place along the transition path. This unanticipated information lays to rest any uncertainty about whether such transitions are possible and how they occur, and in doing so lays a firm foundation for theoretical studies to better understand the transitions between basins that have been little studied but are integrally involved in protein folding and function. Also, the context of one such residue shows that even a designed highly stable protein can harbor substantial unfavorable interactions.