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1.
Proteins ; 89(12): 1959-1976, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34559429

RESUMO

NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana , Modelos Moleculares , Conformação Proteica , Software , Biologia Computacional , Aprendizado de Máquina , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dobramento de Proteína , Análise de Sequência de Proteína
2.
Proteins ; 87(12): 1315-1332, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31603581

RESUMO

CASP13 has investigated the impact of sparse NMR data on the accuracy of protein structure prediction. NOESY and 15 N-1 H residual dipolar coupling data, typical of that obtained for 15 N,13 C-enriched, perdeuterated proteins up to about 40 kDa, were simulated for 11 CASP13 targets ranging in size from 80 to 326 residues. For several targets, two prediction groups generated models that are more accurate than those produced using baseline methods. Real NMR data collected for a de novo designed protein were also provided to predictors, including one data set in which only backbone resonance assignments were available. Some NMR-assisted prediction groups also did very well with these data. CASP13 also assessed whether incorporation of sparse NMR data improves the accuracy of protein structure prediction relative to nonassisted regular methods. In most cases, incorporation of sparse, noisy NMR data results in models with higher accuracy. The best NMR-assisted models were also compared with the best regular predictions of any CASP13 group for the same target. For six of 13 targets, the most accurate model provided by any NMR-assisted prediction group was more accurate than the most accurate model provided by any regular prediction group; however, for the remaining seven targets, one or more regular prediction method provided a more accurate model than even the best NMR-assisted model. These results suggest a novel approach for protein structure determination, in which advanced prediction methods are first used to generate structural models, and sparse NMR data is then used to validate and/or refine these models.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Algoritmos , Simulação por Computador , Cristalografia por Raios X , Reprodutibilidade dos Testes
3.
Nat Methods ; 12(8): 751-4, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26121406

RESUMO

Accurate determination of protein structure by NMR spectroscopy is challenging for larger proteins, for which experimental data are often incomplete and ambiguous. Evolutionary sequence information together with advances in maximum entropy statistical methods provide a rich complementary source of structural constraints. We have developed a hybrid approach (evolutionary coupling-NMR spectroscopy; EC-NMR) combining sparse NMR data with evolutionary residue-residue couplings and demonstrate accurate structure determination for several proteins 6-41 kDa in size.


Assuntos
Biologia Computacional/métodos , Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Algoritmos , Cristalografia por Raios X , Evolução Molecular , Humanos , Hidrodinâmica , Imageamento Tridimensional , Modelos Estatísticos , Conformação Molecular , Conformação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras/química
4.
Adv Exp Med Biol ; 1105: 153-169, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30617828

RESUMO

While 3D structure determination of small (<15 kDa) proteins by solution NMR is largely automated and routine, structural analysis of larger proteins is more challenging. An emerging hybrid strategy for modeling protein structures combines sparse NMR data that can be obtained for larger proteins with sequence co-variation data, called evolutionary couplings (ECs), obtained from multiple sequence alignments of protein families. This hybrid "EC-NMR" method can be used to accurately model larger (15-60 kDa) proteins, and more rapidly determine structures of smaller (5-15 kDa) proteins using only backbone NMR data. The resulting structures have accuracies relative to reference structures comparable to those obtained with full backbone and sidechain NMR resonance assignments. The requirement that evolutionary couplings (ECs) are consistent with NMR data recorded on a specific member of a protein family, under specific conditions, potentially also allows identification of ECs that reflect alternative allosteric or excited states of the protein structure.


Assuntos
Evolução Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas/química , Alinhamento de Sequência
5.
J Biomol NMR ; 62(4): 439-51, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26081575

RESUMO

ASDP is an automated NMR NOE assignment program. It uses a distinct bottom-up topology-constrained network anchoring approach for NOE interpretation, with 2D, 3D and/or 4D NOESY peak lists and resonance assignments as input, and generates unambiguous NOE constraints for iterative structure calculations. ASDP is designed to function interactively with various structure determination programs that use distance restraints to generate molecular models. In the CASD-NMR project, ASDP was tested and further developed using blinded NMR data, including resonance assignments, either raw or manually-curated (refined) NOESY peak list data, and in some cases (15)N-(1)H residual dipolar coupling data. In these blinded tests, in which the reference structure was not available until after structures were generated, the fully-automated ASDP program performed very well on all targets using both the raw and refined NOESY peak list data. Improvements of ASDP relative to its predecessor program for automated NOESY peak assignments, AutoStructure, were driven by challenges provided by these CASD-NMR data. These algorithmic improvements include (1) using a global metric of structural accuracy, the discriminating power score, for guiding model selection during the iterative NOE interpretation process, and (2) identifying incorrect NOESY cross peak assignments caused by errors in the NMR resonance assignment list. These improvements provide a more robust automated NOESY analysis program, ASDP, with the unique capability of being utilized with alternative structure generation and refinement programs including CYANA, CNS, and/or Rosetta.


Assuntos
Automação , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Proteínas/química , Conjuntos de Dados como Assunto
6.
J Struct Funct Genomics ; 15(4): 201-7, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24941917

RESUMO

High-quality solution NMR structures of three homeodomains from human proteins ALX4, ZHX1 and CASP8AP2 were solved. These domains were chosen as targets of a biomedical theme project pursued by the Northeast Structural Genomics Consortium. This project focuses on increasing the structural coverage of human proteins associated with cancer.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação a DNA/química , Proteínas de Homeodomínio/química , Proteínas de Neoplasias/química , Neoplasias/química , Fatores de Transcrição/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína
7.
Nucleic Acids Res ; 40(Web Server issue): W542-6, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22570414

RESUMO

We describe the RPF web server, a quality assessment tool for protein NMR structures. The RPF server measures the 'goodness-of-fit' of the 3D structure with NMR chemical shift and unassigned NOESY data, and calculates a discrimination power (DP) score, which estimates the differences between the fits of the query structures and random coil structures to these experimental data. The DP-score is an accuracy predictor of the query structure. The RPF server also maps local structure quality measures onto the 3D structure using an online molecular viewer, and onto the NMR spectra, allowing refinement of the structure and/or NOESY peak list data. The RPF server is available at: http://nmr.cabm.rutgers.edu/rpf.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Software , Algoritmos , Internet , Multimerização Proteica , Proteínas/química , Controle de Qualidade
8.
bioRxiv ; 2023 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-36712039

RESUMO

Recent advances in molecular modeling of protein structures are changing the field of structural biology. AlphaFold-2 (AF2), an AI system developed by DeepMind, Inc., utilizes attention-based deep learning to predict models of protein structures with high accuracy relative to structures determined by X-ray crystallography and cryo-electron microscopy (cryoEM). Comparing AF2 models to structures determined using solution NMR data, both high similarities and distinct differences have been observed. Since AF2 was trained on X-ray crystal and cryoEM structures, we assessed how accurately AF2 can model small, monomeric, solution protein NMR structures which (i) were not used in the AF2 training data set, and (ii) did not have homologous structures in the Protein Data Bank at the time of AF2 training. We identified nine open source protein NMR data sets for such "blind" targets, including chemical shift, raw NMR FID data, NOESY peak lists, and (for 1 case) 15 N- 1 H residual dipolar coupling data. For these nine small (70 - 108 residues) monomeric proteins, we generated AF2 prediction models and assessed how well these models fit to these experimental NMR data, using several well-established NMR structure validation tools. In most of these cases, the AF2 models fit the NMR data nearly as well, or sometimes better than, the corresponding NMR structure models previously deposited in the Protein Data Bank. These results provide benchmark NMR data for assessing new NMR data analysis and protein structure prediction methods. They also document the potential for using AF2 as a guiding tool in protein NMR data analysis, and more generally for hypothesis generation in structural biology research. Highlights: AF2 models assessed against NMR data for 9 monomeric proteins not used in training.AF2 models fit NMR data almost as well as the experimentally-determined structures. RPF-DP, PSVS , and PDBStat software provide structure quality and RDC assessment. RPF-DP analysis using AF2 models suggests multiple conformational states.

9.
Front Mol Biosci ; 9: 877000, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35769913

RESUMO

Recent advances in molecular modeling using deep learning have the potential to revolutionize the field of structural biology. In particular, AlphaFold has been observed to provide models of protein structures with accuracies rivaling medium-resolution X-ray crystal structures, and with excellent atomic coordinate matches to experimental protein NMR and cryo-electron microscopy structures. Here we assess the hypothesis that AlphaFold models of small, relatively rigid proteins have accuracies (based on comparison against experimental data) similar to experimental solution NMR structures. We selected six representative small proteins with structures determined by both NMR and X-ray crystallography, and modeled each of them using AlphaFold. Using several structure validation tools integrated under the Protein Structure Validation Software suite (PSVS), we then assessed how well these models fit to experimental NMR data, including NOESY peak lists (RPF-DP scores), comparisons between predicted rigidity and chemical shift data (ANSURR scores), and 15N-1H residual dipolar coupling data (RDC Q factors) analyzed by software tools integrated in the PSVS suite. Remarkably, the fits to NMR data for the protein structure models predicted with AlphaFold are generally similar, or better, than for the corresponding experimental NMR or X-ray crystal structures. Similar conclusions were reached in comparing AlphaFold2 predictions and NMR structures for three targets from the Critical Assessment of Protein Structure Prediction (CASP). These results contradict the widely held misperception that AlphaFold cannot accurately model solution NMR structures. They also document the value of PSVS for model vs. data assessment of protein NMR structures, and the potential for using AlphaFold models for guiding analysis of experimental NMR data and more generally in structural biology.

10.
Structure ; 17(2): 151-9, 2009 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-19217386

RESUMO

We describe the proceedings and conclusions from the "Workshop on Applications of Protein Models in Biomedical Research" (the Workshop) that was held at the University of California, San Francisco on 11 and 12 July, 2008. At the Workshop, international scientists involved with structure modeling explored (i) how models are currently used in biomedical research, (ii) the requirements and challenges for different applications, and (iii) how the interaction between the computational and experimental research communities could be strengthened to advance the field.


Assuntos
Pesquisa Biomédica/métodos , Modelos Moleculares , Proteínas/química , Animais , Pesquisa Biomédica/tendências , Química Farmacêutica/métodos , Bases de Dados de Proteínas , Descoberta de Drogas/métodos , Enzimas/química , Diretrizes para o Planejamento em Saúde , Humanos , Conformação Proteica , Engenharia de Proteínas/métodos , Mapeamento de Interação de Proteínas/métodos , Software
11.
J Struct Biol ; 172(1): 21-33, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20688167

RESUMO

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (>97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as>26,000 constructs. Over the past 10 years, more than 16,000 of these expressed protein, and more than 4400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last 5 years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.


Assuntos
Genômica/métodos , Proteínas/metabolismo , Proteômica/métodos , Clonagem Molecular , Bases de Dados de Proteínas , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Proteínas/química , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
12.
Mol Cell Proteomics ; 7(10): 2048-60, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18487680

RESUMO

Structural genomics provides an important approach for characterizing and understanding systems biology. As a step toward better integrating protein three-dimensional (3D) structural information in cancer systems biology, we have constructed a Human Cancer Pathway Protein Interaction Network (HCPIN) by analysis of several classical cancer-associated signaling pathways and their physical protein-protein interactions. Many well known cancer-associated proteins play central roles as "hubs" or "bottlenecks" in the HCPIN. At least half of HCPIN proteins are either directly associated with or interact with multiple signaling pathways. Although some 45% of residues in these proteins are in sequence segments that meet criteria sufficient for approximate homology modeling (Basic Local Alignment Search Tool (BLAST) E-value <10(-6)), only approximately 20% of residues in these proteins are structurally covered using high accuracy homology modeling criteria (i.e. BLAST E-value <10(-6) and at least 80% sequence identity) or by actual experimental structures. The HCPIN Website provides a comprehensive description of this biomedically important multipathway network together with experimental and homology models of HCPIN proteins useful for cancer biology research. To complement and enrich cancer systems biology, the Northeast Structural Genomics Consortium is targeting >1000 human proteins and protein domains from the HCPIN for sample production and 3D structure determination. The long range goal of this effort is to provide a comprehensive 3D structure-function database for human cancer-associated proteins and protein complexes in the context of their interaction networks. The network-based target selection (BioNet) approach described here is an example of a general strategy for targeting co-functioning proteins by structural genomics projects.


Assuntos
Genômica , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Mapeamento de Interação de Proteínas/métodos , Humanos , Proteínas de Neoplasias/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes
13.
Methods Enzymol ; 615: 453-475, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30638538

RESUMO

Cell surface molecules are important for development and function of multicellular organisms. Although several methods are available to identify ligand-receptor pairs, ELISA-based methods are particularly amenable to high-throughput screens. ELISA-based methods have high sensitivity and low false-positive rates for detecting protein-protein interaction (PPI) complexes. Here, we provide a detailed protocol for a 384-well ELISA-based PPI screening protocol for the identification of novel cell surface ligand-receptor interactions, together with considerations for validation of PPIs by biophysical methods. This PPI screen has been developed and tested for discovery of novel ligand-receptor pairs between human synaptic adhesion proteins, believed to play crucial roles in many steps of neurodevelopment, from neuronal maturation, to axon guidance, synapse connectivity, and pruning.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Ligantes , Receptores de Superfície Celular/metabolismo , Cromatografia de Afinidade , Cristalografia por Raios X , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica
14.
Methods Enzymol ; 614: 363-392, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30611430

RESUMO

Accurate protein structure determination by solution-state NMR is challenging for proteins greater than about 20kDa, for which extensive perdeuteration is generally required, providing experimental data that are incomplete (sparse) and ambiguous. However, the massive increase in evolutionary sequence information coupled with advances in methods for sequence covariance analysis can provide reliable residue-residue contact information for a protein from sequence data alone. These "evolutionary couplings (ECs)" can be combined with sparse NMR data to determine accurate 3D protein structures. This hybrid "EC-NMR" method has been developed using NMR data for several soluble proteins and validated by comparison with corresponding reference structures determined by X-ray crystallography and/or conventional NMR methods. For small proteins, only backbone resonance assignments are utilized, while for larger proteins both backbone and some sidechain methyl resonance assignments are generally required. ECs can be combined with sparse NMR data obtained on deuterated, selectively protonated protein samples to provide structures that are more accurate and complete than those obtained using such sparse NMR data alone. EC-NMR also has significant potential for analysis of protein structures from solid-state NMR data and for studies of integral membrane proteins. The requirement that ECs are consistent with NMR data recorded on a specific member of a protein family, under specific conditions, also allows identification of ECs that reflect alternative allosteric or excited states of the protein structure.


Assuntos
Algoritmos , Proteínas de Escherichia coli/química , Escherichia coli/química , Evolução Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Periplásmicas de Ligação/química , Software , Análise de Variância , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Proteínas , Deutério/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Marcação por Isótopo , Modelos Moleculares , Proteínas Periplásmicas de Ligação/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Homologia Estrutural de Proteína , Termodinâmica
15.
J Mol Biol ; 364(1): 80-96, 2006 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-16999976

RESUMO

Tropomyosin is a coiled-coil protein that binds head-to-tail along the length of actin filaments in eukaryotic cells, stabilizing them and providing protection from severing proteins. Tropomyosin cooperatively regulates actin's interaction with myosin and mediates the Ca2+ -dependent regulation of contraction by troponin in striated muscles. The N-terminal and C-terminal ends are critical functional determinants that form an "overlap complex". Here we report the solution NMR structure of an overlap complex formed of model peptides. In the complex, the chains of the C-terminal coiled coil spread apart to allow insertion of 11 residues of the N-terminal coiled coil into the resulting cleft. The plane of the N-terminal coiled coil is rotated 90 degrees relative to the plane of the C terminus. A consequence of the geometry is that the orientation of postulated periodic actin binding sites on the coiled-coil surface is retained from one molecule to the next along the actin filament when the overlap complex is modeled into the X-ray structure of tropomyosin determined at 7 Angstroms. Nuclear relaxation NMR data reveal flexibility of the junction, which may function to optimize binding along the helical actin filament and to allow mobility of tropomyosin on the filament surface as it switches between regulatory states.


Assuntos
Actinas/metabolismo , Tropomiosina/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Tropomiosina/metabolismo
16.
Proteins ; 62(3): 587-603, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16374783

RESUMO

This article formulates the multidimensional nuclear Overhauser effect spectroscopy (NOESY) interpretation problem using graph theory and presents a novel, bottom-up, topology-constrained distance network analysis algorithm for NOESY cross peak interpretation using assigned resonances. AutoStructure is a software suite that implements this topology-constrained distance network analysis algorithm and iteratively generates structures using the three-dimensional (3D) protein structure calculation programs XPLOR/CNS or DYANA. The minimum input for AutoStructure includes the amino acid sequence, a list of resonance assignments, and lists of 2D, 3D, and/or 4D-NOESY cross peaks. AutoStructure can also analyze homodimeric proteins when X-filtered NOESY experiments are available. The quality of input data and final 3D structures is evaluated using recall, precision, and F-measure (RPF) scores, a statistical measure of goodness of fit with the input data. AutoStructure has been tested on three protein NMR data sets for which high-quality structures have previously been solved by an expert, and yields comparable high-quality distance constraint lists and 3D protein structures in hours. We also compare several protein structures determined using AutoStructure with corresponding homologous proteins determined with other independent methods. The program has been used in more than two dozen protein structure determinations, several of which have already been published.


Assuntos
Proteínas/química , Algoritmos , Cristalografia por Raios X , Processamento de Imagem Assistida por Computador , Espectroscopia de Ressonância Magnética , Modelos Teóricos , Conformação Proteica , Estrutura Secundária de Proteína , Reprodutibilidade dos Testes
17.
Methods Enzymol ; 394: 111-41, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15808219

RESUMO

Recent developments provide automated analysis of NMR assignments and three-dimensional (3D) structures of proteins. These approaches are generally applicable to proteins ranging from about 50 to 150 amino acids. In this chapter, we summarize progress by the Northeast Structural Genomics Consortium in standardizing the NMR data collection process for protein structure determination and in building an integrated platform for automated protein NMR structure analysis. Our integrated platform includes the following principal steps: (1) standardized NMR data collection, (2) standardized data processing (including spectral referencing and Fourier transformation), (3) automated peak picking and peak list editing, (4) automated analysis of resonance assignments, (5) automated analysis of NOESY data together with 3D structure determination, and (6) methods for protein structure validation. In particular, the software AutoStructure for automated NOESY data analysis is described in this chapter, together with a discussion of practical considerations for its use in high-throughput structure production efforts. The critical area of data quality assessment has evolved significantly over the past few years and involves evaluation of both intermediate and final peak lists, resonance assignments, and structural information derived from the NMR data. Methods for quality control of each of the major automated analysis steps in our platform are also discussed. Despite significant remaining challenges, when good quality data are available, automated analysis of protein NMR assignments and structures with this platform is both fast and reliable.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Software , Interpretação Estatística de Dados , Estrutura Terciária de Proteína
18.
J Mol Biol ; 327(2): 521-36, 2003 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-12628255

RESUMO

Ribosome-binding factor A (RbfA) from Escherichia coli is a cold-shock adaptation protein. It is essential for efficient processing of 16S rRNA and is suspected to interact with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is a member of a large family of small proteins found in most bacterial organisms, making it an important target for structural proteomics. Here, we describe the three-dimensional structure of RbfADelta25, a 108 residue construct with 25 residues removed from the carboxyl terminus of full-length RbfA, determined in solution at pH 5.0 by heteronuclear NMR methods. The structure determination was carried out using largely automated methods for determining resonance assignments, interpreting nuclear Overhauser effect (NOE) spectroscopy (NOESY) spectra, and structure generation. RbfADelta25 has an alpha+beta fold containing three helices and three beta-strands, alpha1-beta1-beta2-alpha2-alpha3-beta3. The structure has type-II KH-domain fold topology, related to conserved KH sequence family proteins whose betaalphaalphabeta subunits are characterized by a helix-turn-helix motif with sequence signature GxxG at the turn. In RbfA, this betaalphaalphabeta subunit is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, including a strongly conserved Ala residue at position 75 forming an interhelical kink. The electrostatic field distribution about RbfADelta25 is bipolar; one side of the molecule is strongly negative and the opposite face has a strong positive electrostatic field. A "dynamic hot spot" of RbfADelta25 has been identified in the vicinity of a beta-bulge at strongly conserved residue Ser39 by 15N R(1), R(2) relaxation rate and heteronuclear 15N-1H NOE measurements. Analyses of these distributions of electrostatic field and internal dynamics, together with evolutionary implications of fold and sequence conservation, suggest that RbfA is indeed a nucleic acid-binding protein, and identify a potential RNA-binding site in or around the conserved polypeptide segment Ser76-Asp100 corresponding to the alpha3-loop-beta3 helix-loop-strand structure. While the structure of RbfADelta25 is most similar to that of the KH domain of the E.coli Era GTPase, its electrostatic field distribution is most similar to the KH1 domain of the NusA protein from Thermotoga maritima, another cold-shock associated RNA-binding protein. Both RbfA and NusA are regulated in the same E.coli operon. Structural and functional similarities between RbfA, NusA, and other bacterial type II KH domains suggest previously unsuspected evolutionary relationships between these cold-shock associated proteins.


Assuntos
Proteínas de Escherichia coli/química , RNA Ribossômico 16S/genética , Proteínas Ribossômicas/química , Ribossomos/química , Adaptação Fisiológica , Sequência de Aminoácidos , Temperatura Baixa , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fatores de Alongamento de Peptídeos/química , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Ribossômico 16S/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Homologia de Sequência de Aminoácidos , Choque , Fatores de Transcrição/química , Fatores de Elongação da Transcrição
19.
Protein Sci ; 13(3): 727-34, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14767079

RESUMO

The structure of Drosophila LC8 pH-induced monomer has been determined by NMR spectroscopy using the program AutoStructure. The structure at pH 3 and 30 degrees C is similar to the individual subunits of mammalian LC8 dimer with the exception that a beta strand, which crosses between monomers to form an intersubunit beta-sheet in the dimer, is a flexible loop with turnlike conformations in the monomer. Increased flexibility in the interface region relative to the rest of the protein is confirmed by dynamic measurements based on (15)N relaxation. Comparison of the monomer and dimer structures indicates that LC8 is not a domain swapped dimer.


Assuntos
Proteínas de Transporte/química , Proteínas de Drosophila/química , Subunidades Proteicas/química , Animais , Proteínas de Transporte/genética , Drosophila/química , Proteínas de Drosophila/genética , Dineínas , Concentração de Íons de Hidrogênio , Marcação por Isótopo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Proteínas Recombinantes/química , Homologia Estrutural de Proteína
20.
Proteins ; 53(2): 290-306, 2003 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-14517980

RESUMO

TOUCHSTONEX, a new method for folding proteins that uses a small number of long-range contact restraints derived from NMR experimental NOE (nuclear Overhauser enhancement) data, is described. The method employs a new lattice-based, reduced model of proteins that explicitly represents C(alpha), C(beta), and the sidechain centers of mass. The force field consists of knowledge-based terms to produce protein-like behavior, including various short-range interactions, hydrogen bonding, and one-body, pairwise, and multibody long-range interactions. Contact restraints were incorporated into the force field as an NOE-specific pairwise potential. We evaluated the algorithm using a set of 125 proteins of various secondary structure types and lengths up to 174 residues. Using N/8 simulated, long-range sidechain contact restraints, where N is the number of residues, 108 proteins were folded to a C(alpha)-root-mean-square deviation (RMSD) from native below 6.5 A. The average RMSD of the lowest RMSD structures for all 125 proteins (folded and unfolded) was 4.4 A. The algorithm was also applied to limited experimental NOE data generated for three proteins. Using very few experimental sidechain contact restraints, and a small number of sidechain-main chain and main chain-main chain contact restraints, we folded all three proteins to low-to-medium resolution structures. The algorithm can be applied to the NMR structure determination process or other experimental methods that can provide tertiary restraint information, especially in the early stage of structure determination, when only limited data are available.


Assuntos
Algoritmos , Conformação Proteica , Aminoácidos/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/química , Proteína Estafilocócica A/química
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