RESUMEN
NMR spectroscopy is uniquely suited for atomic resolution studies of biomolecules such as proteins, nucleic acids and metabolites, since detailed information on structure and dynamics are encoded in positions and line shapes of peaks in NMR spectra. Unfortunately, accurate determination of these parameters is often complicated and time consuming, in part due to the need for different software at the various analysis steps and for validating the results. Here, we present an integrated, cross-platform and open-source software that is significantly more versatile than the typical line shape fitting application. The software is a completely redesigned version of PINT ( https://pint-nmr.github.io/PINT/ ). It features a graphical user interface and includes functionality for peak picking, editing of peak lists and line shape fitting. In addition, the obtained peak intensities can be used directly to extract, for instance, relaxation rates, heteronuclear NOE values and exchange parameters. In contrast to most available software the entire process from spectral visualization to preparation of publication-ready figures is done solely using PINT and often within minutes, thereby, increasing productivity for users of all experience levels. Unique to the software are also the outstanding tools for evaluating the quality of the fitting results and extensive, but easy-to-use, customization of the fitting protocol and graphical output. In this communication, we describe the features of the new version of PINT and benchmark its performance.
Asunto(s)
Interpretación Estadística de Datos , Espectroscopía de Resonancia Magnética , Programas Informáticos , Espectroscopía de Resonancia Magnética/métodos , Reproducibilidad de los Resultados , Interfaz Usuario-Computador , Navegador WebRESUMEN
The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software?l=en. Source code and binaries for Linux, Mac OS X and Microsoft Windows are available.
Asunto(s)
Algoritmos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Programas Informáticos , Secuencia de Aminoácidos , Estructura Secundaria de ProteínaRESUMEN
More than 100 distinct mutations in the gene CuZnSOD encoding human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS), a fatal neuronal disease. Many studies of different mutant proteins have found effects on protein stability, catalytic activity, and metal binding, but without a common pattern. Notably, these studies were often performed under conditions far from physiological. Here, we have used experimental conditions of pH 7 and 37 °C and at an ionic strength of 0.2 M to mimic physiological conditions as close as possible in a sample of pure protein. Thus, by using NMR spectroscopy, we have analyzed amide hydrogen exchange of the fALS-associated I113T CuZnSOD variant in its fully metalated state, both at 25 and 37 °C, where (15)N relaxation data, as expected, reveals that CuZnSOD I113T exists as a dimer under these conditions. The local dynamics at 82% of all residues have been analyzed in detail. When compared to the wild-type protein, it was found that I113T CuZnSOD is particularly destabilized locally at the ion binding sites of loop 4, the zinc binding loop, which results in frequent exposure of the aggregation prone outer ß-strands I and VI of the ß-barrel, possibly enabling fibril or aggregate formation. A similar study (Museth, A. K., et al. (2009) Biochemistry, 48, 8817-8829) of amide hydrogen exchange at pH 7 and 25 °C on the G93A variant also revealed a selective destabilization of the zinc binding loop. Thus, a possible scenario in ALS is that elevated local dynamics at the metal binding region can result in toxic species from formation of new interactions at local ß-strands.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Mutación Puntual , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/metabolismo , Sitios de Unión , Cobre/metabolismo , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Estructura Secundaria de Proteína , Superóxido Dismutasa/metabolismo , Zinc/metabolismoRESUMEN
A selective isotope labeling scheme based on the utilization of [2-(13)C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state (13)Cα chemical shifts using Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-(13)C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state (13)Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s(-1), despite the small fraction of (13)Cα-(13)Cß spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using (13)Cα spin probes.
Asunto(s)
Isótopos de Carbono/metabolismo , Glicerol/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteínas/metabolismo , Isótopos de Carbono/análisis , Isótopos de Carbono/química , Redes y Vías Metabólicas , Sensibilidad y EspecificidadRESUMEN
The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. By nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we show that although Myc occupies a very heterogeneous conformational space, we find transiently structured regions in residues 22-33 and in the Myc homology box I (MBI; residues 45-65); both these regions are conserved in other members of the Myc family. Binding of Bin1 to Myc-1-88 as assayed by NMR and surface plasmon resonance (SPR) revealed primary binding to the S62 region in a dynamically disordered and multivalent complex, accompanied by population shifts leading to altered intramolecular conformational dynamics. These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.
Asunto(s)
Proteínas Proto-Oncogénicas c-myc/química , Transactivadores/química , Proteínas Supresoras de Tumor/química , Sitios de Unión , Humanos , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Dominios Homologos srcRESUMEN
Calcium dependent protein kinases are unique to plants and certain parasites and comprise an N-terminal segment and a kinase domain that is regulated by a C-terminal calcium binding domain. Since the proteins are not found in man they are potential drug targets. We have characterized the calcium binding lobes of the regulatory domain of calcium dependent protein kinase 3 from the malaria parasite Plasmodium falciparum. Despite being structurally similar, the two lobes differ in several other regards. While the monomeric N-terminal lobe changes its structure in response to calcium binding and shows global dynamics on the sub-millisecond time-scale both in its apo and calcium bound states, the C-terminal lobe could not be prepared calcium-free and forms dimers in solution. If our results can be generalized to the full-length protein, they suggest that the C-terminal lobe is calcium bound even at basal levels and that activation is caused by the structural reorganization associated with binding of a single calcium ion to the N-terminal lobe.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Fenómenos Biofísicos , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Calmodulina/química , Calmodulina/metabolismo , Calorimetría , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Plasmodium falciparum/genética , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Proteínas Quinasas/química , Proteínas Quinasas/genética , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genéticaRESUMEN
MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 Å) presented here is closely similar to wild-type MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members.
Asunto(s)
Proteínas Bacterianas/química , ADN Bacteriano/química , Regulación Bacteriana de la Expresión Génica , Mutación , Pseudomonas aeruginosa/genética , Proteínas Represoras/química , Regulación Alostérica , Sitio Alostérico , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Simulación de Dinámica Molecular , Operón , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Dispersión del Ángulo Pequeño , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Difracción de Rayos XRESUMEN
We present the software CDpal that is used to analyze thermal and chemical denaturation data to obtain information on protein stability. The software uses standard assumptions and equations applied to two-state and various types of three-state denaturation models in order to determine thermodynamic parameters. It can analyze denaturation monitored by both circular dichroism and fluorescence spectroscopy and is extremely flexible in terms of input format. Furthermore, it is intuitive and easy to use because of the graphical user interface and extensive documentation. As illustrated by the examples herein, CDpal should be a valuable tool for analysis of protein stability.
Asunto(s)
Biología Computacional/métodos , Proteínas/química , Dicroismo Circular , Modelos Moleculares , Desnaturalización Proteica , Estabilidad Proteica , Programas Informáticos , Espectrometría de Fluorescencia , TemperaturaRESUMEN
The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBP's concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Modelos Moleculares , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Factores Asociados con la Proteína de Unión a TATA/química , Proteína de Unión a TATA-Box/química , Factor de Transcripción TFIID/química , Transcripción Genética/fisiología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Cristalización , Unión Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID/metabolismo , Factor de Transcripción TFIIIB/química , Factor de Transcripción TFIIIB/metabolismoRESUMEN
The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations-stability, domain interactions, and internal hydrophobic surfaces-are also critical for the regulation of MexR DNA binding.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Genes Reguladores/genética , Mutación , Pseudomonas aeruginosa/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Operón , Pliegue de Proteína , Proteínas Represoras/metabolismoRESUMEN
To characterize the sites on the protein surface that are involved in the adsorption to silica nanoparticles and the subsequent rearrangements of the protein/nanoparticle interaction, a novel approach has been used. After incubation of protein with silica nanoparticles for 2 or 16 h, the protein was cleaved with trypsin and the peptide fragments were analyzed with mass spectrometry. The nanoparticle surface area was in 16-fold excess over available protein surface to minimize the probability that the initial binding would be affected by other protein molecules. When the fragment patterns obtained in the presence and absence of silica nanoparticles were compared, we were able to characterize the protein fragments that interact with the surface. This approach has allowed us to identify the initial binding sites on the protein structure and the rearrangement of the binding sites that occur upon prolonged incubation with the surface.
Asunto(s)
Anhidrasas Carbónicas , Dióxido de Silicio , Adsorción , Humanos , Nanopartículas/química , Fragmentos de Péptidos/química , Proteolisis , Dióxido de Silicio/químicaRESUMEN
Ro52 is one of the major autoantigens targeted in the autoimmune disease Sjögren syndrome. By sequence similarity, Ro52 belongs to the RING-B-box-coiled-coil (RBCC) protein family. Disease-related antibodies bind Ro52 in a conformation-dependent way both in the coiled-coil region and in the Zn2+-binding Ring-B-box region. Primarily associated with Sjögren syndrome, Ro52 autoantibodies directed to a specific, partially structured epitope in the coiled-coil region may also induce a congenital heart block in the fetus of pregnant Ro52-positive mothers. To improve our understanding of the pathogenic effects of autoantibody binding to the Zn2+-binding region, a multianalytical mapping of its structural, biophysical, and antigenic properties is presented. Structure content and ligand binding of subregions, dissected by peptide synthesis and subcloning, were analyzed by fluorescence and circular dichroism spectroscopy. A novel matrix-assisted laser desorption ionization time-of-flight mass spectrometry strategy for time-resolved proteolysis experiments of large protein domains was developed to facilitate analysis and to help resolve the tertiary arrangement of the entire RBCC subregion. The linker region between the RING and B-box motifs is crucial for full folding, and Zn2+ affinity of the RING-B-box region is further protected in the entire RBCC region and appears to interact with the coiled-coil region. Murine monoclonal antibodies raised toward the RING-B-box region were primarily directed toward the linker, further supporting a highly functional role for the linker in a cellular environment. Taken together with our previous analysis of autoantigenic epitopes in the coiled-coil region, localization of autoantigenic epitopes in Ro52 appears closely related to molecular functionalities.
Asunto(s)
Autoantígenos/inmunología , Epítopos , Ribonucleoproteínas/química , Ribonucleoproteínas/inmunología , Zinc/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Autoantígenos/análisis , Autoantígenos/química , Autoantígenos/genética , Biofisica/métodos , Dicroismo Circular , Ensayo de Inmunoadsorción Enzimática , Humanos , Hidrólisis , Cinética , Ligandos , Modelos Biológicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ribonucleoproteínas/genética , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Dedos de ZincRESUMEN
Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.