RESUMEN
Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins with selective isotope-labeling of selected amino acid residues on a perdeuterated background for studies of enzyme active sites or for ligand screening in drug discovery projects, as well as the synthesis of perdeuterated polypeptides for NMR spectroscopy with large supra-molecular structures. As an illustration, it is demonstrated that the 800-kDa chaperonine GroEL synthesized with the D-S30 cell-free system had a uniform deuteration level of about 95% and assembled into its biologically active oligomeric form.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Biosíntesis de Proteínas , Chaperonina 60/biosíntesis , Chaperonina 60/genética , Chaperonina 60/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Marcaje Isotópico/métodos , Proteínas de Unión a Tacrolimus/biosíntesis , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/aislamiento & purificaciónRESUMEN
Cell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear (1)H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-alpha-helix bundle with an up-down-up topology and a 3(10)-helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters.
Asunto(s)
Adaptación Fisiológica , Frío , Euplotes/química , Resonancia Magnética Nuclear Biomolecular/métodos , Feromonas/química , Animales , Regiones Antárticas , Euplotes/fisiología , Modelos MolecularesRESUMEN
The NMR structures of the unphosphorylated Thermotoga maritima protein TM1442 at pH 4.8 and of the phosphorylated TM1442 (TM1442-P) at pH 7.0 are presented, and a functional interaction of TM1442 with TM0733 is characterized. Although the NMR spectra of TM1442-P at pH 7.0 are of high quality, detailed NMR studies of unphosphorylated TM1442 could be performed only at slightly acidic pH values and high salt concentration. TM1442 is a putative anti-sigma-factor antagonist related to the sigmaF and sigmaB regulation systems in Bacillus subtilis, which is the component in this system that can be phosphorylated. The kinase TM0733, which shows sequence similarity to the GHKL ATPase/kinase superfamily, was identified as the possible anti-sigma-factor of TM1442 using a bioinformatics analysis. Phosphorylation of TM1442 by TM0733 was confirmed by NMR, mass spectroscopy and native gel electrophoresis, and Ser59 was identified as the phosphorylation site using site-directed mutational analysis. The solution structure of TM1442-P at pH 7.0 has the same global fold as free TM1442 at pH 4.8, with an alpha/beta topology consisting of a central four-stranded beta sheet and three alpha helices, but the regular secondary structure elements wrapping the hydrophobic core of the protein undergo subtle conformational changes upon phosphorylation.
Asunto(s)
Proteínas Bacterianas/química , Factor sigma/antagonistas & inhibidores , Thermotoga maritima/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Biología Computacional , Análisis Mutacional de ADN , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Conformación Proteica , Pliegue de Proteína , Soluciones/químicaRESUMEN
A fully automated, NOE-based NMR structure determination of a uniformly 13C,15N-labeled protein was achieved in crude cell-extract, without purification of the overexpressed protein. Essentially complete sequence-specific assignments were obtained using triple resonance experiments, based on the high intensity of the resonances from the overexpressed protein relative to those of the background. For the collection of NOE distance constraints, efficient discrimination between NOE cross peaks from the target protein and background signals was achieved using the programs ATNOS and CANDID. In the iterative ATNOS/CANDID procedure, the identification of the desired protein NOEs is initially guided by the self-consistency of the protein NOE-network. Although the intensities of the signals in this network vary over a wide range, and are in many instances comparable to or smaller than those of the background, the first cycle of calculations resulted in the correct global polypeptide fold, and the structure was then refined in six subsequent cycles using the intermediate NMR structures for additional guidance. The experience gained with this work demonstrates that the ATNOS/CANDID procedure for automatic protein structure determination is highly robust and reliable in the presence of intense background signals, and might thus also represent a platform for future protein structure determinations in physiological fluids.
Asunto(s)
Proteínas Bacterianas/química , Extractos Celulares/química , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Algoritmos , Proteínas Bacterianas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Thermotoga maritima/químicaRESUMEN
In large molecular structures, the magnetization of all hydrogen atoms in the solute is strongly coupled to the water magnetization through chemical exchange between solvent water and labile protons of macromolecular components, and through dipole-dipole interactions and the associated "spin diffusion" due to slow molecular tumbling. In NMR experiments with such systems, the extent of the water polarization is thus of utmost importance. This paper presents a formalism that describes the propagation of the water polarization during the course of different NMR experiments, and then compares the results of model calculations for optimized water polarization with experimental data. It thus demonstrates that NMR spectra of large molecular structures can be improved with the use of paramagnetic spin relaxation agents which selectively enhance the relaxation of water protons, so that a substantial gain in signal-to-noise can be achieved. The presently proposed use of a relaxation agent can also replace the water flip-back pulses when working with structures larger than about 30 kDa. This may be a valid alternative in situations where flip-back pulses are difficult to introduce into the overall experimental scheme, or where they would interfere with other requirements of the NMR experiment.
Asunto(s)
Modelos Teóricos , Resonancia Magnética Nuclear Biomolecular/métodos , Solventes/química , Agua/química , Chaperonina 60/química , Peso MolecularAsunto(s)
Proteínas Bacterianas/química , Espectroscopía de Resonancia Magnética/métodos , Thermotoga maritima/química , Secuencia de Aminoácidos , Clonación Molecular , Biología Computacional/métodos , Cristalografía por Rayos X , Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Proteómica/métodos , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
This paper describes the NMR screening of 141 small (<15 kDa) recombinant Thermotoga maritima proteins for globular folding. The experimental data shows that approximately 25% of the screened proteins are folded under our screening conditions, which makes this procedure an important step for selecting those proteins that are suitable for structure determination. A comparison of screening based either on 1D 1H NMR with unlabeled proteins or on 2D [1H,15N]-COSY with uniformly 15N-labeled proteins is presented, and a comprehensive analysis of the 1D 1H NMR screening data is described. As an illustration of the utility of these methods to structural proteomics, the NMR structure determination of TM1492 (ribosomal protein L29) is presented. This 66-residue protein consists of a N-terminal 3(10)-helix and two long alpha-helices connected by a tight turn centered about glycine 35, where conserved leucine and isoleucine residues in the two alpha-helices form a small hydrophobic core.