RESUMO
Methods based on pulse electron paramagnetic resonance allow measurement of the electron-electron dipolar coupling between two spin labels. Here we compare the most popular technique, Double Electron-Electron Resonance (DEER or PELDOR), with the dead-time free 5-pulse Relaxation-Induced Dipolar Modulation Enhancement (RIDME) method for Gd(iii)-Gd(iii) distance measurements at W-band (94.9 GHz, ≈3.5 T) using Gd(iii) tags with a small zero field splitting (ZFS). Such tags are important because of their high EPR sensitivity arising from their narrow central transition. Two systems were investigated: (i) a rigid model compound with an inter-spin distance of 2.35 nm, and (ii) two mutants of a homodimeric protein, both labeled with a DOTA-based Gd(iii) chelate and characterized by an inter-spin distance of around 6 nm, one having a narrow distance distribution and the other a broad distribution. Measurements on the model compound show that RIDME is less sensitive to the complications arising from the failure of the weak coupling approximation which affect DEER measurements on systems characterized by short inter-spin distances between Gd(iii) tags having a narrow central transition. Measurements on the protein samples, which are characterized by a long inter-spin distance, emphasize the complications due to the appearance of harmonics of the dipolar interaction frequency in the RIDME traces for S > 1/2 spin systems, as well as enhanced uncertainties in the background subtraction. In both cases the sensitivity of RIDME was found to be significantly better than DEER. The effects of the experimental parameters on the RIDME trace are discussed.
RESUMO
High-resolution proton nuclear magnetic resonance studies of protein hydration in aqueous solution show that there are two qualitatively different types of hydration sites. A well-defined, small number of water molecules in the interior of the protein are in identical locations in the crystal structure and in solution, and their residence times are in the range from about 10(-2) to 10(-8) second. Hydration of the protein surface in solution is by water molecules with residence times in the subnanosecond range, even when they are located in hydration sites that contain well-ordered water in the x-ray structures of protein single crystals.
Assuntos
Ocitocina/química , Conformação Proteica , Inibidores da Tripsina/química , Animais , Bovinos , Cristalografia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Pâncreas/enzimologia , Soluções , Inibidores da Tripsina/ultraestrutura , Água , Difração de Raios XRESUMO
The three-dimensional structure of the homeodomain, as determined by nuclear magnetic resonance spectroscopy, reveals the presence of a helix-turn-helix motif, similar to the one found in prokaryotic gene regulatory proteins. Isolated homeodomains bind with high affinity to specific DNA sequences. Thus, the structure-function relationship is highly conserved in evolution.
Assuntos
Drosophila/genética , Genes Homeobox , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , DNA/genética , DNA/metabolismo , Drosophila/anatomia & histologia , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Enzymes frequently recognize substrates and pharmaceutical drugs through specific binding interactions in deep pockets on the protein surface. We show how the specificity-determining substrate binding site of hen egg-white lysozyme (HEWL) can be readily identified in aqueous solution by nuclear magnetic resonance spectroscopy using small organic solvent molecules as detection probes. Exchange of magnetization between the 1H nuclei of the protein and the ligands through dipole-dipole interactions is observed which allows the modeling of their position and orientation at the binding site. Combined with site-specific binding constants measured by titration experiments with different organic solvents, the method can provide important information for rational drug design. In addition, the lifetime of nonspecific interactions of HEWL with organic solvents is shown to be in the sub-nanosecond time range.
Assuntos
Muramidase/metabolismo , Solventes/química , Animais , Sítios de Ligação , Galinhas , Cristalografia por Raios X , Ligantes , Espectroscopia de Ressonância Magnética , Muramidase/química , Ligação Proteica , Solventes/metabolismo , Ureia/química , Ureia/metabolismoRESUMO
BACKGROUND: ERp29 is a ubiquitously expressed rat endoplasmic reticulum (ER) protein conserved in mammalian species. Fold predictions suggest the presence of a thioredoxin-like domain homologous to the a domain of human protein disulfide isomerase (PDI) and a helical domain similar to the C-terminal domain of P5-like PDIs. As ERp29 lacks the double-cysteine motif essential for PDI redox activity, it is suggested to play a role in protein maturation and/or secretion related to the chaperone function of PDI. ERp29 self-associates into 51 kDa dimers and also higher oligomers. RESULTS: 3D structures of the N- and C-terminal domains determined by NMR spectroscopy confirmed the thioredoxin fold for the N-terminal domain and yielded a novel all-helical fold for the C-terminal domain. Studies of the full-length protein revealed a short, flexible linker between the two domains, homodimerization by the N-terminal domain, and the presence of interaction sites for the formation of higher molecular weight oligomers. A gadolinium-based relaxation agent is shown to present a sensitive tool for the identification of macromolecular interfaces by NMR. CONCLUSIONS: ERp29 is the first eukaryotic PDI-related protein for which the structures of all domains have been determined. Furthermore, an experimental model of the full-length protein and its association states was established. It is the first example of a protein where the thioredoxin fold was found to act as a specific homodimerization module, without covalent linkages or supporting interactions by further domains. A homodimerization module similar as in ERp29 may also be present in homodimeric human PDI.
Assuntos
Retículo Endoplasmático/química , Proteínas de Choque Térmico/química , Modelos Moleculares , Chaperonas Moleculares/química , Tiorredoxinas/química , Sequência de Aminoácidos , Dimerização , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: DnaB is the primary replicative helicase in Escherichia coli. Native DnaB is a hexamer of identical subunits, each consisting of a larger C-terminal domain and a smaller N-terminal domain. Electron-microscopy data show hexamers with C6 or C3 symmetry, indicating large domain movements and reversible pairwise association. RESULTS: The three-dimensional structure of the N-terminal domain of E. coli DnaB was determined by nuclear magnetic resonance (NMR) spectroscopy. Structural similarity was found with the primary dimerisation domain of a topoisomerase, the gyrase A subunit from E. coli. A monomer-dimer equilibrium was observed for the isolated N-terminal domain of DnaB. A dimer model with C2 symmetry was derived from intermolecular nuclear Overhauser effects, which is consistent with all available NMR data. CONCLUSIONS: The monomer-dimer equilibrium observed for the N-terminal domain of DnaB is likely to be of functional significance for helicase activity, by participating in the switch between C6 and C3 symmetry of the helicase hexamer.
Assuntos
Proteínas de Bactérias , DNA Helicases/química , Escherichia coli/enzimologia , Sequência de Aminoácidos , Sequência Conservada , DnaB Helicases , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Alinhamento de SequênciaRESUMO
The solution structure of the N-terminal DNA-binding domain of the P22 c2 repressor (residues 1 to 76) was determined by nuclear magnetic resonance (NMR) spectroscopy. The structure determination was based on nearly complete sequence-specific resonance assignments for 1H, 13C and 15N, and tables of the chemical shifts for all three nuclei are included here. A group of 20 conformers was calculated from the NMR constraints using the program DIANA, and energy-minimized using an implementation of the AMBER force field in the program OPAL. The core of the protein formed by residues 5 to 68 is structurally well defined, with an average of 0.7 A for the root-mean-square deviations calculated for the backbone atoms of the individual conformers relative to the mean coordinates. The N-terminal tetrapeptide segment and the C-terminal octapeptide segment are flexibly disordered. The molecular architecture includes five alpha-helical segments with residues 6 to 17, 21 to 28, 32 to 39, 47 to 57 and 61 to 65. The length and relative orientation of these helices are closely similar to the arrangement of corresponding regular secondary structures in the DNA-binding domain of the 434 repressor, with the sole exception of the fourth helix, which is one turn longer at its amino-terminal end than the corresponding helix in the 434 repressor. This extension of the fourth helix implies that the DNA-binding mode of the P22 c2 repressor must be somewhat different from that observed for the 434 repressor. Exact superposition of two P22 c2 repressor DNA-binding domains for best fit of corresponding polypeptide backbone atoms onto the two 434 repressor DNA-binding domains in the crystal structure of the 434 repressor-DNA complex would result in a model of the P22 c2 repressor-DNA complex which could not accommodate the fourth helices because of steric overlap.
Assuntos
Bacteriófago P22/química , Proteínas de Ligação a DNA/química , Proteínas Repressoras/química , Proteínas Virais/química , Amidas/química , Sequência de Aminoácidos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes , Soluções , Solventes , Proteínas Virais Reguladoras e AcessóriasRESUMO
The structure and hydration of the DNA duplex d-(AGCGTACTAGTACGCT)2 corresponding to the trp operator fragment used in the crystal structure of the half site complex (PDB entry 1TRR) was studied by a 1.4 ns molecular dynamics simulation in water. The simulation, starting from a B-DNA conformation, used a non-bonded cutoff of 1.4 nm with a reaction field correction and resulted in a stable trajectory. The average DNA conformation obtained was closer to the ones found in the crystal structures of the complexes (PDB entries 1TRO and 1TRR) than to the crystal structure of unbound trp operator (Nucleic Acid Database entry BDJ061). The DNA hydration was characterized in terms of hydrogen bond percentages and corresponding residence times. The residence times of water molecules within 0.35 nm of the DNA non-exchangeable protons were calculated for comparison with NMR measurements of intermolecular water-DNA NOEs and nuclear magnetic relaxation dispersion measurements. No significant difference was found between major and minor groove hydration. The DNA donors and acceptors were hydrogen bonded to water molecules for 77(+/-19)% of the time on average. The average residence time of the hydrogen bonded water molecules was 11(+/-11) ps with a maximum of 223 ps. When all water molecules within NOE distance (0.35 nm) of non-exchangeable protons were considered, the average residence times increased to an average of 100(+/-4) ps and a maximum of 608 ps. These results agree with the experimental NMR results of Sunnerhagen et al. which did not show any evidence for water molecules bound with more than 1 ns residence time on the DNA surface. The exchange of hydration water from the DNA occurred in the major groove primarily through direct exchange with the bulk solvent, while access to and from the minor groove frequently proceeded via pathways involving ribose O3' and O4' and phosphate O2P oxygen atoms. The most common water diffusion pathways in the minor groove were perpendicular to the groove direction. In general, water molecules visited only a limited number of sites in the DNA grooves before exiting. The hydrogen bonding sites, where hydrogen bonds could be formed with donor and acceptor groups of the DNA, were filled with water molecules with an average B-factor value of 0.58 mn2. No special values were observed at any of the sites, where water molecules were observed both in the trp repressor/operator co-crystals and in the crystal structure of unbound DNA.
Assuntos
Proteínas de Bactérias , Simulação por Computador , DNA/química , DNA/metabolismo , Regiões Operadoras Genéticas/genética , Triptofano/genética , Água/metabolismo , Sítios de Ligação , Cristalização , DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Bacterianos/genética , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Prótons , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Solventes , Termodinâmica , Água/químicaRESUMO
The three-dimensional solution structure of porcine PEC-60, a 60 amino acid residue protein of the Kazal type family of proteinase inhibitors, was determined by nuclear magnetic resonance (NMR) spectroscopy. The structure determination is based on nearly complete 1H, 13C and 15N resonance assignments including stereospecific 1H resonance assignments for 40 pairs of methylene protons and isopropyl methyl groups. The stereospecific resonance assignments of the beta-protons were supported by heteronuclear long-range correlation experiments recorded at natural 13C and 15N isotopic abundances. A group of 20 conformers were calculated using the experimentally derived NMR constraints with the program DIANA, and energy-minimized in a 4 A water shell using the program OPAL. The average of the root-mean-square deviations relative to the mean structure of the 20 conformers selected to represent the solution structure of PEC-60 is 0.55 A for the backbone atoms of residues 6 to 10 and 24 to 60. Disordered conformations are observed for the amino-terminal pentapeptide and the polypeptide segment containing residues 11 to 23. The NMR structure confirms the structural similarity of PEC-60 to the Kazal type family of proteinase inhibitors which had been previously suggested on the basis of amino acid homology. The well-defined part of PEC-60 contains a short three-stranded anti-parallel beta-sheet involving the residues 27 to 29, 33 to 35 and 53 to 56 with a beta-bulge at residue 55, a type I turn comprising residues 29 to 32, and an alpha-helix involving the residues 37 to 48. T1(13C) relaxation measurements of the alpha-carbons and linewidth measurements of the amide proton signals indicate substantially increased mobility on the pico- to nanosecond time-scale for the amino-terminal pentapeptide as well as within the loop comprising residues 11 to 23. The structure of PEC-60 is compared to the X-ray crystal structures of homologous Kazal type proteinase inhibitors and the dynamic properties of PEC-60 are discussed with respect to the observed lack of any substantial trypsin inhibiting activity.
Assuntos
Peptídeos/química , Conformação Proteica , Inibidor da Tripsina Pancreática de Kazal/química , Sequência de Aminoácidos , Animais , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/genética , Soluções , Suínos , Inibidor da Tripsina Pancreática de Kazal/genéticaRESUMO
Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of protein disulfide groups and glutathione-containing mixed disulfide groups via an active site Grx-glutathione mixed disulfide (Grx-SG) intermediate. The NMR solution structure of the Escherichia coli Grx3 mixed disulfide with glutathione (Grx3-SG) was determined using a C14S mutant which traps this intermediate in the redox reaction. The structure contains a thioredoxin fold, with a well-defined binding site for glutathione which involves two intermolecular backbone-backbone hydrogen bonds forming an antiparallel intermolecular beta-bridge between the protein and glutathione. The solution structure of E. coli Grx3-SG also suggests a binding site for a second glutathione in the reduction of the Grx3-SG intermediate, which is consistent with the specificity of reduction observed in Grxs. Molecular details of the structure in relation to the stability of the intermediate and the activity of Grx3 as a reductant of glutathione mixed disulfide groups are discussed. A comparison of glutathione binding in Grx3-SG and ligand binding in other members of the thioredoxin superfamily is presented, which illustrates the highly conserved intermolecular interactions in this protein family.
Assuntos
Proteínas de Bactérias/química , Escherichia coli/química , Espectroscopia de Ressonância Magnética , Oxirredutases , Conformação Proteica , Proteínas/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Catálise , Cisteína/química , Escherichia coli/genética , Glutarredoxinas , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleotídeo Redutases/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The present NMR study investigates the residence times of the hydration water molecules associated with uncomplexed trp operator DNA in solution by measuring intermolecular nuclear Overhauser effects (NOE) between water and DNA protons, and the nuclear magnetic relaxation dispersion (NMRD) of the water 2H and 17O resonances. Both methods indicate that the hydration water molecules exchange with bulk water on the sub-nanosecond time scale at 4 degreesC. No evidence was obtained for water molecules bound with longer residence times. In particular, the water molecules at the sites of interfacial hydration in the trp repressor/operator complex do not seem kinetically stabilized in the uncomplexed DNA. Analysis of the crystal structures of two different trp repressor/operator complexes shows very similar structural environments for the water molecules mediating specific contacts between the protein and the DNA, whereas much larger variations are observed for the location of corresponding water molecules detected in the crystal structure of an uncomplexed trp operator DNA duplex. Therefore, it appears unlikely that the hydration characteristics of the uncomplexed DNA target would be a major determinant of trp repressor/operator recognition.
Assuntos
Proteínas de Bactérias , DNA/metabolismo , Regiões Operadoras Genéticas/genética , Triptofano/genética , Água/metabolismo , Sequência de Bases , Sítios de Ligação , Cristalização , DNA/química , DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Bacterianos/genética , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Netropsina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Prótons , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Solventes , Água/químicaRESUMO
A 1:1 complex formed by the mutant Antennapedia(C39S) homeodomain and a 14 base-pair DNA duplex (molecular weight approximately 18,000) was prepared in which the protein was uniformly 13C-labeled. Using two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy with 13C(omega 1, omega 2)-double-half-filter and three-dimensional 13C-correlated NOE spectroscopy, nearly complete sequence-specific resonance assignments were obtained for both the protein and the DNA in the complex. On this basis conformational constraints needed for a three-dimensional structure determination were collected. Using 855 intramolecular distance constraints as input, the structure of the DNA-bound Antp(C39S) homeodomain was calculated with the program DIANA, followed by restrained energy minimization with the program OPAL. A group of 20 conformers characterizes a well-defined structure for residues 8 to 56, with an average of 0.5 A of the pairwise root-mean-square deviations calculated for the backbone atoms of the individual conformers relative to the mean coordinates. The quality of the resulting structure is comparable to the one for the free protein, and the global fold of the free Antp(C39S) homeodomain was found to be conserved in the DNA complex. The structure of the DNA-bound protein was the starting point for the subsequent structure determination of the complete Antp(C39S) homeodomain-DNA complex in solution.
Assuntos
Proteínas de Ligação a DNA/química , Desoxirribonucleoproteínas/química , Genes Homeobox , Proteínas de Homeodomínio , Proteínas Nucleares , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Proteína do Homeodomínio de Antennapedia , Proteínas de Drosophila , Drosophila melanogaster , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas RecombinantesRESUMO
The nuclear magnetic resonance (NMR) solution structure of a complex formed by the mutant Antennapedia homeodomain with Cys39 replaced by Ser, Antp(C39S), and a 14 base-pair DNA duplex containing the BS2 operator sequence was determined using uniform 13C and 15N-labeling of the protein. Two-dimensional nuclear Overhauser enhancement spectroscopy ([1H,1H]NOESY) with 15N(omega 2)-half-filter and 13C(omega 1, omega 2)-double-half-filter, and three-dimensional heteronuclear-correlated [1H,1H]NOESY yielded a total of 855 intramolecular NOE upper distance constraints in the homeodomain, 151 upper distance constraints within the DNA duplex, and 39 intermolecular protein-DNA upper distance constraints. These data were used as the input for the structure calculation with simulated annealing followed by molecular dynamics in a water bath and energy refinement. A group of 16 conformers was thus generated which represent the solution structure of the Antp(C39S) homeodomain-DNA complex. The new structure determination confirms the salient features reported previously from a preliminary investigation of the same complex, in particular the location of the recognition helix in the major groove with the turn of the helix-turn-helix motif outside the contact area with the DNA, and the N-terminal arm of the homeodomain contacting the minor groove of the DNA. In addition, distinct amino acid side-chain-DNA contacts could be identified, and evidence was found that the invariant residue Asn51 (and possibly also Gln50) is in a slow dynamic equilibrium between two or several different DNA contact sites. The molecular dynamics calculations in a water bath yielded structures with hydration water molecules in the protein-DNA interface, which coincides with direct NMR observations of hydration waters. In the Appendix the experimental data obtained with the Antp(C39S) homeodomain-DNA complex and the techniques used for the structure calculation are evaluated using a simulated input data set derived from the X-ray crystal structure of a DNA complex with a homologous homeodomain. This study indicates that a nearly complete set of NOE upper distance constraints for the Antp(C39S) homeodomain and the protein-DNA interface was presently obtained. It further shows that the structure calculation used here yields a precise reproduction of the crystal structure from the simulated input data, and also results in hydration of the protein-DNA interface in the recalculated complex.
Assuntos
Proteínas de Ligação a DNA/química , Desoxirribonucleoproteínas/química , Genes Homeobox , Proteínas de Homeodomínio , Proteínas Nucleares , Fatores de Transcrição , Animais , Proteína do Homeodomínio de Antennapedia , Proteínas de Drosophila , Drosophila melanogaster , Ligação de Hidrogênio , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , SoluçõesRESUMO
The structure of a mutant Antennapedia homeodomain, Antp(C39----S), from Drosophila melanogaster was determined using a set of new programs introduced in the accompanying paper. An input dataset of 957 distance constraints and 171 dihedral angle constraints was collected using two-dimensional n.m.r. experiments with the 15N-labeled protein. The resulting high quality structure for Antp(C39----S), with an average root-mean-square deviation of 0.53 A between the backbone atoms of residues 7 to 59 in 20 energy-refined distance geometry structures and the mean structure, is nearly identical to the previously reported structure of the wild-type Antp homeodomain. The only significant difference is in the connection between helices III and IV, which was found to be less kinked than was indicated by the structure determination for Antp. The main emphasis of the presentation in this paper is on a detailed account of the practical use of a novel strategy for the computation of nuclear magnetic resonance structures of proteins with the combined use of the programs DIANA, CALIBA, HABAS and GLOMSA.
Assuntos
Proteínas de Ligação a DNA/ultraestrutura , Proteínas de Homeodomínio , Espectroscopia de Ressonância Magnética/métodos , Proteínas Nucleares , Conformação Proteica , Software , Fatores de Transcrição , Algoritmos , Sequência de Aminoácidos , Animais , Proteína do Homeodomínio de Antennapedia , Cisteína , Proteínas de Ligação a DNA/química , Proteínas de Drosophila , Drosophila melanogaster , Genes Homeobox , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Serina , Estereoisomerismo , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The determination of the three-dimensional structure of the Antennapedia homeodomain from Drosophila in solution is described. The techniques used are 1H nuclear magnetic resonance spectroscopy for the data collection, and calculation of the protein structure with the program DISMAN followed by restrained energy minimization with a modified version of the program AMBER. A group of 19 conformers characterizes a well-defined structure for residues 7 to 59, with an average root-mean-square distance from the backbone atoms of 0.6 A relative to the mean of the 19 structures. The structure contains a helix from residues 10 to 21, a helix-turn-helix motif from residues 28 to 52, which is similar to those reported for several prokaryotic repressor proteins, and a somewhat flexible fourth helix from residues 53 to 59, which essentially forms an extension of the presumed recognition helix, residues 42 to 52. The helices enclose a structurally well-defined molecular core of hydrophobic amino acid side-chains.
Assuntos
Proteínas de Ligação a DNA , Drosophila/genética , Proteínas de Homeodomínio , Proteínas Nucleares , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Proteína do Homeodomínio de Antennapedia , Proteínas de Drosophila , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação Proteica , PrótonsRESUMO
A high precision NMR structure of oxidized glutaredoxin 3 [C65Y] from Escherichia coli has been determined. The conformation of the active site including the disulphide bridge is highly similar to those in glutaredoxins from pig liver and T4 phage. A comparison with the previously determined structure of glutaredoxin 3 [C14S, C65Y] in a complex with glutathione reveals conformational changes between the free and substrate-bound form which includes the sidechain of the conserved, active site tyrosine residue. In the oxidized form this tyrosine is solvent exposed, while it adopts a less exposed conformation, stabilized by hydrogen bonds, in the mixed disulfide with glutathione. The structures further suggest that the formation of a covalent linkage between glutathione and glutaredoxin 3 is necessary in order to induce these structural changes upon binding of the glutathione peptide. This could explain the observed low affinity of glutaredoxins for S-blocked glutathione analogues, in spite of the fact that glutaredoxins are highly specific reductants of glutathione mixed disulfides.
Assuntos
Escherichia coli/química , Ressonância Magnética Nuclear Biomolecular , Oxirredutases , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Glutarredoxinas , Glutationa/análogos & derivados , Glutationa/metabolismo , Glutationa/farmacologia , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Oxigênio/metabolismo , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Substâncias Redutoras/metabolismo , Substâncias Redutoras/farmacologia , Alinhamento de Sequência , Solventes , Especificidade por Substrato , Termodinâmica , Tirosina/metabolismoRESUMO
A 1 ns trajectory from a molecular dynamics study of 1.4 ns total length was used for a detailed analysis of the residence times of water molecules located near 227 selected bovine pancreatic trypsin inhibitor (BPTI) atoms. The simulation was performed using the GROMOS force field, with apolar hydrogen atoms treated as united atoms, and the SPC/E water model. The system consisted of 568 BPTI atoms and 2371 water molecules. The theoretical results are in good agreement with experimental data available from nuclear magnetic resonance spectroscopy. The residence times of individual water molecules coming near a given BPTI atom, as obtained from the simulation, vary greatly and range between 10 and 500 ps. The effective residence time, calculated using a correlation function technique from the presence of all individual water molecules visiting the hydration shell of a given BPTI atom, never exceeds 200 ps. The average residence time near backbone and side-chain atoms is approximately 39 ps and 24 ps, respectively. The shortest residence times, on average, are found near charged atoms (19 ps), whereas near non-polar and polar side-chain atoms the residence times are 25 ps and 36 ps, respectively. There is no apparent correlation between the residence times of the hydration water molecules of solvent-accessible residues and their location in different regular or non-regular secondary structures.
Assuntos
Aprotinina/química , Sequência de Aminoácidos , Simulação por Computador , Modelos Químicos , Dados de Sequência Molecular , Termodinâmica , Água/químicaRESUMO
Quantitative cysteine-independent ligation of a Gd(3+) tag to genetically encoded p-azido-L-phenylalanine via Cu(I)-catalyzed click chemistry is shown to deliver an exceptionally powerful tool for Gd(3+)-Gd(3+) distance measurements by double electron-electron resonance (DEER) experiments, as the position of the Gd(3+) ion relative to the protein can be predicted with high accuracy.
Assuntos
Proteínas de Transporte/síntese química , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/química , Gadolínio , Glutamatos/química , Fenilalanina/análogos & derivados , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Azidas/química , Proteínas de Transporte/química , Química Click , Mutagênese Sítio-Dirigida , Fenilalanina/química , Fenilalanina/genética , Marcadores de SpinRESUMO
The design, synthesis and evaluation of four novel lanthanide-binding tags for paramagnetic NMR spectroscopy are reported. Each tag is based on the ((2S,2'S,2''S,2'''S)-1,1',1'',1'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetrakis(propan-2-ol)) scaffold, featuring small chiral alcohol coordinating pendants to minimise the size and hydrophobic character of each tag. The tags feature different linkers of variable length for conjugation to protein via a single cysteine residue. Each tag's ability to induce pseudocontact shifts (PCS) was assessed on a ubiquitin A28C mutant. Two enantiomeric tags of particular note, C7 and C8, produced significantly larger Δχ-tensors compared to a previously developed tag, C1, attributed to the extremely short linker utilised, limiting the mobility of the bound lanthanide ion. The C7 and C8 tags' capacity to induce PCSs was further demonstrated on GB1 Q32C and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) S112C/C80A mutants. Whilst factors such as the choice of lanthanide ion, pH and site of conjugation influence the size of the PCSs obtained, the tags represent a significant advance in the field.
RESUMO
The complex formed between the anticancer drug 4-[p-[p-(4-quinolylamino)benzamido]anilino]pyridine (SN 6999) and the decadeoxyribonucleoside nonaphosphate d-(GCATTAATGC)2 was investigated using two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) with a 13C(omega 1)-half-filter. The two quaternary methyl groups in SN 6999 had been labeled with 13C for these experiments. The simplified subspectra of [1H,1H]-NOESY obtained with this procedure greatly facilitate the identification and assignment of intermolecular NOEs. Quite generally, the combined use of isotope labeling and heteronuclear filters in [1H,1H]-NOESY provides an improved experimental basis for structural studies of drug/DNA complexes.