RESUMEN
Scalar coupling constants and magnetic shieldings in the imino hydrogen-bonding region of Hoogsteen-Watson-Crick T.A-T and C(+).G-C triplets have been calculated as a function of the distance between proton donor and acceptor nitrogen atoms. The Fermi contact contributions to (h2)J((15)N-H...(15)N), (1)J((15)N-(1)H), and (h1)J((1)H...(15)N) were computed using density functional theory/finite perturbation theory (DFT/FPT) methods for the full base triplets at the unrestricted B3PW91/6-311G level. Chemical shifts delta((1)H) and delta((15)N) were obtained at the same level using the gauge including atomic orbital (GIAO) method for magnetic shielding. All three scalar couplings and all three chemical shifts are strongly interrelated and exhibit monotonic changes with base pair separation. These correlations are in conformity with experimental data for a 32-nucleotide DNA triplex. The results suggest that both chemical shifts and coupling constants can be used to gain information on H-bond donor-acceptor distances in nucleic acids. In addition to the DFT/FPT calculations, a simple three-orbital model of the N-H...H bond and a sum-over-states analysis is presented. This model reproduces the basic features of the H-bond coupling effect. In accordance with this model and the DFT calculations, a positive sign for the (h2)J(NN) coupling is determined from an E.COSY experiment.
Asunto(s)
ADN/química , Enlace de Hidrógeno , Emparejamiento Base , Espectroscopía de Resonancia Magnética/métodos , Modelos Teóricos , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
Helix formation of an S-peptide analog, comprising the first 20 residues of Ribonuclease A and two additional N-terminal residues, was studied by measuring hydrogen bond (H-bond) (h3)J(NC') scalar couplings as a function of 2,2,2-trifluoroethanol (TFE) concentration. The (h3)J(NC') couplings give direct evidence for the closing of individual backbone N-H***O = C H-bonds during the TFE-induced formation of secondary structure. Whereas no (h3)J(NC') correlations could be detected without TFE, alpha-helical (i,i +4) H-bond correlations were observed for the amides of residues A5 to M15 in the presence of TFE. The analysis of individual coupling constants indicates that alpha-helix formation starts at the center of the S-peptide around residue E11 and proceeds gradually from there to both peptide ends as the TFE concentration is increased. At 60% to 90% TFE, well-formed alpha-helical H-bonds were observed for the amides hydrogens of residues K9 to Q13, whereas H-bonds of residues T5 to A8, H14, and M15 are affected by fraying. No intramolecular backbone H-bonds are present at and beyond the putative helix stop signal D16. As the (h3)J(NC') constants represent ensemble averages and the dependence of (h3)J(NC') on H-bond lengths is very steep, the size of the individual (h3)J(NC') coupling constants can be used as a measure for the population of a closed H-bond. These individual populations are in agreement with results derived from the Lifson-Roig theory for coil-to-helix transitions. The present work shows that the closing of individual H-bonds during TFE-induced helix formation can be monitored by changes in the size of H-bond scalar couplings.
Asunto(s)
Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Trifluoroetanol/farmacología , Amidas/química , Amidas/metabolismo , Secuencia de Aminoácidos , Relación Dosis-Respuesta a Droga , Estabilidad de Enzimas/efectos de los fármacos , Enlace de Hidrógeno/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína , Estructura Secundaria de Proteína/efectos de los fármacos , Ribonucleasa Pancreática/química , TermodinámicaRESUMEN
We have recently shown that an energy penalty for the incorporation of residual tensorial constraints into molecular structure calculations can be formulated without the explicit knowledge of the Saupe orientation tensor (Moltke and Grzesiek. J. Biomol. NMR, 1999, 15, 77-82). Here we report the implementation of such an algorithm into the program X-PLOR. The new algorithm is easy to use and has good convergence properties. The algorithm is used for the structure refinement of the HIV-1 Nef protein using 252 dipolar coupling restraints. The approach is compared to the conventional penalty function with explicit knowledge of the orientation tensor's amplitude and rhombicity. No significant differences are found with respect to speed, Ramachandran core quality or coordinate precision.
Asunto(s)
Productos del Gen nef/química , VIH-1/química , Resonancia Magnética Nuclear Biomolecular/métodos , Algoritmos , Cristalografía por Rayos X , Conformación Proteica , Temperatura , Termodinámica , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Changes in the molecular conformation of proteins can result from a variety of perturbations, and can play crucial roles in the regulation of biological activity. A new solution NMR method has been applied to monitor ligand-induced changes in hydrogen bond geometry in the chicken c-Src SH3 domain. The structural response of this domain to ligand binding has been investigated by measuring trans-hydrogen bond (15)N-(13)C' scalar couplings in the free state and when bound to the high affinity class I ligand RLP2, containing residues RALPPLPRY. A comparison between hydrogen bonds in high resolution X-ray structures of this domain and those observed via (h3)J(NC') couplings in solution shows remarkable agreement. Two backbone-to-side-chain hydrogen bonds are observed in solution, and each appears to play a role in stabilization of loop structure. Reproducible ligand-induced changes in trans-hydrogen bond scalar couplings are observed across the domain that translate into changes in hydrogen bond length ranging between 0.02 to 0.12 A. The observed changes can be rationalized by an induced fit mechanism in which hydrogen bonds across the protein participate in a compensatory response to forces imparted at the protein-ligand interface. Upon ligand binding, mutual intercalation of the two Leu-Pro segments of the ligand between three aromatic side-chains protruding from the SH3 surface wedges apart secondary structural elements within the SH3 domain. This disruption is transmitted in a domino-like effect across the domain through networks of hydrogen bonded peptide planes. The unprecedented resolution obtained demonstrates the ability to characterize subtle structural rearrangements within a protein upon perturbation, and represents a new step in the endeavor to understand how hydrogen bonds contribute to the stabilization and function of biological macromolecules.
Asunto(s)
Pollos , Resonancia Magnética Nuclear Biomolecular , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Sitios de Unión , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , ProtonesRESUMEN
Scalar coupling correlations across hydrogen bonds with carbonyl groups as acceptors have been observed in a variety of proteins, but not in nucleic acids. Here we present a pulse scheme that allows such an observation and quantification of trans-hydrogen bond 3hJ(NC)' correlations in nucleic acid base pairs, between the imino nitrogen 15N1 and the carbonyl 13C6 nuclei within the guanine quartets of the Oxy-1.5 DNA-quadruplex. Intra- and internucleotide N-H...O=C connectivities can be traced around each guanine quartet, allowing the hydrogen bonding partners to be unambiguously assigned. Absolute values of the 3hJ(NC)' couplings are approximately 0.2 Hz as quantified by a selective long-range H(N)CO experiment and are thus on average smaller than the analogous 3hJ(NC)' couplings observed in proteins. In addition, an improved version of the pseudo-heteronuclear H(N)N-COSY [Majumdar et al. (1999) J. Biomol. NMR, 14, 67-70] is presented which allows simultaneous detection of the 15N-donor and 15N-acceptor resonances connected by 2hJ(NN) couplings in hydrogen bonds involving amino groups. Using this experiment, values ranging between 6 and 8 Hz are determined for the 2hJNN couplings between 15N2 and 15N7 nuclei in the guanine quartet. These values are not strongly influenced by the presence of a significant amount of chemical exchange broadening due to amino group rotations.
Asunto(s)
Guanosina/química , Resonancia Magnética Nuclear Biomolecular/métodos , Emparejamiento Base , Isótopos de Carbono , Dimerización , Guanosina/metabolismo , Enlace de Hidrógeno , Magnetismo , Isótopos de Nitrógeno , Conformación de Ácido NucleicoRESUMEN
Few structures of viral serine proteases, those encoded by the Sindbis and Semliki Forest viruses, hepatitis C virus (HCV) and cytomegalovirus, have been reported. In the life cycle of HCV a crucial role is played by a chymotrypsin-like serine protease encoded at the N-terminus of the viral NS3 protein, the solution structure of which we present here complexed with a covalently bound reversible inhibitor. Unexpectedly, the residue in the P2 position of the inhibitor induces an effective stabilization of the catalytic His-Asp hydrogen bond, by shielding that region of the protease from the solvent. This interaction appears crucial in the activation of the enzyme catalytic machinery and represents an unprecedented observation for this family of enzymes. Our data suggest that natural substrates of this serine protease could contribute to the enzyme activation by a similar induced-fit mechanism. The high degree of similarity at the His-Asp catalytic site region between HCV NS3 and other viral serine proteases suggests that this behaviour could be a more general feature for this category of viral enzymes.
Asunto(s)
Aminobutiratos/metabolismo , Dominio Catalítico , Dipéptidos/metabolismo , Hepacivirus/enzimología , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Proteínas no Estructurales Virales/química , Aminobutiratos/química , Sitios de Unión , Catálisis , Dipéptidos/química , Activación Enzimática , Estabilidad de Enzimas , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Inhibidores de Serina Proteinasa/química , Solventes , Relación Estructura-Actividad , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismoRESUMEN
Rhodopsin-containing disks, isolated from rod outer segments of bovine retina, align at high magnetic fields with their membrane normal parallel to the magnetic field. After light-activation of rhodopsin, transient binding of the C-terminal transducin undecapeptide, selectively labeled with 15N at Leu5 and Gly9, results in residual dipolar contributions to the 1J(NH) splittings for these two residues. Both residues show 1J(NH) splittings which are smaller than in the dark-adapted or rhodopsin-free sample, and return to their isotropic values at a rate determined by the decay of the meta II state of rhodopsin. The dipolar couplings indicate that in the bound state, N-H vectors of Leu5 and Gly9 make angles of 48+/-4 degrees and 40+/-8 degrees, respectively, with the disk normal. These 'transferred' dipolar couplings potentially offer a useful method for studying the conformation and orientation of flexible, low affinity ligands when bound to oriented integral membrane receptors.
Asunto(s)
Transducina/química , Secuencia de Aminoácidos , Fenómenos Químicos , Química Física , Difusión , Ligandos , Luz , Membrana Dobles de Lípidos , Magnetismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/efectos de la radiación , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fotoquímica , Unión Proteica , Radiación , Rodopsina/química , Rodopsina/metabolismo , Rodopsina/efectos de la radiación , Electricidad Estática , Transducina/metabolismoRESUMEN
The diffusive properties of biomacromolecules within the aqueous phase of polyacrylamide gels are described. High quality NMR spectra can be obtained under such conditions. As compared to water, a fivefold reduction in the translational diffusion constant, but only a 1.6-fold decrease (1.4-fold increase) in amide-15N T2 (T1) are observed for human ubiquitin within a 10% acrylamide gel. Weak alignment of the solute macromolecules can be achieved within such gels by vertical or radial compression or by the embedding of magnetically oriented purple membrane fragments. The methods are applied to deriveresidual dipolar couplings for human HIV-1 Nef and ubiquitin.
Asunto(s)
Resinas Acrílicas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Resinas Acrílicas/farmacología , Anisotropía , Difusión/efectos de los fármacos , Productos del Gen nef/química , VIH-1/química , Humanos , Isótopos de Nitrógeno , Membrana Púrpura , Proteínas de los Retroviridae/química , Soluciones , Ubiquitinas/química , Agua/farmacología , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
It is demonstrated that J connectivity between amide protons and hydrogen-bond-accepting carbonyl carbons can be observed in perdeuterated human ubiquitin. A selective pulse scheme is used to detect these small 2hJHC' interactions in the presence of the much larger through-covalent-bond 2JHC' and 3JHC' couplings. The ratio of the observed through-H-bond correlation intensity and the 2JHC' connectivity observed in a reference spectrum indicates 2hJHC' values of ca. 0.4-0.6 Hz, which are only slightly smaller than the corresponding 3hJNC' values. However, for technical reasons, 2hJHC' couplings are more difficult to measure than 3hJNC'.
Asunto(s)
Espectroscopía de Resonancia Magnética , Ubiquitinas/química , Amidas/química , Aminoácidos/química , Carbono/química , Deuterio , Humanos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética/métodos , ProtonesRESUMEN
It is shown that the recently developed quantitative J(NN)HNN-COSY experiment can be used for the direct identification of hydrogen bonds in non-canonical base pairs in RNA. Scalar(2h)J(NN)couplings across NH.N hydrogen bonds are observed in imino hydrogen bonded GA base pairs of the hpGA RNA molecule, which contains a tandem GA mismatch, and in the reverse Hoogsteen AU base pairs of the E-loop of Escherichia coli 5S rRNA. These scalar couplings correlate the imino donor(15)N nucleus of guanine or uridine with the acceptor N1 or N7 nucleus of adenine. The values of the corresponding(2h)J(NN)coupling constants are similar in size to those observed in Watson-Crick base pairs. The reverse Hoogsteen base pairs could be directly detected for the E-loop of E.coli 5S rRNA both in the free form and in a complex with the ribosomal protein L25. This supports the notion that the E-loop is a pre-folded RNA recognition site that is not subject to significant induced conformational changes. Since Watson-Crick GC and AU base pairs are also readily detected the HNN-COSY experiment provides a useful and sensitive tool for the rapid identification of RNA secondary structure elements.
Asunto(s)
Emparejamiento Base/genética , Resonancia Magnética Nuclear Biomolecular , ARN/química , Disparidad de Par Base/genética , Secuencia de Bases , Sitios de Unión , Escherichia coli/genética , Hidrógeno/química , Hidrógeno/metabolismo , Enlace de Hidrógeno , ARN/genética , ARN/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico 5S/química , ARN Ribosómico 5S/genética , ARN Ribosómico 5S/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Secuencias Repetidas en Tándem/genéticaRESUMEN
A method is described which permits detection of 3hJNC' scalar couplings across hydrogen bonds in larger, perdeuterated proteins. The experiment is demonstrated for the uniformly 2H/13C/15N-enriched 30 kDa ribosome inactivating protein MAP30. The 3hJNC' interactions are smaller than 1 Hz, but their detection in an HNCO experiment is made possible through the use of constructive interference between the 15N chemical shift anisotropy and 1H-15N dipole-dipole relaxation mechanisms in a manner similar to that of recently proposed TROSY schemes. Sensitivity of the HNCO experiment depends strongly on the 15N transverse relaxation rate of the downfield 15N multiplet component and on the amide proton T1. In perdeuterated MAP30 at 40 degrees C, the average TROSY T2 was 169 ms at 750 MHz 1H frequency, and a wide range of longitudinal relaxation rates was observed for the amide protons.
Asunto(s)
Estructura Secundaria de Proteína , Proteínas/química , Isótopos de Carbono , Deuterio , Enlace de Hidrógeno , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Inactivadoras de Ribosomas , Proteínas Inactivadoras de Ribosomas Tipo 2RESUMEN
Calbindin D28k, a member of the troponin C superfamily of calcium-binding proteins, contains six putative EF hand domains but binds only four calcium-atoms: one at a binding site of very high affinity and three calcium-atoms at binding sites of lower affinity. The high-affinity site could be located within domain I while domains III, IV, and V bind calcium less tightly. The recombinant protein construct calb I-II (residues 1-93) comprising the first two EF hands affords a unique opportunity to study a pair of EF hands with one site binding calcium tightly and the second site empty. A series of heteronuclear 2D, 3D and 4D high-resolution NMR experiments were applied to calb I-II, and led to the complete assignment of the 1H, 13C and 15N resonances. The secondary structure of the protein was deduced from the size of the 3JHN-Halpha coupling constants, the chemical shift indices of 1Etaalpha, 13Calpha, 13C' and 13Cbeta nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major alpha-helices are identified: Ala13-Phe23, Gly33-Ala50, Leu54-Asp63, Val76-Leu90, while residues Ala2-Leu6 form a fifth, flexible helical segment. Two short beta-strands (Tyr30-Glu32, Lys72-Gly74) are found preceding helices B and D and are arranged in an anti-parallel interaction. Based on these data a structural model of calb I-II was constructed that shows that the construct adopts a tertiary structure related to other well-described calcium-binding proteins of the EF-hand family. Surprisingly, the protein forms a homodimer in solution, as was shown by its NMR characterization, size-exclusion chromatography and analytical ultra-centrifugation studies.
Asunto(s)
Proteína G de Unión al Calcio S100/química , Secuencia de Aminoácidos , Animales , Química Encefálica , Calbindina 1 , Calbindinas , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , Estructura Secundaria de Proteína , RatasRESUMEN
The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the interleukin-6-type cytokines. It is a member of the cytokine-receptor superfamily predicted to consist of six domains in its extracellular part. The second and third domain constitute the cytokine-binding module defined by a set of four conserved cysteines and a WSXWS motif, respectively. The three-dimensional structure of the carboxy-terminal domain of this region was determined by multidimensional NMR. The domain consists of seven beta-strands constituting a fibronectin type III-like topology. The structure reveals that the WSDWS motif of gp130 is part of an extended tryptophan/arginine zipper which modulates the conformation of the CD loop.
Asunto(s)
Antígenos CD/química , Glicoproteínas de Membrana/química , Receptores de Citocinas/química , Secuencia de Aminoácidos , Receptor gp130 de Citocinas , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
A simple, sensitivity-enhanced HSQC experiment is described which separates the upfield and downfield components in the indirect dimension into different subspectra. The sequence is similar to the generalized TROSY scheme; however, decoupling of the X-nucleus is used during detection. A detailed analysis of relaxation effects, precision and sensitivity of the method is presented. The approach is demonstrated in a two-dimensional water flip-back 1H-15N HSQC which measures 1JHN splittings in isotropic and oriented samples of ubiquitin and the hepatitis C protease. The results are in excellent agreement with splittings obtained from a conventional 1H-coupled HSQC.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Matemática , ProtonesRESUMEN
Structural restraints from residual tensorial couplings in high resolution NMR are usually incorporated into molecular structure calculation programs by an energy penalty function which depends on the knowledge of the alignment tensor. Here, we show that the alignment tensor enters in linear form into such a function. Therefore, the explicit appearance of the alignment tensor can be eliminated from the penalty function. This avoids the necessity of a determination of magnitude and rhombicity of the alignment tensor in the absence of structural information. The price for this procedure is a slightly shallower energy landscape. Simulations in the vicinity of the energy minimum for the backbone of human ubiquitin show that the reduction in curvature is on the order of a few percent.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/química , Estructura Secundaria de Proteína , Factores de Transcripción , Secuencia de Aminoácidos , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Factores de Transcripción NFI , Proteínas Nucleares , Proteínas Recombinantes de Fusión/química , Proteína 1 de Unión a la Caja YAsunto(s)
Espectroscopía de Resonancia Magnética/métodos , Conformación Proteica , Proteínas/química , Calcineurina/química , Productos del Gen nef/química , VIH-1/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
The tendency of HIV-1 Nef to form aggregates in solution, particularly at pH values below 8, together with its large fraction of highly mobile residues seriously complicated determination of its three-dimensional structure, both for heteronuclear solution NMR (Grzesiek et al., 1996a, Nat Struct Biol 3:340-345) and for X-ray crystallography (Lee et al., 1996, Cell 85:931-942). Methods used to determine the Nef structure by NMR at pH 8 and 0.6 mM concentration are presented, together with a detailed description of Nef's secondary and tertiary structure. The described techniques have general applicability for the NMR structure determination of proteins that are aggregating and/or have limited stability at low pH values. Extensive chemical shift assignments are reported for backbone and side chain 1H, 13C, and 15N resonances of the HIV-1 Nef deletion mutants NEF delta 2-39, NEF delta 2-39, delta 159-173, and of NEF delta 2-39, delta 159-173 in complex with the SH3 domain of the Hck tyrosine protein kinase. Besides a type II polyproline helix, Nef's structure consists of three alpha-helices, a 3(10) helix, and a five-stranded anti-parallel beta-sheet. The analysis of 15N relaxation parameters of the backbone amide sites reveals that all the secondary structure elements are non-mobile on the picosecond to nanosecond and on the millisecond time scale. A large number of slowly exchanging amide protons provides evidence for the stability of the Nef core even on the time scale of hours. Significant internal motions on the ps to ns time scale are detected for residues 60 to 71 and for residues 149 to 180, which form solvent-exposed loops. The residues of the HIV-1 protease cleavage site (W57/L58) do not exhibit large amplitude motions on the sub-nanosecond time scale, and their side chains insert themselves into a hydrophobic crevice formed between the C-terminus of helix 1 and the N-terminus of helix 2. A refined structure has been determined based on additional constraints for side-chain and backbone dihedral angles derived from a large number of three-bond J-coupling and ROE data.