RESUMO
DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pKa of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 1HN, 15N, 13C(alpha) 13CO, 1H(alpha), and 13Cbeta NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety-nine percent of the frequencies were assigned using a combination of triple (1H-13C-15N) and double resonance (1H-15N or 1H-13C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, 3(J)H(N)H(alpha) and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro 151, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large 13C(alpha) chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.
Assuntos
Escherichia coli/enzimologia , Isomerases de Dissulfetos de Proteínas/química , Proteínas de Bactérias/química , Sítios de Ligação , Dissulfetos/metabolismo , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Oxirredução , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
The backbone dynamics of the major coat protein (gVIIIp) of the filamentous bacteriophage M13, solubilized in detergent micelles, have been studied using 15N nuclear magnetic resonance spectroscopy at three frequencies. Motional parameters and overall and internal correlation times were derived with the model-free approach. It was also checked whether these parameters had to be modified due to anisotropic motion of the protein/micelle complex. Reduced spectral density mapping was used to calculate the spectral densities at J(O), J(omegaN), and [J(omegaH)]. The spectral densities were interpreted by mapping a linear or scaled linear combination of two Lorentzians onto a J(O)-J(omega) plot. The major coat protein of bacteriophage M13 consists of two alpha-helices, one of which is hydrophobic and located within the micelle, while the other is amphipathic and located on the surface of the micelle. Our results indicate that the motion of the hydrophobic helix is restricted such that it corresponds to the overall tumbling of the protein/micelle complex. The interpretation of the relaxation data of the amphipathic helix by means of the model-free approach and the reduced spectral density mapping indicate that in addition to the overall motion all residues in this helix are subject to motion on the fast nanosecond and picosecond time scales. The motions of the vectors in the low nanosecond range are characterized by similar values of the spectral densities and correlation times and represent the motion of the amphipathic helix on and away from the surface of the micelle. The relaxation data of the residues in the hinge region connecting the helices show that there is an abrupt change from highly restricted to less restricted motion. Both the C-terminal and N-terminal residues are very mobile.
Assuntos
Bacteriófago M13/química , Proteínas do Capsídeo , Capsídeo/química , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Detergentes , Matemática , Micelas , Fosforilcolina/análogos & derivados , Estrutura Secundária de Proteína , Dodecilsulfato de SódioRESUMO
Backbone dynamics of Savinase, a subtilisin of 269 residues secreted by Bacillus lentus, have been studied using 15N relaxation measurements derived from proton-detected dimensional 1H-15N-NMR spectroscopy. 15N spin-lattice rate constants (R1), spin-spin relaxation-rate constants(R2), and 1H-15N nuclear Overhauser effects (NOE) were determined for 84% of the backbone amide 15N nuclei. The model-free formalism [Lipari, G. & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559] was used to derive values for a generalized order parameter, S2, interpretable as a measure of the amplitude of motion on the picosecond-nanosecond timescale, for each N-H bond vector. Additional terms used to fit the data include an effective correlation time for internal motions (taue) and an exchange term (Rex) to account for exchange contributions to R2. The overall rotational correlation time (taum) is 9.59 +/- 0.02 ns; the average order parameter (S2) is 0.90 +/- 0.07, indicative of a rigid structure consistent with Savinase's high degree of secondary structure and compact tertiary fold. Residues S125-S128, located in the substrate-binding region, represent the longest stretch of protein which exhibits disorder on the picosecond-nanosecond timescale. These residues also exhibit significant exchange terms, possibly indicative of motion on the microsecond-millisecond timescale, which could also be influenced by the proximity of the phenyl ring of the substituted aryl boronic acid inhibitor used in this study. S103 and G219 in the substrate-binding region, represent the longest stretch of protein which exhibits disorder on the picosecond-nanosecond timescale. These residues also exhibit significant exchange terms, possibly indicative of motion on the microsecond-millisecond timescale, which could also be influenced by the proximity of the phenyl ring of the substituted aryl boronic acid inhibitor used in this study. S103 and G219 in the substrate-binding region also show flexibility on the picosecond-nanosecond timescale. There is also significant motion in the turn, G258-T260, of a small solvent-exposed loop region which may make the protein vulnerable autolysis at that point. Some residues in both calcium-binding sites and nearby also show mobility.
Assuntos
Serina Endopeptidases/química , Bacillus/enzimologia , Sítios de Ligação , Cálcio/metabolismo , Espectroscopia de Ressonância Magnética , Isótopos de Nitrogênio , Inibidores de Serina Proteinase/farmacologiaRESUMO
1H, 13C and 15N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques. With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date. Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure. The HNCO, HN(CO)-CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size. It was necessary to use several experiments to unambiguously determine a majority of the alpha-protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, 15N TOCSY-HMQC and 15N NOESY-HMQC experiments provided the H alpha chemical shifts. Correlations for glycine protons were absent from most of the spectra. A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al. [(1990) J. Am. Chem. Soc., 112, 9020-9022] in the seminal paper on heteronuclear backbone assignment. A major impediment to the linking process was the amount of overlap in the C alpha and H alpha frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C alpha chemical shifts and 'TOCSY ladders'. Ninety-four percent of the backbone resonances are reported for this subtilisin. The secondary structure was analyzed using 3D 15N NOESY-HMQC data and C alpha secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427-445] shows no major differences.
Assuntos
Bacillus/enzimologia , Conformação Proteica , Estrutura Secundária de Proteína , Serina Endopeptidases/química , Sequência de Aminoácidos , Isótopos de Carbono , Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Isótopos de Nitrogênio , Difração de Raios X/métodosRESUMO
The major coat protein (gVIIIp) of bacteriophage M13 complexed with SDS detergent micelles was used as a model system to study the lipid-bound conformation of the protein. Conditions were found that allowed the recording of good quality of NMR spectra. By making extensive use of three-dimensional heteronuclear (13C, 15N) NMR, we obtained a complete set of resonance assignments for 1HN, 1H alpha, 1H beta, 13C alpha, CO, and 15N and partially assigned the rest of the 1H spectrum. Analysis of NOE and chemical shift data reveals that gVIIIp is composed of two alpha-helical domains, one ranging from Pro-6 to Glu20 and the other ranging from Tyr-24 all the way to the C-terminus Ser-50. In contrast to the results reported by Henry and Sykes [Henry, G.D., & Sykes, B.D. (1992) Biochemistry 31, 5285-5297], at a high SDS to protein ratio the protein appears to be monomeric.
Assuntos
Proteínas do Capsídeo , Capsídeo/química , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Sequência de Aminoácidos , Bacteriófago M13/química , Bacteriófago M13/genética , Capsídeo/metabolismo , Escherichia coli/genética , Substâncias Macromoleculares , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Micelas , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Dodecilsulfato de Sódio , SolubilidadeRESUMO
The synthesis of an oligonucleotide labeled with 13C at the thymine methyl and 15N at the exocyclic amino groups of the cytosines is described. 13CH3I and 15NH4OH were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNA-binding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Espectroscopia de Ressonância Magnética , Oligodesoxirribonucleotídeos/síntese química , Receptores de Glucocorticoides/metabolismo , Animais , Sequência de Bases , Isótopos de Carbono , Marcação por Isótopo , Dados de Sequência Molecular , Isótopos de Nitrogênio , Oligodesoxirribonucleotídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Receptores de Glucocorticoides/genéticaRESUMO
The complex of the rat glucocorticoid receptor (GR) DNA binding domain (DBD) and half-site sequence of the consensus glucocorticoid response element (GRE) has been studied by two-dimensional 1H NMR spectroscopy. The DNA fragment is a 10 base-pair oligonucleotide, 5'd(GCTGTTCTGC)3'.5'd-(GCAGAACAGC)3', containing the stronger binding GRE half-site hexamer, with GC base pairs at each end. The 93-residue GR-DBD contains an 86-residue segment corresponding to residues 440-525 of the rat GR. Eleven NOE cross peaks between the protein and DNA have been identified, and changes in the chemical shift of the DNA protons upon complex formation have been analyzed. Using these protein-DNA contact points, it can be concluded that (i) the "recognition helix" formed by residues C460-E469 lies in the major groove of the DNA; (ii) the GR-DBD is oriented on the GRE half-site such that residues A477-D481, forming the so-called D-loop, are available for protein-protein interaction in the GR-DBD dimer on the intact consensus GRE; and (iii) the 5-methyl of the second thymine in the half-site and valine 462 interact, confirming indirect evidence [Truss et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7180-7184; Mader et al. (1989) Nature 338, 271-274] that both play an important role in GR-DBD DNA binding. These findings are consistent with the model proposed by Härd et al. [(1990) Science 249, 157-160] and the X-ray crystallographic complex structure determined by Luisi et al. [(1991) Nature 352, 497-505].
Assuntos
DNA/metabolismo , Receptores de Glucocorticoides/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Modelos Moleculares , Dados de Sequência Molecular , Difração de Raios XRESUMO
Sequential assignments and secondary structural analysis have been accomplished for the 113-residue apoprotein of the antitumor drug neocarzinostatin (NCS) from Streptomyces carzinostaticus. A total of 98% of the main-chain and 77% of the side-chain resonances have been sequence specifically assigned by use of information from coherence transfer experiments and by sequential and interstrand NOEs. Because of the complexity of the NCS spectrum, several sequential assignment strategies were employed to complete the analysis. Apo-NCS consists of three antiparallel beta-sheeted domains by NMR analysis. There is an extensive four-strand antiparallel beta-sheet, and two two-stranded domains. One of the two-strand domains is contiguous, S72-N87, with chain reversal occurring through the region L77-R82. The other two-stranded domain has the section G16-A24 antiparallel with respect to the region S62-R70. This secondary structure is consistent with the crystal structure of holo-NCS at 2.8-A resolution.
Assuntos
Apoproteínas/química , Zinostatina/química , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , SoluçõesRESUMO
Using two-dimensional isotropic mixing spectroscopy all 5'/5" proton resonances of the EcoRI restriction site DNA dodecamer [d(CGCGAATTCGCG)]2 have been assigned. This completes the previous assignments of 1'H to 4'H resonances of the deoxyribose spin systems (Hare et al., 1983). With mixing times of up to 500 ms, many of these resonances showed connectivities of 5'/5" protons in the two-dimensional isotropic mixing spectrum. Relying only on through-bond connectivities makes these assignments independent of assumptions about the conformation of the DNA oligonucleotide. The assignment of the 5'H/5"H resonances will allow the interpretation of intra- and interresidue NOEs to these protons, providing information about the DNA backbone conformation.
Assuntos
Sequência de Bases , DNA , Desoxirribonuclease EcoRI , Conformação de Ácido Nucleico , Desoxirribose , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , PrótonsRESUMO
Reversed-phase high-performance liquid chromatography has been used to purify to homogeneity two different lymphokines. Human IL-2 was purified on a C8 reversed-phase column in pyridine-acetate-propanol followed by chromatography on a C18 reversed-phase column in trifluoroacetic acid-acetonitrile. Protein sequence analysis of in situ-generated cyanogen bromide peptides obtained from this preparation established the homogeneity of this material and confirmed the amino acid sequence predicted from the published DNA sequence. Murine CSF-2 alpha was purified on a C18 reversed-phase column in trifluoroacetic acid-acetonitrile followed by chromatography on the same column in pyridine-acetate-propanol. The final preparation yielded a single band on a sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 24,500.