首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Using proton magnetic resonance, we have investigated the structure and the base-pair opening kinetics of the d-(AATTGCAATT) self-complementary duplex. All the non-exchangeable (except H5',5") and most exchangeable proton resonances have been assigned. The structure belongs to the B family. Imino proton exchange, measured by line broadening, longitudinal relaxation and magnetization transfer from water, is catalyzed by proton acceptors. The base-pair lifetimes, obtained by extrapolation of the exchange times to infinite concentration of ammonia are 2 and 3 milliseconds for internal A.Ts and 18 ms for G.C at 15 degrees C. In the absence of added catalysts, the imino proton of the first A.T base pair exchanges faster than that of the unpaired thymidine of the duplex formed by the sequence d-(AATTGCAATTT). This gives strong evidence for intrinsic exchange catalysis. The exchange of adenine amino protons from the closed state has been observed. Hence amino proton exchange is ill-suited for the investigation of base-pair opening kinetics.  相似文献   

2.
We present the results of studies of an aqueous sample of a highly {15N,2H} enriched protein, the SH3 domain from Fyn. Measurements of 1H relaxation and interactions between H2O solvent and exchangeable protons are given, as well as a method for increasing the effective longitudinal relaxation of solvent exchangeable proton resonances. The long-range isotope shifts are measured, for 1H and 15N, which arise due to perdeuteration. Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time series of 2D NOESY datasets for some resonances. The agreement between experiment and simulation suggest that, with this 1H dilute sample, relatively long mixing times (up to 1.2 s) can be used to detect specific dipolar interactions between amide protons up to about 7Å apart. A set of 155 inter-amide NOEs and 7 side chain NOEs were thus identified in a series of 3D HSQC-NOESY-HSQC experiments. These data, alone and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR. These results are compared to the available X-ray and NMR structures of the Fyn SH3 domain.  相似文献   

3.
TROSY-type optimization of liquid-state NMR experiments is based on the preservation of unique coherence transfer pathways with distinct transverse relaxation properties. The broadband decoupling of the 1H spins interchanges the TROSY and anti-TROSY magnetization transfer pathways and thus is not used in TROSY-type triple resonance experiments or is replaced with narrowband selective decoupling. To achieve the full advantage of TROSY, the uniform deuteration of proteins is usually required. Here we propose a new and general method for 1H broadband decoupling in TROSY NMR, which does not compromise the relaxation optimization in the 15N–1H moieties, but uniformly and efficiently refocuses the 1 J CH scalar coupling evolution in the 13C–1H moieties. Combined with the conventional 2H decoupling, this method enables obtaining high sensitivity TROSY-type triple resonance spectra with partially deuterated or fully protonated 13C,15N labeled proteins.  相似文献   

4.
The effect of deuteration on the 13C linewidths of U-13C, 15N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in 2H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by <0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the C delta, C gamma 1, and C gamma 2 Ile 13C signals observed in the direct, 21-39 ppm, and indirect, 9-17 ppm, dimensions). The measured 13C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies. 1) decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power 1H decoupling is required.  相似文献   

5.
The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.  相似文献   

6.
J D Puglisi  J R Wyatt  I Tinoco 《Biochemistry》1990,29(17):4215-4226
The hairpin conformation adopted by the RNA sequence 5'GCGAUUUCUGACCGCC3' has been studied by one- and two-dimensional NMR spectroscopy. Exchangeable imino spectra in 60 mM Na+ indicate that the hairpin has a stem of six base pairs (indicated by boldface type) and a loop of three nucleotides. NOESY spectra of nonexchangeable protons confirm the formation of the stem region. The duplex has an A-conformation and contains an A.C apposition; a G.U base pair closes the loop region. The stem nucleotides have C3'-endo sugar conformations, as expected of an A-form duplex, whereas the three loop nucleotides adopt C2'-endo sugar puckers. Stacking within the loop, C8 upon the sugar of U7, stabilizes the structure. The pH dependence of both the exchangeable and nonexchangeable NMR spectra is consistent with the formation of an A+.C base pair, protonated at the N1 position of adenine. The stability of the hairpin was probed by using absorbance melting curves. The hairpin structure with the A+.C base pair is about +2 kcal/mol less stable in free energy at 37 degrees C than the hairpin formed with an A.U pair replacing the A+.C pair.  相似文献   

7.
The effect of deuteration on the 13C linewidths of U-13C, 15N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in 2H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by < 0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the Cδ, Cγ1, and Cγ2 Ile 13C signals observed in the direct, 21-39 ppm, and indirect, 9-17 ppm, dimensions). The measured 13C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies. 1H decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power 1H decoupling is required.  相似文献   

8.
The 1H, 13C, and 15N high field nuclear magnetic resonance spectra of the cyclic peptide viomycin have been fully assigned using homo- and heteronuclear double resonance experiments and pH effects. In addition it is shown how the two- and three-bond H-D isotope effects upon carbonyl resonances may assist in their assignment. The resistance to exchange with solvent water of the amide proton involved in the transannular hydrogen bond is observed directly in the 1H spectra, via the isotope effect on a carbonyl resonance in the 13C spectra, and via the one-bond 1H couppling in the 15N spectra.  相似文献   

9.
A 500-MHz 1H-NMR study on a double-stranded non-self-complementary DNA undecamer comprising a portion of the specific target site for the cyclic AMP receptor protein in the gal operon is presented. Using pre-steady-state nuclear Overhauser effect (NOE) measurements, all exchangeable imino, non-exchangeable base, methyl, and H1', H2' and H2" sugar proton resonances are assigned in a sequential manner. In addition, some of the H3' sugar proton resonances are also assigned and some of the exchangeable amino proton resonances identified. The relative magnitudes of the intranucleotide and internucleotide NOEs are indicative of a right-handed B-type conformation for the duplex undecamer in solution.  相似文献   

10.
A De Marco  M Llinás 《Biochemistry》1979,18(18):3846-3854
Polypeptides and proteins in native conformation exhibit 13C NMR spectra which are highly nondegenerate. Assignment of resonances to carbons in particular residues is hence a prerequisite for a structural analysis of the spectroscopic data. For nonprotonated carbonyl carbons, the assignment can be achieved by selective (1H alpha)13C' 2J decoupling. Using this method, we have assigned the Orn1 and Gly2 carbonyl resonances in alumichrome at 67.9 MHz. We show that a single off-resonance experiment with the decoupling frequency centered in the aliphatic proton spectrum is sufficient to assign unequivocally all the protonated carbon resonances via analysis of the reduced 1J heteronuclear splittings. Alumichrome thus becomes the first complex polypeptide spin system whose 1H, 15N, and now 13C nuclear resonances have been fully identified to date. 13C chemical shifts and 1H--13C spin--spin couplings are discussed in terms of structural strain leading to specific orbital hybridizations and on the basis of polarization effects due to electron density shifts toward hydrogen-bonding and metal-binding sites. A number of 3J(13C--C--C--1H) coupling constants measured on selected multiplets after resolution enhancement were used to derive the x-related Karplus relationship 3J(theta) = (10.2 cos2 theta -- 1.3 cos theta + 0.2) Hz.  相似文献   

11.
High-resolution proton nuclear magnetic resonance spectroscopy and nuclear Overhauser effects for the low-field exchangeable proton resonances of human normal adult hemoglobin in aqueous solvents are being used to confirm and extend the assignments of these resonances to specific protons at the intersubunit interfaces of the molecule. Most of these exchangeable proton resonances of human normal adult hemoglobin have been found to be absent in the spectra of isolated alpha or beta subunits. This finding indicates that they are specific spectral markers for the quaternary structure of the hemoglobin tetramer. Based on the nuclear Overhauser effect results, we have assigned the exchangeable proton resonance at +7.4 ppm downfield from H2O to the hydrogen-bonded proton between alpha 103(G10)His and beta 108(G10)Asn at the alpha 1 beta 1 interface. The nuclear Overhauser effect results have also confirmed the assignments of the exchangeable proton resonances at +9.4 and +8.2 ppm downfield from H2O previously proposed by workers in this laboratory based on a comparison of human normal adult hemoglobin and appropriate mutant hemoglobins. This independent confirmation of previously proposed assignments is necessary in view of the possible long-range conformational effects of single amino-acid substitutions in mutant hemoglobin molecules.  相似文献   

12.
The secondary structure of a recently identified ATP-binding RNA aptamer consists of apurine-rich 11-residue internal loop positioned opposite a single guanine bulge flanked oneither side by helical stem segments. The ATP ligand targets the internal loop and bulgedomains, inducing a structural transition in this RNA segment on complex formation.Specifically, 10 new slowly exchanging proton resonances in the imino, amino and sugarhydroxyl chemical shift range are observed on AMP–RNA aptamer complex formation.This paper outlines site-specific labeling approaches to identify slowly exchanging imino(guanine) and amino (guanine and adenine) protons in internal loop and bulge segments ofcompact RNA folds such as found in the AMP–RNA aptamer complex. One approachincorporates 15N-labeled guanine (N1 imino and N2 amino positions) and 15N-labeledadenine (N6 amino position), one residue at a time, in the AMP-binding RNA aptamer, withlabeling incorporation through chemical synthesis facilitated by generating the aptamer fromtwo separate strands. The unambiguous assignments deduced from the 15N labeling studieshave been verified from an independent labeling strategy where individual guanines in theinternal loop have been replaced, one at a time, by inosines and assignments were made onthe basis of the large 2 ppm downfield shift of the guanine imino protons on inosinesubstitution. The strengths and limitations of the inosine-for-guanine substitution approachemerge from our studies on the AMP–RNA aptamer complex. The assignment of theinternal loop and bulge imino and amino protons was critical in our efforts to define thesolution structure of the AMP–RNA aptamer complex since these slowly exchangingprotons exhibit a large number of long-range intramolecular NOEs within the RNA, as wellas intermolecular NOEs to the AMP in the complex. The current application of specific 15Nand inosine labeling approaches for exchangeable imino and amino proton assignments in thenonhelical segments of an RNA aptamer complex in our laboratory complements selective 2Hand 13C approaches to assign nonexchangeable base and sugar protons in RNA andligand–RNA complexes reported in the literature.  相似文献   

13.
The assignment of amide resonances in the two-dimensional PISEMA (Polarization Inversion with Spin Exchange at the Magic Angle) spectrum of uniformly 15N labeled M2 peptide corresponding to the channel-lining segment of the acetylcholine receptor in oriented phospholipid bilayers is described. The majority of the resonances were assigned through comparisons with spectra from selectively 15N labeled recombinant peptides and specifically 15N labeled synthetic peptides. Some resonances were assigned to specific amino acid residues by means of homonuclear 15N spin-exchange spectroscopy. A modification to the conventional spin-exchange pulse sequence that significantly shortens the length of the experiments by combining the intervals for 15 N spin-exchange and 1H magnetization recovery is described.  相似文献   

14.
Fast magic-angle spinning and partial sample deuteration allows direct detection of 1H in solid-state NMR, yielding significant gains in mass sensitivity. In order to further analyze the spectra, 1H detection requires assignment of the 1H resonances. In this work, resonance assignments of backbone HN and Hα are presented for HET-s(218–289) fibrils, based on the existing assignment of Cα, Cβ, C’, and N resonances. The samples used are partially deuterated for higher spectral resolution, and the shifts in resonance frequencies of Cα and Cβ due to the deuterium isotope effect are investigated. It is shown that the deuterium isotope effect can be estimated and used for assigning resonances of deuterated samples in solid-state NMR, based on known resonances of the protonated protein.  相似文献   

15.
A technique for proton labelling of selected amino acids in deuterated calbindin D9K, heterologously expressed in E. coli, was developed in order to simplify and obtain higher resolution in 1H-NMR spectra. The spectra from two double-labelling experiments, Val plus Ser and Val plus Leu, when compared to the uniformly protonated protein showed a dramatically simpler pattern with low background signals and gave considerably sharper resonances due to reduced relaxation rates in the deuterated proteins. The selective proton labelling technique will enable detailed and rapid analysis of interesting domains of proteins and will also make the analysis of larger proteins feasible.  相似文献   

16.
Summary A triple resonance HNC-TOCSY-CH experiment is described for correlating the guanosine imino proton and H8 resonances in 13C-/15N-labeled RNAs. Sequential assignment of the exchangeable imino protons in Watson-Crick base pairs is generally made independently of the assignment of the nonexchangeable base protons. This H(NC)-TOCSY-(C)H experiment makes it possible to unambiguously link the assignment of the guanosine H8 resonances with sequential assignment of the guanosine imino proton resonances. 2D H(NC)-TOCSY-(C)H spectra are presented for two isotopically labeled RNAs, a 30-nucleotide lead-dependent ribozyme known as the leadzyme, and a 48-nucleotide hammerhead ribozyme-RNA substrate complex. The results obtained on these two RNAs demonstrate that this HNC-TOCSY-CH experiment is an important tool for resonance assignment of isotopically labeled RNAs.  相似文献   

17.
A Lapidot  C S Irving 《Biochemistry》1979,18(4):704-714
The proton-decoupled 9.12 MHz 15N NMR spectra of 15N-labeled Bacillus subtilis, Bacillus licheniformis, Staphylococcus auresu, Streptococcus faecalis, and Micrococcus lysodeikticus intact cells, isolated cells walls, and cell wall digests have been examined. The general characteristics of Gram-positive bacteria 15N NMR spectra and described and spectral assignments are provided, which allow in vivo 15N NMR to be applied to a wide range of problems in bacterial cell wall research. The qualitative similarity of the intact cell and cell wall spectra found in each bacteria allowed the 15 N resonances observed in the proton broad-band noise-decoupled 15N NMR spectra of intact cells to be assigned to cell wall components. Each of the five Gram-positive bacteria displayed a unique set of cell wall 15N resonances, which reflected variations in the primary structure of peptidoglycans and the amounts of teichoic acid and teichuronic acid in the cell wall, as well as the dynamic properties of the cell wall polymers. Spectral assignments of cell wall 15 N resonances assigned to teichoic D-Ala residues, teichuronic acid and acetamido groups, and peptidoglycan acetamido, amide, peptide, and free amino groups have been made on the basis of specific isotopic labeling and dilution experiments, comparison of chemical shifts to literature values, determination of pH titration shifts, cell wall fractionation experiments, and comparative analysis of the cell wall lysozyme digest spectra in terms of the known primary sequences of peptide chains. All the peptidoglycan 15N peptide resonances observed in the intact cells and isolated cell walls could be accounted for by residues in the bridge or crossbar regions of the peptide chains, which indicated that only the cross-linking groups had a high degree of motional freedom. Thermal- and pH-induced conformational changes around the cross-linking D-Ala residues were detected in the B. licheniformis cell wall lysozyme digest products. Comparison of the proton broad-band noise-decoupled and gated decoupled intact cell and cell wall 15N spectra indicated that broad-band proton decoupling resulted in nulling of cytoplasmic resonances and enhancement of the cell wall resonances by the 15N [1H5 nuclear Overhauser effect.  相似文献   

18.
As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin (Mr 12,000) uniformly labeled with 15N has been performed. The 15N chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using 15N-1H correlated and two-dimensional heteronuclear single-quantum coherence (HSQC) TOCSY and NOESY spectra. The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear 1H spectra. The differences in the 15N chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin.  相似文献   

19.
Summary The advent of methods for preparing 15N- and 13C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studies by NMR spectroscopy. A practical limitation is the expense of the 13C label. It may therefore sometimes be desirable to prepare a relatively inexpensive 15N-labeled sample only. Here we show that the two-bond 1H-15N HSQC experiment can be used on 15N-labeled RNA to correlate the intranucleotide H1 and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected 1H-15N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RNA oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1 and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.  相似文献   

20.
15N and 1H NMR studies of Rhodospirillum rubrum cytochrome c2   总被引:1,自引:0,他引:1  
L P Yu  G M Smith 《Biochemistry》1988,27(6):1949-1956
15N-Enriched cytochrome c2 was purified from Rhodospirillum rubrum that had been grown on 15NH4Cl, and the diamagnetic iron(II) form of the cytochrome was studied by 15N and 1H NMR spectroscopy. 15N resonances of the four pyrrole nitrogens, the ligand histidine nitrogens, the highly conserved tryptophan indole nitrogen, and some proline nitrogens are assigned. The resonances of the single nonligand histidine are observed only at low pH because of severe broadening produced by proton tautomerization. The resonances of exchangeable protons bonded to the nitrogens of the ligand histidine, the tryptophan, and some amide groups are also assigned. The exchange rates of the nitrogen-bound protons vary greatly: most have half-lives of less than minutes, the indolic NH of Trp-62 exchanges with a half-time of weeks, and the ligand histidine NH proton exchanges with a half-time of months. The latter observation is indicative of extreme exclusion of solvent from the area surrounding the ligand histidine and lends credence to theories implicating the degree of hydrophobicity in this region as an important factor in adjusting the midpoint potential. The dependence of the 15N and 1H NMR spectra of ferrocytochrome c2 on pH indicates neither the Trp-62 nor the ligand His side chains become deprotonated to any appreciable extent below pH 9.5. The His-18 NH remains hydrogen bonded, presumably to the Pro-19 carboxyl group, throughout the pH titrations. Because neither deprotonated nor non-hydrogen-bonded forms of His-18 are observed in spectra of the ferrocytochrome, the participation of such forms in producing a heterogeneous population having different g tensor values seems unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号