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1.
Two-dimensional NMR investigation of a bent DNA fragment: assignment of the proton resonances and preliminary structure analysis. 总被引:11,自引:8,他引:3
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The resonances of all the non-exchangeable protons (except 5'H and 5"H) of d(CGAAAAATCGG) + d(CCGATTTTTCG), a putatively bent DNA duplex, have been assigned using 1H two-dimensional nuclear magnetic resonance methods. The nuclear Overhauser effect data indicate an overall B-form structure for this double-helical DNA undecamer. However, several features of the NMR data such as some unusually weak C8/C6 proton to C1' proton NOE cross-peaks, the presence of relatively intense C2H to C1'H NOE cross-peaks, and unusual chemical shifts of some 2", 2', and 1' protons suggest a substantial perturbation of the helix structure at the junctions and along the length of the tract of A residues. These structural deviations are considered in terms of models of DNA bending. 相似文献
2.
The B and Z forms of the d(m5C-G)3 and d(br5C-G)3 hexamers in solution. A 300-MHz and 500-MHz two-dimensional NMR study 总被引:3,自引:0,他引:3
L P Orbons G A van der Marel J H van Boom C Altona 《European journal of biochemistry》1986,160(1):131-139
The non-exchangeable proton resonances of the hexadeoxynucleoside pentakisphosphates d(m5C-G)3 and d(br5C-G)3 in the B form as well as in the Z form were assigned by means of two-dimensional correlated spectroscopy and two-dimensional nuclear Overhauser enhancement spectroscopy. The complete proton NMR spectrum of the B form of the methylated compound was assigned in a pure 2H2O solution as well as in a 2H2O/C2H3O2H mixed solvent, containing 5 mM MgCl2. In the latter solvent the B form occurs in slow equilibrium (on the NMR time scale) with the Z form, the resonances of which also were fully assigned. The proton resonances of the B and Z forms of the brominated fragment were assigned in a 2H2O/C2H3O2H solution containing 5 mM MgCl2. A new and general method is described for the sequential assignment of the non-exchangeable proton resonances of oligonucleotides in the Z form. 相似文献
3.
J Kemmink R Boelens T M Koning R Kaptein G A van der Marel J H van Boom 《European journal of biochemistry》1987,162(1):37-43
In order to obtain insight into the repair mechanism of DNA containing thymine photo-dimer, the conformation of the duplex d(GCGTTGCG) x d(CGCAACGC) with a thymine dimer incorporated has been studied by proton NMR and the results are compared with NMR data of the parent octamer. Two-dimensional nuclear Overhauser enhancement (2D NOE) spectroscopy and two-dimensional homonuclear Hartmann-Hahn spectroscopy have been applied to assign all the non-exchangeable base protons and most of the deoxyribose protons of both duplexes. From these experiments it is clear that indeed a cis-syn cyclobutane-type thymine photodimer is formed by the irradiation of this oligonucleotide with ultraviolet light. Comparison of 2D NOE spectra and the 1H chemical shifts of the damaged and the intact DNA duplexes reveals that formation of a thymine dimer induces small distortions of the B-DNA structure, the main conformational change occurring at the site of the thymine dimer. 相似文献
4.
J G Collins 《Biochemistry international》1988,16(5):819-828
The non-exchangeable base and sugar protons of the dodecanucleotide d(CAATCCGGATTG) have been assigned by the combined use of one and two-dimensional NMR spectroscopy. The sequential assignments of the base and sugar protons have been obtained using two dimensional nuclear Overhauser effect and homonuclear shift correlated spectra. By analysis of the nuclear Overhauser effect data it was determined that the dodecanucleotide assumes a right handed B-type helix in the aqueous medium used in this study. 相似文献
5.
Induction of B-A transitions of deoxyoligonucleotides by multivalent cations in dilute aqueous solution. 总被引:4,自引:1,他引:3
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Circular dichroism (CD) spectra of d(CCCCGGGG) in the presence of Co(NH3)6(3+) are very similar to spectra of r(CCCCGGGG). In contrast, B-form characteristics are observed for d(CCCCGGGG) in the presence of Na+ and Mg2+, even at high salt concentrations. Spermidine induces modest changes of the CD of d(CCCCGGGG). The NMR chemical shifts of the nonexchangeable protons of d(CCCCGGGG) in the absence and presence of Co(NH3)6(3+) were assigned by proton two-dimensional (2D) NOESY and COSY measurements. The chemical shifts of the GH8 protons of d(CCCCGGGG) move upfield upon titration with Co(NH3)6Cl3. The sums of the sugar H1' coupling constants decrease with added Co(NH3)6Cl3. Cross peak intensities in the 2D proton NOESY spectra show a transformation from B-DNA to A-DNA characteristics upon the addition of Co(NH3)6Cl3. The temperature-dependent 59Co transverse and longitudinal relaxation rates demonstrate that Co(NH3)6(3+) is site-bound to the oligomer. Such localization is not a general feature of Co(NH3)6(3+) binding to oligonucleotides. 59Co NMR relaxation and CD measurements demonstrate chiral discrimination by d(CCCCGGGG) for the two stereoisomers of Co(en)3(3+). Both stereoisomers bind tightly as judged by 59Co NMR, and both cause large (but nonequivalent) changes in the CD of this oligomer. 相似文献
6.
1H NMR studies of tris(phenanthroline) metal complexes bound to oligonucleotides: characterization of binding modes 总被引:5,自引:0,他引:5
The binding of Ru(phen)3(2+), Rh(phen)3(3+), and Co(phen)3(3+) to the oligonucleotides d(GTGCAC)2 and 5'-pd(CGCGCG)2 has been examined by 1H NMR spectroscopy as a function of temperature, concentration, and chirality of the metal complex. The duplex oligonucleotides act as chiral shift reagents for the metal complexes; phenanthroline protons associated with each enantiomer are resolved upon binding to the oligomer. The spectral titrations, consistent with photophysical studies, indicate that the complexes bind to the oligomer through two modes: one assigned as intercalation favoring the delta-isomer, and the other assigned as the surface-bound interaction favoring the lambda-isomer. The ligand protons are perturbed in a manner that implies sensitivity of particular protons to binding mode; specifically, the H4,7 protons appear to be altered most for the lambda-enantiomer while the H5,6 protons are perturbed more for the delta-enantiomer. The NMR chemical shift variations appear particularly sensitive to this surface-bound interaction, which, on the basis of a comparison of binding and photophysical parameters for Ru(phen)3(2+), appears more prominant in binding to oligonucleotides than that to polynucleotides. With respect to oligonucleotide proton shifts, the adenine H2 proton, positioned in the minor groove of the helix, shows the largest upfield shifts with metal binding, and more dramatically with lambda-isomers. The major groove thymine methyl protons (TMe) shift downfield to a lesser extent, and more so for delta-isomers. The different binding modes also differ with respect to their dynamics of association; the longitudinal relaxation rates of delta- and lambda-4,7 phenanthroline protons of Rh(phen)3(3+) are 0.88 and 1.14 s, respectively, in the presence of d(GTGCAC)2.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
J M Schmidt H Thüring A Werner H Rüterjans R Quaas U Hahn 《European journal of biochemistry》1991,197(3):643-653
Uniformly 15N-enriched ribonuclease T1 (RNase T1) was obtained from Escherichia coli by recombinant techniques. Heteronuclear 1H, 15N-shift correlation spectra were recorded utilizing proton detection. Direct 1H, 15N connectivities were established applying the heteronuclear multiple-quantum coherence technique. Additional 1H, 1H-TOCSY or 1H, 1H-NOESY transfer steps allowed for sequential assignments. Nitrogen atoms without directly bonded protons were detected by means of the heteronuclear multiple-bond correlation experiment. Signals emerging from 15NH and 15NH2 groups were distinguished by heteronuclear triple-quantum filtering methods. 119 nitrogen resonances out of the expected 127 were assigned unambiguously; in addition, previously obtained proton assignments were extended. Preliminary 1H, 15N NMR investigation were performed on the RNase-T1-3'GMP inhibitor complex. Results were interpreted with respect to nucleotide binding. 相似文献
8.
Davies DB Eaton RJ Baranovsky SF Veselkov AN 《Journal of biomolecular structure & dynamics》2000,17(5):887-901
500 MHz NMR spectroscopy has been used to investigate the complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary deoxytetranucleotides, 5'-d(CGCG), 5'-d(GCGC), 5'-d(TGCA), 5'-d(ACGT) and 5'-d(AGCT), of different base sequence in aqueous salt solution. 2D homonuclear 1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H - 31P NMR spectroscopy (HMBC) have been used for complete assignment of the non-exchangeable protons and the phosphorus resonance signals, respectively, and for a qualitative determination of the preferred binding sites of the drug. Analysis shows that DAU intercalates preferentially into the terminal sites of each of the tetranucleotides and that the aminosugar of the antibiotic is situated in the minor groove of the tetramer duplex, partly eclipsing the third base pair. A quantitative determination of the complexation of DAU with the deoxytetranucleotides has been made using the experimental concentration and temperature dependences of the drug proton chemical shifts; these have been analysed in terms of the equilibrium reaction constants, limiting proton chemical shifts and thermodynamical parameters (enthalpies deltaH, entropies deltaS) of different drug-DNA complexes (1:1, 1:2, 2:1, 2:2) in aqueous solution. It is found that DAU interacts with sites containing three adjacent base pairs but does not show any significant sequence specificity of binding with either single or double-stranded tetranucleotides, in contrast with other intercalating drugs such as proflavine, ethidium bromide and actinomycin D. The most favourable structures of the 1:2 complexes have been derived from the induced limiting proton chemical shifts of the drug in the intercalated complexes with the tetranucleotide duplex, in conjunction with 2D NOE data. It has been found that the conformational parameters of the double helix and the orientation of the DAU chromophore in the intercalated complexes depend on base sequence at the binding site of the tetramer duplexes in aqueous solution. 相似文献
9.
J H den Hartog C Altona J H van Boom G A van der Marel C A Haasnoot J Reedijk 《Journal of biomolecular structure & dynamics》1985,2(6):1137-1155
The structural distortion of a single- and a double-stranded decadeoxynucleotide upon binding of cis-PtCl2(NH3)2 was studied by 1H-NMR. After selective platination of d(T-C-T-C-G-G-T-C-T-C) (I) at the central d(-GpG-) site (resulting in I-Pt), several non-exchangeable base protons as well as H1', H2', H2" and H3' protons could be assigned by means of conventional NMR double-resonance techniques. Addition of the complementary decamer strand to I and I-Pt yielded the double-stranded III and III-Pt, respectively. All non-exchangeable base, H1', and most of the H2' and H2" protons in the two double stranded compounds could be assigned using 2D-chemical shift correlation (COSY) and nuclear Overhauser enhancement (NOESY) techniques. The double stranded compound III appears to adopt a B-DNA like structure. Comparison of NOEs and proton-proton coupling constants in the d(-GpG-).cisPt part in I-Pt and III-Pt reveals that their structure displays large similarity. Significant chemical shift changes (i.e. larger than 0.1 ppm) between III and III-Pt are restricted to the central four base pairs. It follows that the outer three base pairs, located on either side of the central four base pairs in III-Pt are likely to adopt a regular B-DNA type helix. The observed large upfield and downfield chemical shifts in the d(-CpGpG-) part of III with respect to III-Pt can be rationalized by describing the distortion of the double helix as a kink. A discussion of the observed physical effects upon platination of a double-stranded oligonucleotide is presented. 相似文献
10.
The molecular structure of 3,3-difluoro-5 alpha-androstane-17 beta-ol acetate was analyzed by 1H, 13C, and 19F nuclear magnetic resonance (NMR) techniques; two-dimensional NMR was used to assigned 1H and 13C resonances. The 1H NMR spectrum in deuterated chloroform shows three sharp singlets (delta = 0.74, 0.79, and 2.00 ppm) integrating for three protons each, an isolated triplet at 4.55 ppm integrating for one proton, and overlapping multiplets between 0.72 and 2.12 ppm integrating for 31 protons. The 13C spectrum shows 18 resonances between 10 and 55 ppm, and three additional resonances at 82.9, 124.0, and 171.5 ppm. The 19F[1H] spectrum shows two sets of doublets (observed 2J = 150 Hz) at 5.00 and -4.80 ppm. Multiplets arising from 19F-13C J-coupling provide the starting assignment for all resonances by means of 1H homonuclear correlation (COSY) and 1H-13C heteronuclear correlation spectroscopy. 相似文献
11.
The self-association of deoxyribohexanucleoside pentaphosphates 5'-d(CpGpTpApCpG) and 5'-d(CpGpCpGpCpG) in aqueous salt solutions was studied by 1 D- and 2 D homonuclear PMR and heteronuclear 1H-31P-spectroscopies. Signals from nonexchangeable protons of hexamers in NMR spectra were assigned using the available 2M-TOCSY, 2M-NOESY, and 1H-31P-(HMBS) spectra. The dependences of proton chemical shifts of deoxyhexanucleotides on concentration and temperature were measured. In terms of the two-states model (monomer-duplex), constants and thermodynamic parameters of self-association of hexamer molecules in solution were obtained based on these dependences. The values obtained correlate well with theoretical values calculated using the model of the "nearest neighbor" for the formation of duplexes of sequences d(CGTACG) and d(CGCGCG). 相似文献
12.
D Norman D Live M Sastry R Lipman B E Hingerty M Tomasz S Broyde D J Patel 《Biochemistry》1990,29(11):2861-2875
Two-dimensional homonuclear and heteronuclear NMR and minimized potential energy calculations have been combined to define the structure of the antitumor agent mitomycin C (MC) cross-linked to deoxyguanosines on adjacent base pairs in the d(T1-A2-C3-G4-T5-A6).d(T7-A8-C9-G10-T11-A12) duplex. The majority of the mitomycin and nucleic acid protons in the MC-X 6-mer complex have been assigned from through-bond and through-space two-dimensional proton NMR studies in aqueous solution at 5 and 20 degrees C. The C3.G10 and G4.C9 base pairs are intact at the cross-link site and stack on each other in the complex. The amino protons of G4 and G10 resonate at 9.36 and 8.87 ppm and exhibit slow exchange with solvent H2O. The NMR experimental data establish that the mitomycin is cross-linked to the DNA through the amino groups of G4 and G10 and is positioned in the minor groove. The conformation of the cross-link site is defined by a set of NOEs between the mitomycin H1" and H2" protons and the nucleic acid imino and amino protons of G4 and the H2 proton of A8 and another set of NOEs between the mitomycin geminal H10" protons and the nucleic acid imino and amino protons of G10 and the H2 proton of A2. Several phosphorus resonances of the d(T-A-C-G-T-A) duplex shift dramatically on mitomycin cross-link formation and have been assigned from proton-detected phosphorus-proton two-dimensional correlation experiments. The proton chemical shifts and NOEs establish fraying at the ends of the d(T-A-C-G-T-A) duplex, and this feature is retained on mitomycin cross-link formation. The base-base and base-sugar NOEs exhibit similar patterns for symmetry-related steps on the two nucleic acid strands in the MC-X 6-mer complex, while the proton and phosphorus chemical shifts are dramatically perturbed at the G10-T11 step on cross-link formation. The NMR distance constraints have been included in minimized potential energy computations on the MC-X 6-mer complex. These computations were undertaken with the nonplanar five-membered ring of mitomycin in each of two pucker orientations. The resulting low-energy structures MX1 and MX2 have the mitomycin cross-linked in a widened minor groove with the chromophore ring system in the vicinity of the G10-T11 step on one of the two strands in the duplex.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
13.
S-[2-(N7-Guanyl)ethyl]glutathione is the major adduct derived from modification of DNA with 1,2-dibromoethane in biological systems and is postulated to be a mutagenic lesion [Humphreys, W. G., Kim, D.-H., Cmarik, J. L., Shimada, T., & Guengerich, F. P. (1990) Biochemistry 29, 10342-10350]. Oligonucleotides containing this modified base were prepared by treatment of oligonucleotides with S-(2-chloroethyl)glutathione and purified by chromatography. The self-complementary oligonucleotide d(ATGCAT), when thus modified at the single guanine, appeared to associate with itself as judged by UV measurements, but CD and NMR measurements indicated a lack of hybridization, with a decrease in the melting temperature of greater than 10 degrees C. The same lack of self-association was noted when d(ATGCAT) was modified to contain an N-acetyl-S-[2-(N7-guanyl)ethyl]cysteine methyl ester moiety. The oligomer d-(C1A2T3G4C5C6T7) was modified to contain a single S-[2-(N7-guanyl)ethyl]glutathione moiety at the central position, and UV, CD, and 1H NMR studies indicated that this oligomer hybridized to its normal complement d(A8G9G10C11A12T13G14), although the binding was considerably weakened by adduction (imino proton NMR spectroscopy in the presence of H2O indicated that the hydrogen bond signals seen in the oligomer were all broadened upon modification). All proton resonances were identified using two-dimensional 1H NMR spectroscopy. Adduct formation affected the chemical shifts of the base and 1', 2', and 2" protons of T3 and C5, the 2" proton of C6, and the 8 and 1' protons of C11, while little effect was observed on other protons. No cross-peaks were detected between the glutathione and oligomer moieties in two-dimensional nuclear Overhauser enhanced NMR studies. These results suggest that a rather local structural perturbation occurs in the DNA oligomer upon modification and that the glutathione moiety appears to be relatively unperturbed by its placement in the duplex. When the cytosine in the normal d(AGGCATG) complement to d-(CATGCCT) was changed to each of the other three potential bases at the central position, no hybridization with the oligomer d(CATGCCT) containing S-[2-(N7-guanyl)ethyl]glutathione was detected. We conclude that these N7-guanyl derivatives destabilize hybridization and that bases other than cytosine do not appear to show preferential thermodynamic bonding to these adducts, at least in the sequences examined to date.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
14.
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
15.
A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model. 相似文献
16.
The non-detectability of NH...N hydrogen bonds in nucleic acids due to exchange broadened imino/amino protons has recently been addressed via the use of non-exchangeable protons for detecting internucleotide 2hJ(NN) couplings. In these applications, the appropriate non-exchangeable proton is separated by two bonds from the NH...N bond. In this paper, we extend the scope of this approach to protons which are separated by four bonds from the NH...N moiety. Specifically, we consider the case of the commonly occurring sheared G x A mismatch alignment, in which we use the adenine H2 proton to report on the (A)N6H6(1.2)...N3(G) hydrogen bond, in the presence of undetectable, exchange broadened N6H6(1.2) protons. Two sequences, the 'straight-through' (H6)N6N3H2 and 'out-and-back' H2N6N3 experiments, are presented for observing these correlations in H2O and D2O solution, respectively. The sequences are demonstrated on two uniformly 15N,13C labelled DNA samples: d(G1G2G3T4T5C6A7G8G9)2, containing a G3 x (C6-A7) triad involving a sheared G3 x A7 mismatch, and d(G1G2G3C4A5G6G7T8)4, containing an A5 x (G3 x G6 x G3 x G6) x A5 hexad involving a sheared G3 x A5 mismatch. 相似文献
17.
Oligodeoxynucleotides are reversibly deuteriated at the purine C8 and cytosine C5 positions with deuterioammonium bisulfite at pD 7.8. The exchange reaction is complete after 48 h at 65 degrees C. When an oligomer deuteriated under these conditions is analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy, the purine H8 and cytosine H5 proton signals are selectively removed from the spectrum. A non-self-complementary oligodeoxynucleotide that has been deuteriated in this manner may be annealed with its complement and the resulting heteroduplex analyzed by two-dimensional nuclear Overhauser enhancement (NOESY) spectroscopy. NOE cross-peaks arising from pyrimidine H6-deoxyribose H1' dipolar interactions in both strands are observed, but purine H8-deoxyribose H1' and purine H8-deoxyribose H2',H2" dipolar interactions are only observed for the nondeuteriated strand. The intense cytosine H5-H6 cross-peaks are also removed from the spectrum of the deuteriated strand, which further simplifies interpretation since these strong cross-peaks often interfere with less intense NOE cross-peaks arising from dipolar coupling between purine H8 or pyrimidine H6 and deoxyribose anomeric protons. The resulting spectral simplification allows unambiguous assignments to be made on NOEs that otherwise may be difficult to distinguish. The deuteration procedure is demonstrated with the sequence d(CGTTATAATGCG).d(CGCATTATAACG), which has previously been assigned by traditional NOESY methods [Wemmer, D. E., Chou, S.-H., Hare, D. R., & Reid, B. R. (1984) Biochemistry 23, 2262-2268]. Although the assignment of this dodecadeoxynucleotide may be completed without deuteriation, several NOEs must be assigned indirectly because of degeneracies in the chemical shift of the purine H8 protons.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
Determination of the high resolution solution structure of a protein using nuclear magnetic resonance (NMR) spectroscopy requires
that resonances observed in the NMR spectra be unequivocally assigned to individual nuclei of the protein. With the advent
of modern, two-dimensional NMR techniques arose methodologies for assigning the1H resonances based on 2D, homonuclear1H NMR experiments. These include the sequential assignment strategy and the main chain directed strategy. These basic strategies
have been extended to include newer 3D homonuclear experiments and 2D and 3D heteronuclear resolved and edited methods. Most
recently a novel, conceptually new approach to the problem has been introduced that relies on heteronuclear, multidimensional
so-called triple resonance experiments for both backbone and sidechain resonance assignments in proteins. This article reviews
the evolution of strategies for the assignment of resonances of proteins. 相似文献
19.
20.
Mark J.S. Kelly Cornelia Krieger Linda J. Ball Yihua Yu Gerald Richter Peter Schmieder Adelbert Bacher Hartmut Oschkinat 《Journal of biomolecular NMR》1999,14(1):79-83
NMR investigations of larger macromolecules (>20 kDa) are severely hindered by rapid 1H and 13C transverse relaxation. Replacement of non-exchangeable protons with deuterium removes many efficient 1H-1H and 1H-13C relaxation pathways. The main disadvantage of deuteration is that many of the protons which would normally be the source of NOE-based distance restraints are removed. We report the development of a novel labeling strategy which is based on specific protonation and 14N-labeling of the residues phenylalanine, tyrosine, threonine, isoleucine and valine in a fully deuterated, 15N-labeled background. This allows the application of heteronuclear half-filters, 15N-editing and 1H-TOCSY experiments to select for particular magnetization transfer pathways. Results from investigations of a 47 kDa dimeric protein labeled in this way demonstrated that the method provides useful information for the structure determination of large proteins. 相似文献