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1.
A novel triple-resonance NMR method is presented for the measurement of the protein backbone dihedral angle based on differential multiple-quantum relaxation induced by relaxation interference between 1H(i)-13C(i) dipolar and 13C(i–1) (carbonyl) chemical shift anisotropy mechanisms. The method employs a simultaneous transfer of 15N magnetization to the inter- and intra-residue 13C carbons as well as the directly attached carbonyl carbon 13C. Results obtained on 13C,15N-labeled ubiquitin demonstrate the potential of the method. 相似文献
2.
Frank Löhr Markus Blümel Jürgen M. Schmidt Heinz Rüterjans 《Journal of biomolecular NMR》1997,10(2):107-118
A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly13C/15N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the3J(Ci–1,Hi
), 3J(Ci–1,Ci
) and3J(Ci–1,Ci)coupling constants in the indirectly detected 13Cdimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone -angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for3J(Ci–1,Hi
),3J(Ci–1,Ci
) and3J(Ci–1,Ci),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings. 相似文献
3.
Permi P 《Journal of biomolecular NMR》2003,27(4):341-349
Novel NMR pulse schemes for simultaneous measurement of 1
D
CHand 2
D
NHresidual dipolar couplings in proteins is presented. We show that 2
D
NHcoupling can be very useful for protein structure determination. The 2
D
NHcoupling can be measured from 15N dimension with good accuracy on a slowly relaxing TROSY resonance, utilizing HNCA-TROSY-based experiments, which concomitantly supply large 1
D
CHcoupling. The dynamic range of 2
D
NHcoupling is comparable to 1
D
NC coupling, but instead, it also serves non-redundant information on the course of protein backbone, thanks to rotational degree of freedom with respect to peptide bond. The HNCA-TROSY-based experiments are optimal for measuring residual dipolar couplings at high magnetic fields owing to absence of rapid transverse relaxation of carbonyl carbon. The reliability of the proposed approach was tested on 15N/13C human ubiquitin. A very good correlation with ubiquitin solution as well as crystal structure, for both 1
D
CHand 2
D
NHcouplings, was obtained. 相似文献
4.
We introduce a solid-state NMR technique for selective detection of a residue pair in multiply labeled proteins to obtain site-specific structural constraints. The method exploits the frequency-offset dependence of cross polarization to achieve 13COi 15Ni 13Ci transfer between two residues. A 13C, 15N-labeled elastin mimetic protein (VPGVG)n is used to demonstrate the method. The technique selected the Gly3 C signal while suppressing the Gly5 C signal, and allowed the measurement of the Gly3 C chemical shift anisotropy to derive information on the protein conformation. This residue-pair selection technique should simplify the study of protein structure at specific residues. 相似文献
5.
Summary We have examined the 13C and 13C chemical shifts of a number of proteins and found that their values at the N-terminal end of a helix provide a good predictor for the presence of a capping box. A capping box consists of a hydrogen-bonded cycle of four amino acids in which the side chain of the N-cap residue forms a hydrogen bond with the backbone amide of the N3 residue, whose side chain in turn may accept a hydrogen bond from the amide of the N-cap residue. The N-cap residue exhibits characteristic values for its backbone torsion angles, with and clustering around 94±15° and 167±5°, respectively. This is manifested by a 1–2 ppm upfield shift of the 13C resonance and a 1–4 ppm downfield shift of the 13C resonance, relative to their random coil values, and is mainly associated with the unusually large value of . The residues following the N-cap residue exhibit downfield shifts of 1–3 ppm for the 13C resonances and small upfield shifts for the 13C ones, typical of an -helix. 相似文献
6.
The continuous control of the maximum position of the dye absorption band (the zero of the derivative dD
()/d of the cell's optical density D
()) in a nematic matrix is demonstrated experimentally, as a result of changing the angle between the optical axis of a planar-oriented sample and the plane of polarization of absorbed light incident normal to the optical axis. The theory proposed describes quantitatively the experimental dependence (). The rotation of the polarizer with given frequency results in the spectral position modulation of the solute band maximum () within (=0°)–(90°)=700 cm–1. 相似文献
7.
Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A
adenosine
-
U
uridine
-
Im
imidazole
-
MeIm
1-methyl-imidazole
-
EDTA
ethylenediaminetetraacetic acid
-
pA
adenosine 5-phosphate
-
pU
uridine 5-phosphate
-
Ap
adenosine cyclic 2:3-phosphate
-
ATP
adenosine 5-triphosphate
-
AppA
P1,P2-diadenosine 5-diphosphate
-
pNp (N = A,U)
nucleotide 2(3), 5-diphosphate
-
ImpA
adenosine 5-phosphoreimidazolide
-
ImpU
uridine 5-phosphorimidazolide
-
A
2pA
adenylyl-[25]-adenosine
-
A
3pA
adenylyl-[35]-adenosine
-
pA
2pA
5-phospho-adenylyl-[25]-adenosine
-
pA
3pA
5-phospho-adenylyl-[35]-adenosine
-
pUpU
5-phospho-uridylyl-uridine
-
pApU
5-phospho-adenylyl-uridine
-
pUpA
5-phospho-uridylyladenine
-
(pA)n (n, 2,3,4,)
oligoadenylates with 5 terminal phosphate
-
ImpApA
5-phosphorimidazolide of adenylyl adenosine
-
(pA)
5+
pentamer and higher oligoadenylates with 5 terminal phosphate
-
(Ap)nA (n = 2,3,4)
oligoadenylates without terminal phosphates
In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage 相似文献
8.
The major barrier for xenotransplantation in humans is the presence of (1–3) Galactosyl epitopes (Gal) in xenogeneic tissue and the vast quantities of natural antibodies (Ab) produced by humans against this epitope. The binding of anti-Gal Ab to cells expressing Gal triggers a complement-mediated hyperacute rejection of target cells. The hyperacute rejection of whole cancer cells, modified to express Gal epitopes, could be exploited as a new cancer vaccine to treat human cancers. We tested this hypothesis in Galactosyltransferase knockout (GT KO) mice which, like humans, do not express Gal on their cell surfaces and can produce anti-Gal Ab. Forty-five percent of mice with preexisting anti-Gal Ab rejected Gal positive melanoma cells (B16Gal). These mice remained tumor-free for more than 90 days. The majority of control mice injected with B16Null, Gal negative cells succumbed to melanoma. The rejection of B16Gal induced strong long-lasting antitumor immunity against B16Null measured by the expansion of cytotoxic T lymphocytes. In addition, mice rejecting B16Gal were protected against melanoma since they survived a second rechallenge with B16Null. Protected mice developed antitumor immunity in the absence of autoimmune depigmentation (vitiligo). These results show that rejection of Gal positive melanoma cells can efficiently boost the immune response to other tumor associated antigens present in Gal negative melanoma cells. This study supports the concept of a novel anticancer vaccine to treat human malignancies. 相似文献
9.
Giesen AW Bae LC Barrett CL Chyba JA Chaykovsky MM Cheng MC Murray JH Oliver EJ Sullivan SM Brown JM Homans SW 《Journal of biomolecular NMR》2002,22(1):21-26
The implementation of [13C,13C,15N,2H] labelled amino acids into proteins allows the acquisition of high resolution triple resonance experiments. We present for the first time resonance assignments facilitated by this new labelling strategy. The absence of 1JC,C couplings enables us to measure 1JC,C scalar and 1DC,C residual dipolar coupling constants using modified HNCA experiments which do not suffer from sensitivity losses characteristic for 13C constant time experiments. 相似文献
10.
A novel automated approach for the sequence specific NMR assignments of 1HN, 13C, 13C, 13C/1H and 15N spins in proteins, using triple resonance experimental data, is presented. The algorithm, TATAPRO (Tracked AuTomated Assignments in Proteins) utilizes the protein primary sequence and peak lists from a set of triple resonance spectra which correlate 1HN and 15N chemical shifts with those of 13C, 13C and 13C/1H. The information derived from such correlations is used to create a `master_list' consisting of all possible sets of 1HN
i, 15Ni, 13C
i, 13C
i, 13Ci/1H
i, 13C
i–1, 13C
i–1 and 13Ci–1/ 1H
i–1 chemical shifts. On the basis of an extensive statistical analysis of 13C and 13C chemical shift data of proteins derived from the BioMagResBank (BMRB), it is shown that the 20 amino acid residues can be grouped into eight distinct categories, each of which is assigned a unique two-digit code. Such a code is used to tag individual sets of chemical shifts in the master_list and also to translate the protein primary sequence into an array called pps_array. The program then uses the master_list to search for neighbouring partners of a given amino acid residue along the polypeptide chain and sequentially assigns a maximum possible stretch of residues on either side. While doing so, each assigned residue is tracked in an array called assig_array, with the two-digit code assigned earlier. The assig_array is then mapped onto the pps_array for sequence specific resonance assignment. The program has been tested using experimental data on a calcium binding protein from Entamoeba histolytica (Eh-CaBP, 15 kDa) having substantial internal sequence homology and using published data on four other proteins in the molecular weight range of 18–42 kDa. In all the cases, nearly complete sequence specific resonance assignments (> 95%) are obtained. Furthermore, the reliability of the program has been tested by deleting sets of chemical shifts randomly from the master_list created for the test proteins. 相似文献
11.
A new pulse sequence exploiting double- and zero-quantum evolution of two-spin 15N–13C coherence is proposed for the accurate measurements of 2J
N(
i
),C(
i -
1) coupling constants. Application of the new experiment is presented for 13C,15N-labeled ubiquitin sample. The density functional theory calculations of 2J
N(
i
),C(
i -
1) coupling constants have been performed to study their dependence on both (i - 1) and (i - 1) angle in model peptides, and the results exhibit a good correlation with experimental data.Supplementary material to this paper is available in electronic form at
http://dx.doi.org/10.1007/s10858-004-7563-7This revised version was published online in April 2005. In the previous version, the name of the last author, Joanna Sadlej, was missing. 相似文献
12.
The simultaneous interpretation of a suite of dipole-dipole and dipole-CSA cross-correlation rates involving the backbone nuclei 13C, 1H,13CO, 15N and 1HN can be used to resolve the ambiguities associated with each individual cross-correlation rate. The method is based on the transformation of experimental cross-correlation rates via calculated values based on standard peptide plane geometry and solid-state 13CO CSA parameters into a dihedral angle probability surface. Triple resonance NMR experiments with improved sensitivity have been devised for the quantification of relaxation interference between 1H(i)-13C(i)/15N(i)-1HN(i) and 1H(i–1)-13C(i–1)/15N(i)-1HN(i) dipole-dipole mechanisms in 15N,13C-labeled proteins. The approach is illustrated with an application to 13C,15N-labeled ubiquitin. 相似文献
13.
A suite of experiments are presented for the measurement of H–C, C–C, C–C and HN–N couplings from uniformly 15N, 13C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional HN–N spectra equivalent to the common 1H–15N–HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H–C, C–C, and HN–N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C–C data collection required 10 h. The resulting couplings were measured with an average error of ±0.06, ±0.05, ±0.04 and ±0.10 Hz, respectively. This study also shows H–C and C–C couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C–C couplings and increased precision compared to IPAP measurements for HN–N couplings are demonstrated. Minor modifications allow for acquisition of modulated HN–C 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded 1H–15N resonances. 相似文献
14.
Summary The metabolic formation of either,-dodecanedioic acid or,-tridecanedioic acid from the individual n-alkane, n-alcohol, n-monoacid and,-diol with corresponding carbon chain length using K-carrageenan entrapped mutants S76 ofCandida tropicalis was studied. The immobilized cells of S76 could also directly produce-hydroxy acid and,-dioic acid from,-diol. With n-alcohol and n-monoacid as substrate, the amount of-hydroxy acid and,-dioic acid produced was also a function of the incubation time.The results demonstrated that in the immobilized cells of S76 the formation of,-dioic acid from n-alcohol can also run both via n-monoacid and via,-diol as well as in the normal cells of S76. 相似文献
15.
Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of 13C and 13C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of 13C and 13C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of 13C and 13C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides. 相似文献
16.
A rapid and one-pot synthesis of phthaloyl derivatives of -amino carboxamides is described. In dichloromethane, -amino carboxamides react with mono-methylphthalate in the presence of BOP and i-Pr2NEt to afford the intermediate N
-[(o-methoxycarbonyl)benzoyl]amino carboxamides which undergo cyclization in dichloromethane/water in the presence of aqueous sodium hydroxide and tetrabutylammonium bromide catalyst to afford the corresponding N
-phthaloyl amides in excellent yields. 相似文献
17.
Bernardo Celda Clelia Biamonti Maria Jose Arnau Roberto Tejero Gaetano T. Montelione 《Journal of biomolecular NMR》1995,5(2):161-172
Summary A large portion of the 13C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific 13C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting 1H–13C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these 1H–13C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC
heteronuclear single-quantum coherence spectroscopy
- PFG
pulsed-field gradient
- TOCSY
1H-1H total correlation spectroscopy
- EGF
epidermal growth factor
- mEGF
murine EGF
- hEGF
human EGF
- hTGF
human type- transforming growth factor
- DIPSI
spm-locking pulse sequence
- NOE
nuclear Overhauser effect
- HNOE
heteronuclear Overhauser effect 相似文献
18.
Quantitative analysis of type IV collagen alpha chains in the basement membrane of human urogenital epithelium 总被引:2,自引:0,他引:2
Kiyofuji MA Iyama K Kitaoka M Sado Y Ninomiya Y Ueda S 《The Histochemical journal》2002,34(10):479-486
Type IV collagen is a major component of the basement membrane (BM), which consists of six genetically distinct (IV) chains. In this study the expression of these six (IV) chains was demonstrated immunohistochemically. In addition, the 2(IV) and 5(IV) chains were analysed quantitatively by confocal laser scanning microscopy in human urogenital epithelial BM. The 1/2(IV) and 5/6(IV) chains were immunoreactive in the epithelial BM, whereas, 3/4(IV) chains were not. The quantitative analysis revealed that the amount of 2(IV) and 5(IV) chains differed in each urogenital epithelial BM. The content of 5(IV) chains in the epithelial BM of the bladder was differentially high, and that of the foreskin was differentially low. It is concluded that the elasticity of epithelial BM of the bladder may be structurally related to the high content of 5/6(IV) chains. 相似文献
19.
A 4D TROSY-based triple resonance experiment, 4D-HNCOi–1CAi, is presented which correlates intra-residue 1HN, 15N, 13 C chemical shifts with the carbonyl (13C) shift of the preceding residue. The experiment is best used in concert with recently described 4D TROSY-HNCOCA and -HNCACO experiments [Yang, D. and Kay, L.E. (1999) J. Am. Chem. Soc., 121, 2571–2575]. In cases where degeneracy of (1HN,15N) spin pairs precludes assignment using the HNCOCA and HNCACO, the HNCOi–1CAi often allows resolution of the ambiguity by linking the 13C and 13C spins surrounding the (1HN,15N) pair. The experiment is demonstrated on a sample of 15N, 13C, 2 H labeled maltose binding protein in complex with -cyclodextrin that tumbles with a correlation time of 46 ns. 相似文献
20.
1H(i)-15N(i)-13C(i) dipole-chemical shift anisotropy (CSA) relaxation interference was quantified for the 13C,15N labeled zinc-finger protein qCRP2(LIM2). The cross-correlation rates obtained for residues located in the metal coordination sites of qCRP2(LIM2) show a high degree of correlation with the peptide plane torsion angles and taken from the solution structure. 1H(i)-15N(i)-13C(i) as well as 13C(i)-1H(i)-13C(i) dipole-CSA cross-correlation rates were subsequently used to improve the geometry of the metal binding site. The optimized dihedral angles of the two zinc-binding sites in qCRP2(LIM2) are in better agreement with values obtained from crystal structures of other zinc-finger proteins and thus establish the utility of this approach to improve the metal-binding site geometry of zinc-finger proteins studied by NMR spectroscopy in solution. 相似文献