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1.
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   

2.
(1) Extensive studies on proton-translocating ATPase (H+-ATPase) revealed that H+-ATPase is an energy transforming device universally distributed in membranes of almost all kinds of cells. (2) Crystallization of the catalytic portion (F1) of H+-ATPase showed that F1 is a hexagonal molecule with a central hole. The diameter of F1 is about 90 Å and its molecular weight is about 380,000. (3) Use of thermophilic F1 permits the complete reconstitution of F1 from its five subunits (, , , , and ) and demonstration of the gate function of the -complex, the catalytic function of (supported by and ), and the H+-translocating functions of all five subunits. (4) Studies using purified thermostable F0 showed that F0 is an H+-channel portion of H+-ATPase. The direct measurement of H+-flux through F0, sequencing of DCCD-binding protein, and isolation of F1-binding protein are described. (5) The subunit stoichiometry of F1 may be 33. (6) Reconstitution of stable H+-ATPase-liposomes revealed that ATP is directly synthesized by the flow of H+ driven by an electrochemical potential gradient and that H+ is translocated by ATP hydrolysis. This rules out functions for all the hypothetical components that do not belong to H+-ATPase in H+-driven ATP synthesis. The roles of conformation change and other phenomena in ATP synthesis are also discussed.  相似文献   

3.
Summary We present ab initio calculations of the Fermi contact term and experimental correlations of six coupling constants, 3JH N H , 1JC H , 2JCH , 1JC N, 2JC N and 1JCN, in a peptide as functions of the backbone dihedral angles, and . Given estimates of experimental uncertainties, we find semiquantitative experimental correlations for 3JH N H , 1JC N and 2JC N, qualitative correlations for 1JC H and 2JCH , but no experimental correlations of practical utility for 1JCN, owing to its complex dependence on at least four dihedral angles. Errors in the estimation of dihedral angles from X-ray crystallographic data for proteins, which result from uncertainties in atom-to-atom distances, place substantial limitations on the quantitative reliability of coupling constant calculations fitted to such data. In the accompanying paper [Edison, A.S. et al., J. Biomol. NMR, 4, 543–551] we apply the results of the coupling constant calculations presented here to the estimation of and angles in staphylococcal nuclease from experimental coupling constants.Abbreviations AO atomic orbital - BPTI basic pancreatic trypsin inhibitor (bovine) - CI-2 chymotrypsin inhibitor 2 - E.COSY exclusive correlation spectroscopy (Griesinger et al., 1986) - nJAB single bond (n=1), geminal (n=2), or vicinal (n=3) coupling constant between nuclei A and B - LCAO linear combination of atomic orbitals - NBO natural bond orbital - n lone pair orbitals - bonding orbitals - * antibonding orbitals - dihedral angle or molecular orbital wave function; r2, correlation coefficient - RHF restricted Hartree-Fock; rmsd, root-mean-square deviation - 3-21G and 6-31G* molecular orbital basis set designations (Hehre et al., 1986)  相似文献   

4.
The Ca2+ channel 1B subunit is a pore-forming component capable of generating N-type Ca2+ channel activity. Although N-type Ca2+ channel plays a role in a variety of neuronal functions, 1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca2+ channel 1 or subunit. In this study, we studied the levels of 1A, 1C, 1D, 1E, 1, 2, 3 and 4 mRNAs in adrenal gland of 1B-deficient mice. The 1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of 1C, 1D, 1E, 1, 2, 3 and 4 was found among wild, heterozygous and homozygous mice. The protein level of 1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of 1A gene, we examined lacZ expression in 1B-deficient × 1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of 1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous 1B-deficient × 1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of 1B-deficient mice is that compensation by 1A subunit occurs and that 6.3-kb 5-upstream region of 1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)  相似文献   

5.
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   

6.
Summary 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 -protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, 15N TOCSY-HMQC and 15N NOESY-HMQC experiments provided the H 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 and H frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C 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 secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427–445] shows no major differences.Supplementary material available from F.J.M. van de Ven: the source code (PASCAL) for the computer program described in this paper.  相似文献   

7.
The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Leb and B Leb healthy subjects, were each found to be contaminated by a minor component when analysed by1H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and1H/13C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Leb determinant  相似文献   

8.
Three transverse relaxation optimised NMR experiments (TROSY) for the measurement of scalar and dipolar couplings suitable for proteins dissolved in aqueous iso- and anisotropic solutions are described. The triple-spin-state-selective experiments yield couplings between 1HN-13C, 15N-13C, 1HN-13C i–1, 15N-13C i–1, 1HN-13Ci–1, 15N-13Ci–1, and 13Ci–1-13C i–1 without introducing nonessential spectral crowding compared with an ordinary two-dimensional 15N-1H correlation spectrum and without requiring explicit knowledge of carbon assignments. This set of /-J-TROSY experiments is most useful for perdeuterated proteins in studies of structure–activity relationships by NMR to observe, in addition to epitopes for ligands, also conformational changes induced by binding of ligands.  相似文献   

9.
Summary 15N-C and15N-C J couplings were measured for the backbone of staphylococcal nuclease, uniformly enriched with15N and13C. It is found that theIJC'N coupling is similar for -sheet, J=14.8 ± 0.5 and for -helix, J = 14.8 ± 0.4 but tends to be larger for the unstructured N- and C-terminal ends of the protein (J=15.6 ± 0.5). On average,1JNC are smaller for -helical residues (J=9.6 ± 0.3 Hz) compared to -sheet (J=10.9 ± 0.8 Hz) and a substantial difference is observed for2JNC in -helices (J=6.4 ± 0.4 Hz) and -sheets (J=8.3 ± 0.8 Hz).Dedicated to the memory of Professor V.F. Bystrov  相似文献   

10.
Interspecies hybrids of HbA and Hb from mouse C57BL/10 [ 2 M 2 H and 2 H 2 M (H=human, M=mouse)], representing 19 and 27 sequence differences per dimers (as compared with human dimer) have been generatedin vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O2 binding. The intrinsic O2 affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybrid 2 M 2 H has high O2 affinity (compared to either human or mouse Hb), while the interspecies hybrid 2 H 2 M exhibits a very low O2 affinity. These results suggest that the mouse chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse and chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse chain. DPG binds the tetramer in the central cavity formed by the two subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of the chain present in the tetramer. The mouse chain reduces considerably the DPG right shift of the O2 affinity of the human-chain containing hybrid. Sequence analysis suggest that perturbations of the 1 1 (not the 1 2) are communicated to the DPG binding pocket in the presence of the alien subunit, and are the primary determinant of the ligand binding properties. The results have implications for the design of Hb-based blood substitutes and understanding of the inhibitory potential of mouse chains in transgenic mouse expressing human S chains.  相似文献   

11.
By the formation of cGMP the NO-sensitive guanylyl cyclase plays a key role within the NO/cGMP signaling cascade involved in vascular regulation and neurotransmission. The prosthetic heme group of the enzyme acts as the NO sensor, and binding of NO induces conformational changes leading to an up to 200-fold activation of the enzyme. The unexpected fast dissociation half-life of NO of a few seconds is fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogues acting as NO sensitizers uncovered a new pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme.Two existing isoforms of the heterodimeric guanylyl cyclase (11, 21) are known that are functionally indistinguishable. Up to now, the NO-sensitive guanylyl cyclase has been considered as a soluble enzyme. However, recent evidence about the 21 isoform interacting with a PDZ domain of the postsynaptic scaffold protein PSD-95 suggests that the 2 subunit directs a membrane association of this isoform. The interaction with PSD-95 locates the 21 isoform in close proximity to the NO-generating NO synthase thereby enabling the NO sensor to respond to locally raised NO concentrations.  相似文献   

12.
The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (11 and 22). The subunit corresponding to the 11 pair is present inH. petiolaris and in the three populations ofH. annuus studied. The 2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific 2a2 and 44 pairs andH. annuus a specific 33 pair. InH. argophyllus 11 33 or 44 are never observed but are replaced by 13 and 31 pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.  相似文献   

13.
Summary Simple pseudo-3D modifications to the constant-time HSQC and HCACO experiments are described that allow accurate (±0.5 Hz) measurement of one bond JCH coupling constants in proteins that are uniformly enriched with 13C. An empirical ,-surface is calculated which describes the deviation of 1JCH from its random coil value, using 203 1JCH values measured for residues in the proteins calmodulin, staphylococcal nuclease, and basic pancreatic trypsin inhibitor, for which and are know with good precision from previous X-ray crystallographic studies. Residues in -helical conformation exhibit positive deviations of 4–5 Hz, whereas deviations in -sheet are small and, on average, slightly negative. Data indicate that 1JCH depends primarily on , and that 1JCH may be useful as a qualitative probe for secondary structure. Comparison of 1JCH coupling constants measured in free calmodulin and in its complex with a 26-aminoacid peptide fragment of myosin light-chain kinase confirm that the calmodulin secondary structure is retained upon complexation but that disruption of the middle part of the central helix is even more extensive than in free calmodulin. Supplementary material available from the authors: One table listing 352 1JCH and 1J-values, together with ,-values for 203 residues of known conformation. Two figures showing (a) a Ramachandran plot of the ,-values of 203 residues used in deriving 1J(,), and (b) the r.m.s.d. 1J(,) distribution.  相似文献   

14.
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   

15.
The metabolism of GA29 in maturing seeds of Pisum sativum cv. Progress No. 9 was further investigated, and the utility of 2H-labelled GAs in conjuction with GC-MS is illustrated. Using [2-2H1]GA29 as an internal standard, endogenous GA29 was shown to reach a maximal level (ca. 10 g/seed) 27 days from anthesis, and to decline to ca. 1.6 g/seed in mature seeds. In a time-course feed the metabolism of [2-2H1] [2-3H1]GA29 applied to 27 day old seeds, and of endogenous GA29, was compared from the 1H:2H ratios in the recovered GA29. Although both [2-2H1] [2-3H1]GA29 and endogenous GA29 were metabolised to the same limited extent to a putative conjugate, in the main metabolic process endogenous GA29 was preferentially converted to an untraceable (i.e. unlabelled) metabolite. In contrast, endogenous GA29 and [1,3-2H2] [1,3-3H2]GA29, derived from [1,3-2H2] [1,3-3H2]GA20 in a time-course feed, were metabolised in an identical manner. In the latter case isotope loss precluded identification of the metabolite. The structure (8) has been assigned to a GA catabolite present in maturing seeds and seedlings of pea. The isotope data are consistent with this compound being the hitherto untraced metabolite of GA29 in pea.Abbreviations GAn gibberellin An - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - M+ molecular ion - Me methyl ester - RT retention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethylsilyl ether  相似文献   

16.
ATP synthase (F0F1) is driven by an electrochemical potential of H+ (H+). F0F1 is composed of an ion-conducting portion (F0) and a catalytic portion (F1). The subunit composition of F1 is 33. The active 33 oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F1 (TF1). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F0. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of 33. The torque of the rotation is estimated to be 420 pN·å from the H+ and H+-current through F0F1. The angular velocity () of is the rate-limiting step, because H+ increased theV max of H+ current through F0, but not theK m (ATP). The rotational unit of F0 (=ab2c10) is /5, while that in 33 is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN3-ATP), and tight Mg-ATP binding in 33 was shown by ESR. The rotational relaxation of TF1 is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of 33 measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF1) and F0-d to hold free rotation of 33.  相似文献   

17.
Postnatal development of Go isoforms in rat cerebral cortex was studied by SDS-PAGE and immunoblotting. When rat cerebral cortical membranes were resolved on separating gels containing 9% acrylamide and 8 M urea, three electrophoretically distinct Go-immunoreactive proteins were evident. Comparison of their electrophoretic mobilities and partial tryptic digest pattern with recombinant Go1 or Go1-specific antibody revealed that the slowest and intermediate-migrating bands represent unmodified and fatty acylated forms of Go1 protein, respectively. The fastestmigrating band corresponds to Go2. While the fatty acylated form of Go1 is the predominant species, its appearance paralleled that observed for Go2 in developing rat cortex. Perinatal hypothyroidism induced by methimazole treatment did not significantly alter the appearance of cerebral cortical Go1 and Go2 between days 1 and 22 postpartum. Our findings support the earlier idea that heterogeneity of Go proteins in mammalian brain is likely the result of different co- or post-translational processings of each splice variant of Go. While the appearance of Go isoforms is developmentally regulated, they likely do not play an obligatory role in neonatal brain development. Alternatively, the expression of Go isoforms in developing rat cortex may be controlled by an intrinsic signal(s) that is independent of the thyroid status.  相似文献   

18.
Various species of bruchid beetles including Callosobruchus chinensis, C. maculatus and C. analis cause postharvest damage of azuki bean seeds, an important East Asian grain legume. The -amylase in the midguts of these insects is inhibited by the -amylase inhibitor (AI) present in common bean seeds. Transformation of azuki bean with the AI gene driven by the promoter of phytohemagglutinin results in high levels of AI in the seeds and the complete block of bruchid development on the seeds. Zabrotes subfasciatus, a South and Central American bruchid that is a storage pest of common bean, develops normally on the transgenic azuki bean.  相似文献   

19.
Subfamilies of voltage-activated K+ channels (Kv1-4) contribute to controlling neuron excitability and the underlying functional parameters. Genes encoding the multiple subunits from each of these protein groups have been cloned, expressed and the resultant distinct K+ currents characterized. The predicted amino acid sequences showed that each subunit contains six putative membrane-spanning -helical segments (S1-6), with one (S4) being deemed responsible for the channels' voltage sensing. Additionally, there is an H5 region, of incompletely defined structure, that traverses the membrane and forms the ion pore; residues therein responsible for K+ selectivity have been identified. Susceptibility of certain K+ currents produced by the Shaker-related subfamily (Kv1) to inhibition by -dendrotoxin has allowed purification of authentic K+ channels from mammalian brain. These are large (Mr 400 kD), octomeric sialoglycoproteins composed of and subunits in a stoichiometry of ()4()4, with subtypes being created by combinations of subunit isoforms. Subsequent cloning of the genes for 1, 2 and 3 subunits revealed novel sequences for these hydrophilic proteins that are postulated to be associated with the subunits on the inner side of the membrane. Coexpression of 1 and Kv1.4 subunits demonstrated that this auxiliary protein accelerates the inactivation of the K+ current, a striking effect mediated by an N-terminal moiety. Models are presented that indicate the functional domains pinpointed in the channel proteins.  相似文献   

20.
Summary Calculated coupling constants (3JH N H , 1JC H , 2JCH , 1JC N and 2JC N) from our accompanying paper [Edison, A.S. et al. (1994) J. Biomol. NMR, 4, 519–542] have been used to generate error surfaces that can provide estimates of the and angles in proteins. We have used experimental coupling data [3JH N H : Kay, L.E. et al. (1989) J. Am. Chem. Soc., 111, 5488–5490; 1JC H : Vuister, G.W. et al. (1993) J. Biomol. NMR, 3, 67–80; 2JCH : Vuister, G.W. and Bax, A. (1992) J. Biomol. NMR, 2, 401–405; 1JC N and 2JC N: Delaglio, F. et al. (1991) J. Biomol. NMR, 1, 439–446] to create error surfaces for selected residues of the protein staphylococcal nuclease. The residues were chosen to include all those with five experimental couplings, as well as some with four experimental couplings, to demonstrate the relative importance of 3JH N H and 1JC H . For most of the cases, we obtained good agreement between the X-ray structure [Loll, P.J. and Lattman, E.E. (1989) Protein Struct. Funct. Genet., 5, 183–201] and the NMR data.Abbreviations CUPID Contin Uous ProbabIlity Distribution analysis of rotamers - nJAB single-bond (n=1), geminal (n=2), or vicinal (n=3) coupling constant between nuclei A and B - NOE nuclear Overhauser enhancement - r2 correlation coefficient  相似文献   

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