共查询到20条相似文献,搜索用时 31 毫秒
1.
R. Bryn Fenwick David Oyen Henry van den Bedem H. Jane Dyson Peter E. Wright 《Biophysical journal》2021,120(2):296-305
NMR relaxation dispersion measurements report on conformational changes occurring on the μs-ms timescale. Chemical shift information derived from relaxation dispersion can be used to generate structural models of weakly populated alternative conformational states. Current methods to obtain such models rely on determining the signs of chemical shift changes between the conformational states, which are difficult to obtain in many situations. Here, we use a “sample and select” method to generate relevant structural models of alternative conformations of the C-terminal-associated region of Escherichia coli dihydrofolate reductase (DHFR), using only unsigned chemical shift changes for backbone amides and carbonyls (1H, 15N, and 13C′). We find that CS-Rosetta sampling with unsigned chemical shift changes generates a diversity of structures that are sufficient to characterize a minor conformational state of the C-terminal region of DHFR. The excited state differs from the ground state by a change in secondary structure, consistent with previous predictions from chemical shift hypersurfaces and validated by the x-ray structure of a partially humanized mutant of E. coli DHFR (N23PP/G51PEKN). The results demonstrate that the combination of fragment modeling with sparse chemical shift data can determine the structure of an alternative conformation of DHFR sampled on the μs-ms timescale. Such methods will be useful for characterizing alternative states, which can potentially be used for in silico drug screening, as well as contributing to understanding the role of minor states in biology and molecular evolution. 相似文献
2.
Pornthip Boonsri Terrence S. Neumann Andrew L. Olson Sheng Cai Timothy J. Herdendorf Henry M. Miziorko Supa Hannongbua Daniel S. Sem 《Biochemical and biophysical research communications》2013,430(1):313-319
Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using autodock. Promising hits were verified and their affinity measured using NMR-based 1H–15N heteronuclear single quantum coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (Kd). Tight binding compounds with Kd’s ranging from 6–60 μM were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC cross peak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development. 相似文献
3.
The helix to coil transitions of calf thymus and salmon sperm DNA were probed by 31P nuclear magnetic resonance spectroscopy. Both the helical and coil forms were observed in the melting region indicating slow exchange between the two forms with an estimated rate of interconversion ? 36 sec?1 at 70°C. At least three different signals were also observed at temperatures significantly above the Tm, suggesting three classes of conformational states. One of these classes has a significantly lower T1 than the other two indicating considerable residual structure in this form. The four only partially resolved signals indicate that the phosphate residues have very similar chemical shift environments. From the experimentally observed small chemical shift differences between the helical and coil forms, it is concluded that the gauche, gauche, conformation predominates in the coil form as has been found for the helix. 相似文献
4.
Various hydrophobic benzenediacetic esters, the corresponding benzenedipropionic esters, and branched alkyl esters were intercalated into DMPC liposomes, where the molar ratio (n/n) of ester:DMPC was 1:5. In the case of the very long-chain derivatives, double carbonyl peaks were observed in the 13C NMR spectrum. This doubling phenomenon was observed only for the carbonyl peaks, whose chemical shift is most sensitive to solvent polarity, and disappeared when the ester:DMPC molar ratio drops below 1:15. This doubling reflects the presence of two populations in these samples: one group includes those molecules which are intercalated within the liposome and feel the polarity corresponding to the liposomal microenvironment; the other consists of aggregates of these long-chain derivatives located in the extra-liposomal aqueous phase. 相似文献
5.
Androcam is a calmodulin-like protein that acts as a testis-specific light chain to myosin VI during spermatogenesis in D. melanogaster. Modest, localized chemical shift changes that accompany Ca2+ binding to the androcam N-terminal lobe indicate that unlike calmodulin, androcam does not undergo a dramatic conformational change upon binding calcium. Here we report the 1H, 15N and 13C resonances of androcam in the high calcium (10 mM) state and show the extent of chemical shift changes for backbone resonances relative to the low calcium state. 相似文献
6.
The development of “molecular rulers” would allow one to quantitatively locate intercalants within the liposomal bilayer. To this end, we have attempted to correlate the 13C NMR chemical shift of a polarizable “reporter” carbon (e.g., carbonyl) of the intercalant—with the ET(30) polarity it experiences, and with its Angstrom distance from the interface. This requires families of molecules with the same two “reporter carbons” separated by a fixed distance, residing at various depths/polarities within the bilayer. The families studied included 4,4-dialkylcyclohexa-2,5-dienones 1, benzenediacetic esters 15, benzenedipropionic esters 17, 4-alkoxybenzaldehydes 19 and methyl 4-alkoxybenzoates 22. These compounds possessed the following characteristics: (1) a planar backbone; (2) polar/hydrophilic “head” groups; (3) modular hydrophobic tails; (4) large changes in the 13C NMR chemical shift (Δδ) of the reporter atoms with solvent polarity. These studies revealed a fifth requirement, namely: (5) the reporter carbons must not be strongly conjugated, lest it reflect the charge build-up at another site within the conjugated system. 相似文献
7.
Satoru Tuzi Tadashi Komoto Isao Ando Hazime Saito Akira Shoji Takuo Ozaki 《Biopolymers》1987,26(12):1983-1992
13C-nmr spectra of poly(β-benzyl L-aspartate) containing 13C-enriched [3-13C]L -alanine residues in the solid state were recorded by the cross polarization–magic angle spinning method, in order to elucidate the conformation-dependent 13C chemical shifts of L -alanine residues taking various conformations such as the antiparallel β-sheet, the right-handed α-helix, the left-handed α-helix, and the left-handed ω-helix forms obtained by appropriate treatment. The latter two conformations for L -alanine residues are achieved when L -alanine residues are incorporated into poly(β-benzyl L -aspartate). We found that the alanine Cβ carbon show significant 13C chemical shift displacement depending on conformational change, and gave the 13C chemical shift values at about 17 ppm for the left-handed ω-helix, 14 ppm for the left-handed α-helix, 15.5 ppm for the right-handed α-helix, and 21.0 ppm for the antiparallel β-sheet relative to tetramethylsilane. 相似文献
8.
31P Nmr parameters (δ, T1, W1/2, and NOE) were measured for the DNA in nucleosome core particles at three frequencies and compared with similar data for the histone-free DNA. An essentially linear relationship was found between the frequency of observation and line-width for the single broad envelope of 31P resonances of the DNA in the nucleosome cores. We attributed this largely to chemical shift dispersion, with smaller contributions from chemical shift anisotropy and dipolar broadening. These results suggest the presence of different environments for phosphorus atoms in the core particles. However, within the accuracy of the method, no asymmetry in the resonance could be detected, which would tend to rule out any significant degree of DNA “kinking.” To investigate the interactions of the DNA and histones within the core particles we also studied transitions induced by urea and by temperature. Urea caused two stepwise increases in linewidth, which we attributed to conformational changes. A biphasic transition was also observed in the temperature profile, consistent with previous optical studies [Weischet et. al. (1978) Nucleic Acids Res. 5 , 139]. Various models with different types of local mobility were examined by the relaxation theory. A model of isotropic motion having a broad distribution of correlation times gave a fairly good fit to the 31P-nmr data. 相似文献
9.
Prolonged exposure to an oriented line shifts the perceived orientation of a subsequently observed line in the opposite direction, a phenomenon known as the tilt aftereffect (TAE). Here we consider whether the TAE for line stimuli is mediated by a mechanism that integrates the local parts of the line into a single global entity prior to the site of adaptation, or the result of the sum of local TAEs acting separately on the parts of the line. To test between these two alternatives we used the fact the TAE transfers almost completely across luminance contrast polarity [1]. We measured the TAE using adaptor and test lines that (1) either alternated in luminance polarity or were of a single polarity, and (2) either alternated in local orientation or were of a single orientation. We reasoned that if the TAE was agnostic to luminance polarity and was parts-based, we should obtain large TAEs using alternating-polarity adaptors with single-polarity tests. However we found that (i) TAEs using one-alternating-polarity adaptors with all-white tests were relatively small, increased slightly for two-alternating-polarity adaptors, and were largest with all-white or all-black adaptors. (ii) however TAEs were relatively large when the test was one-alternating polarity, irrespective of the adaptor type. (iii) The results with orientation closely mirrored those obtained with polarity with the difference that the TAE transfer across orthogonal orientations was weak. Taken together, our results demonstrate that the TAE for lines is mediated by a global shape mechanism that integrates the parts of lines into whole prior to the site of orientation adaptation. The asymmetry in the magnitude of TAE depending on whether the alternating-polarity lines was the adaptor or test can be explained by an imbalance in the population of neurons sensitive to 1st-and 2nd-order lines, with the 2nd-order lines being encoded by a subset of the mechanisms sensitive to 1st-order lines. 相似文献
10.
Mathias A. S. Hass Ali Yilmaz Hans E. M. Christensen Jens J. Led 《Journal of biomolecular NMR》2009,44(4):225-233
The use of 13C NMR relaxation dispersion experiments to monitor micro-millisecond fluctuations in the protonation states of histidine residues
in proteins is investigated. To illustrate the approach, measurements on three specifically 13C labeled histidine residues in plastocyanin (PCu) from Anabaena variabilis (A.v.) are presented. Significant Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion is observed for 13Cε1 nuclei in the histidine imidazole rings of A.v. PCu. The chemical shift changes obtained from the CPMG dispersion data are in good agreement with those obtained from the
chemical shift titration experiments, and the CPMG derived exchange rates agree with those obtained previously from 15N backbone relaxation measurements. Compared to measurements of backbone nuclei, 13Cε1 dispersion provides a more direct method to monitor interchanging protonation states or other kinds of conformational changes
of histidine side chains or their environment. Advantages and shortcomings of using the 13Cε1 dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics
of the histidine side chains are discussed. 相似文献
11.
Abstract The synthesis of several dinucleoside phosphate derivatives which are linked by phosphoramidate bonds 3′-OP(O)NH-5′ are described. One of these dimer units can be used in automated solid phase DNA synthesis by the phosphoramidite procedure. In order to study the conformational change which is induced on substituting O-P-0 against O-P-N we have also p-repared the fully deprotected dimer analog. The constitution of the dimer units were confirmed by means of 2D-300MHZ homonuclear chemical shift correlation spectroscopy (1H,1H-COSY). 相似文献
12.
Sven Schünke Justin Lecher Matthias Stoldt U. Benjamin Kaupp Dieter Willbold 《Biomolecular NMR assignments》2010,4(2):147-150
Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels play crucial roles in neuronal excitability and signal transduction of sensory cells. These channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). A comparison of the structures of wildtype ligand-free and ligand-bound CNBD is essential to elucidate the mechanism underlying nucleotide-dependent activation of CNBDs. We recently reported the solution structure of the Mesorhizobium loti K1 (MloK1) channel CNBD in complex with cAMP. We have now extended these studies and achieved nearly complete assignments of 1H, 13C and 15N resonances of the nucleotide-free CNBD. A completely new assignment of the nucleotide-free wildtype CNBD was necessary due to the sizable chemical shift differences as compared to the cAMP bound CNBD and the slow exchange behaviour between both forms. Scattering of these chemical shift differences over the complete CNBD suggests that nucleotide binding induces significant overall conformational changes. 相似文献
13.
DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3′-OH/5′-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone 15N spin relaxation and 15N,1H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5′-phosphate. 相似文献
14.
Network of long-range concerted chemical shift displacements upon ligand binding to human angiogenin
Donald Gagné Chitra Narayanan Nicolas Doucet 《Protein science : a publication of the Protein Society》2015,24(4):525-533
Molecular recognition models of both induced fit and conformational selection rely on coupled networks of flexible residues and/or structural rearrangements to promote protein function. While the atomic details of these motional events still remain elusive, members of the pancreatic ribonuclease superfamily were previously shown to depend on subtle conformational heterogeneity for optimal catalytic function. Human angiogenin, a structural homologue of bovine pancreatic RNase A, induces blood vessel formation and relies on a weak yet functionally mandatory ribonucleolytic activity to promote neovascularization. Here, we use the NMR chemical shift projection analysis (CHESPA) to clarify the mechanism of ligand binding in human angiogenin, further providing information on long-range intramolecular residue networks potentially involved in the function of this enzyme. We identify two main clusters of residue networks displaying correlated linear chemical shift trajectories upon binding of substrate fragments to the purine- and pyrimidine-specific subsites of the catalytic cleft. A large correlated residue network clusters in the region corresponding to the V1 domain, a site generally associated with the angiogenic response and structural stability of the enzyme. Another correlated network (residues 40–42) negatively affects the catalytic activity but also increases the angiogenic activity. 15N-CPMG relaxation dispersion experiments could not reveal the existence of millisecond timescale conformational exchange in this enzyme, a lack of flexibility supported by the very low-binding affinities and catalytic activity of angiogenin. Altogether, the current report potentially highlights the existence of long-range dynamic reorganization of the structure upon distinct subsite binding events in human angiogenin. 相似文献
15.
James M. Aramini Toshifumi Hiraoki Michio Yazawa T. Yuan M. Zhang H. J. Vogel 《Journal of biological inorganic chemistry》1996,1(1):39-48
The high-affinity Ca2+–binding sites of carp (pI 4.25) and pike (pI 5.0) parvalbumins, as well as those of mammalian calmodulin (CaM) and its C-terminal
tryptic half-molecule (TR2C), were analyzed by 207Pb NMR spectroscopy. For the parvalbumins, two 207Pb signals were observed ranging in chemical shift from ≈750 to ≈1260 ppm downfield of aqueous Pb(NO3)2, corresponding to 207Pb2+ bound to the two high-affinity helix-loop-helix Ca2+–binding sites in each of these proteins. Four 207Pb signals, which fall in the same chemical shift window, could be discerned for CaM. Experiments on TR2C permitted the assignment of each signal as due to 207Pb2+ occupying a helix-loop-helix site in either the N- or the C-lobe of the intact protein. 207Pb and 1H NMR titration studies on CaM provided evidence that Pb2+ binding to all four sites occurs simultaneously, in contrast to the behavior of this protein in the presence of Ca2+. Titrations of the 207Pb2+–forms of CaM and TR2C with the antipsychotic drug trifluoperazine demonstrated that drug binding to the exposed hydrophobic surfaces in CaM causes
substantial conformational changes and proceeds in a sequential manner – first the C-lobe and subsequently the N-lobe. Finally,
the field dependence of CaM-bound 207Pb signals was examined. The 207Pb signal linewidths exhibited a sharp dependence on the square of the external magnetic field, a trend characteristic of
relaxation via chemical shift anisotropy. Relaxation studies on TR2C demonstrated that chemical exchange also contributes to the observed linewidths. The large chemical shift dispersion observed
for the 207Pb signals of the three proteins studied here illustrates the remarkable sensitivity of this parameter to subtle differences
in the chemical environment of the protein-bound 207Pb nucleus. To our knowledge, the data presented in this article comprise the first ever published example of the application
of 207Pb NMR spectroscopy to metalloproteins. 相似文献
16.
We have previously shown how the location of an intercalant within the lipid bilayer can be qualitatively determined by using the excellent correlation that exists between the 13C NMR chemical shift of a polarizable carbon (e.g., the carbonyl or nitronyl carbon) and the polarity (using the Dimroth-Reichardt's ET(30) parameter) of the microenvironment in which that carbon resides. In a companion paper, we have determined criteria for reporter molecules that will assist us in converting this qualitative polarity data into quantitative Angstrom values. In the present paper, we report on our initial success in quantitatively mapping of the DMPC bilayer by linking two or more vertical points within a bilayer by both distance (in Angstroms) and ET(30) polarity. The results correlated well with the values obtained using the “parallax method” of Erwin London. 相似文献
17.
Maureen Leninger William M. Marsiglia Alexej Jerschow Nathaniel J. Traaseth 《Journal of biomolecular NMR》2018,71(1):19-30
Exchange between conformational states is required for biomolecular catalysis, allostery, and folding. A variety of NMR experiments have been developed to quantify motional regimes ranging from nanoseconds to seconds. In this work, we describe an approach to speed up the acquisition of chemical exchange saturation transfer (CEST) experiments that are commonly used to probe millisecond to second conformational exchange in proteins and nucleic acids. The standard approach is to obtain CEST datasets through the acquisition of a series of 2D correlation spectra where each experiment utilizes a single saturation frequency to 1H, 15N or 13C. These pseudo 3D datasets are time consuming to collect and are further lengthened by reduced signal to noise stemming from the long saturation pulse. In this article, we show how usage of a multiple frequency saturation pulse (i.e., MF-CEST) changes the nature of data collection from series to parallel, and thus decreases the total acquisition time by an integer factor corresponding to the number of frequencies in the pulse. We demonstrate the applicability of MF-CEST on a Src homology 2 (SH2) domain from phospholipase Cγ and the secondary active transport protein EmrE as model systems by collecting 13C methyl and 15N backbone datasets. MF-CEST can also be extended to additional sites within proteins and nucleic acids. The only notable drawback of MF-CEST as applied to backbone 15N experiments occurs when a large chemical shift difference between the major and minor populations is present (typically greater than ~?8 ppm). In these cases, ambiguity may arise between the chemical shift of the minor population and the multiple frequency saturation pulse. Nevertheless, this drawback does not occur for methyl group MF-CEST experiments or in cases where somewhat smaller chemical shift differences occur are present. 相似文献
18.
Channel-forming colicins are bactericidal proteins that spontaneously insert into hydrophobic lipid bilayers. We have used magic-angle spinning solid-state nuclear magnetic resonance spectroscopy to examine the conformational differences between the water-soluble and the membrane-bound states of colicin Ia channel domain, and to study the effect of bound colicin on lipid bilayer structure and dynamics. We detected 13C and 15N isotropic chemical shift differences between the two forms of the protein, which indicate structural changes of the protein due to membrane binding. The Val Cα signal, unambiguously assigned by double-quantum experiments, gave a 0.6 ppm downfield shift in the isotropic position and a 4 ppm reduction in the anisotropic chemical shift span after membrane binding. These suggest that the α-helices in the membrane-bound colicin adopt more ideal helical torsion angles as they spread onto the membrane. Colicin binding significantly reduced the lipid chain order, as manifested by 2H quadrupolar couplings. These results are consistent with the model that colicin Ia channel domain forms an extended helical array at the membrane-water interface upon membrane binding. 相似文献
19.
Domenico Sanfelice Geoff Kelly Stephen Curry Maria R. Conte 《Biomolecular NMR assignments》2008,2(2):107-109
1H, 15N and 13C chemical shift assignments are presented for the N-terminal region of human La protein, in the apo and 5′-UUUU RNA-bound
state. Secondary structure analysis shows conformational changes in the interdomain linker upon complex formation. 相似文献
20.
tRNA molecules are the adaptors in ribosome-based protein biosynthesis and are stabilized by Mg2+. However, the detailed mechanism for the Mg2+ mediated stability is not fully understood. To study the effects of Mg2+ on conformational flexibility of Escherichia coli tRNALeu (CAG) at millisecond timescale, we applied NMR spectroscopic approach to measure proton exchange rates of imino groups in
the presence of different concentration of Mg2+ and correlated them with the corresponding aminoacylation activity of tRNALeu. Here, we report the first part of the above mentioned study, the 1H, 15N chemical shift assignments of the imino groups in all base pairs of Escherichia coli tRNALeu (CAG) based on 2D 1H-15N TROSY, 2D NOESY and 3D NOESY-HMQC experiments. This work laid the foundation for the NMR study of tRNALeu (BMRB deposits with accession number 17078). 相似文献