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1.
Androcam is a D. melanogaster calmodulin-like protein expressed exclusively in the testis that interacts with myosin VI and is critical to spermatogenesis. At micromolar free Ca2+, androcam binds two calcium ions using its C-terminal lobe but its N-terminal lobe is Ca2+-free. We are pursuing structural studies on androcam at physiological (10 μM) and high (10 mM) calcium. Here we report the 1H, 15N, and 13C chemical shifts of androcam at 10 μM free Ca2+ determined using multi-dimensional NMR experiments. 相似文献
2.
The binding of oxygen to hemoglobin has been investigated by 19F-nuclear magnetic resonance spectroscopy. The 19F-nmr spectrum of hemoglobin trifluoroacetonylated at cysteine β 93 exhibits chemical shift changes on binding of ligands, which differ depending on which chains are undergoing complexation. Comparison of these changes to the fractional ligation of all chains, determined concurrently from the fractional change in the visible spectrum, shows that initial oxygen molecules bind preferentially to α-chains. The 19F-nmr spectrum of partially oxygenated hemoglobin contains resonances at the normal chemical shift positions of the oxygenated and deoxy species, in addition to two small resonances at intermediate positions. Analysis of the relativ magnitudes of these four peaks as functions of oxygen pressure permits identification of the intermediate species 相似文献
3.
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
4.
Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca2+-sensitive activation of retinal guanylyl cyclase 1 (RetGC1) upon light activation of photoreceptor cells. Here we present NMR assignments and functional analysis to probe Ca2+-dependent structural changes in GCAP1 that control activation of RetGC. NMR assignments were obtained for both the Ca2+-saturated inhibitory state of GCAP1 versus a GCAP1 mutant (D144N/D148G, called EF4mut), which lacks Ca2+ binding in EF-hand 4 and models the Ca2+-free/Mg2+-bound activator state of GCAP1. NMR chemical shifts of backbone resonances for Ca2+-saturated wild type GCAP1 are overall similar to those of EF4mut, suggesting a similar main chain structure for assigned residues in both the Ca2+-free activator and Ca2+-bound inhibitor states. This contrasts with large Ca2+-induced chemical shift differences and hence dramatic structural changes seen for other NCS proteins including recoverin and NCS-1. The largest chemical shift differences between GCAP1 and EF4mut are seen for residues in EF4 (S141, K142, V145, N146, G147, G149, E150, L153, E154, M157, E158, Q161, L166), but mutagenesis of EF4 residues (F140A, K142D, L153R, L166R) had little effect on RetGC1 activation. A few GCAP1 residues in EF-hand 1 (K23, T27, G32) also show large chemical shift differences, and two of the mutations (K23D and G32N) each decrease the activation of RetGC, consistent with a functional conformational change in EF1. GCAP1 residues at the domain interface (V77, A78, L82) have NMR resonances that are exchange broadened, suggesting these residues may be conformationally dynamic, consistent with previous studies showing these residues are in a region essential for activating RetGC1. 相似文献
5.
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
6.
The complete sequence-specific assignment of resonances in the1H-NMR spectrum of the polypeptide neurotoxin III (Hm III) from the sea anemoneHeteractis macrodactylus is described. Comparison of the chemical shifts and pattern of NOEs for Hm III with those for the related toxin Hp III fromHeteractis paumotensis, which differs only in the substitution of Asn for Tyr at position 11, shows that the overall secondary and tertiary structures are conserved. The largest differences in chemical shift caused by the substitution at position 11 are observed for the NH resonances of Arg-13, Thr-14, Ala-15, Leu-17, and Cys-26. The CαH resonances influenced most are those of ASP-6, Gly-9, Leu-17, and Glu-42, while the most affected CβH resonances are from Leu-17, Glu-28, and Lys-32. The absence of long-range NOEs to the aromatic ring of Tyr-11 as well as the lack of significant chemical shift effects on residues outside the loop comprising residues 7–16 confirm that this part of the loop makes no long-lived contacts with the rest of the molecule. The deviations from random coil shifts of Hm III are compared with those of the related anemone toxins Hp II, Hp III, and toxin I fromStichodactyla helianthus (Sh I). The similarity in deviations in chemical shift as a function of sequence position for these four toxins emphasizes the overall structural homology among these polypeptides. 相似文献
7.
Conformational and metal-binding properties of androcam, a testis-specific, calmodulin-related protein from Drosophila
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Martin SR Lu AQ Xiao J Kleinjung J Beckingham K Bayley PM 《Protein science : a publication of the Protein Society》1999,8(11):2444-2454
Androcam is a testis-specific protein of Drosophila melanogaster, with 67% sequence identity to calmodulin and four potential EF-hand calcium-binding sites. Spectroscopic monitoring of the thermal unfolding of recombinant calcium-free androcam shows a biphasic process characteristic of a two-domain protein, with the apo-N-domain less stable than the apo-C-domain. The two EF hands of the C-domain of androcam bind calcium cooperatively with 40-fold higher average affinity than the corresponding calmodulin sites. Magnesium competes with calcium binding [Ka(Mg) approximately 3 x 10(3) M(-1)]. Weak calcium binding is also detected at one or more N-domain sites. Compared to apo-calmodulin, apo-androcam has a smaller conformational response to calcium and a lower alpha-helical content over a range of experimental conditions. Unlike calmodulin, a tryptic cleavage site in the N-domain of apo-androcam remains trypsin sensitive in the presence of calcium, suggesting an altered calcium-dependent conformational change in this domain. The affinity of model target peptides for androcam is 10(3)-10(5) times lower than for calmodulin, and interaction of the N-domain of androcam with these peptides is significantly reduced. Thus, androcam shows calcium-induced conformational responses typical of a calcium sensor, but its properties indicate calcium sensitivity and target interactions significantly different from those of calmodulin. From the sequence differences and the altered calcium-binding properties it is likely that androcam differs from calmodulin in the conformation of residues in the second calcium-binding loop. Molecular modeling supports the deduction that there are significant conformational differences in the N-domain of androcam compared to calmodulin, and that these could affect the surface, conferring a different specificity on androcam in target interactions related to testis-specific calcium signaling functions. 相似文献
8.
The conformation of a melittin—inhibitor complex was studied by solution NMR, solid-state NMR, and circular dichroism. In solution, binding was studied by titrating inhibitor against melittin in dimethyl sulfoxide, methanol, aqueous buffer, and dodecylphosphocholine micelles. The change in chemical shift of Trp19 resonances and the formation of a precipitate at 1:1 molar ratio indicated that the inhibitor was bound to melittin. Solid-state NMR also showed a change in chemical shift of two labeled carbons of melittin near Pro14 and a change in 1H T 1 relaxation times when complexed with inhibitor. Rotational resonance experiments of melittin labeled in the proline region indicated a change in conformation for melittin complexed with inhibitor. This observation was also supported by circular dichroism measurements, indicating a reduction in α-helical structure for increasing ratios of inhibitor bound to melittin. 相似文献
9.
Yanni K.-Y. Chin Stephen Headey Biswaranjan Mohanty Jonas Emsley Jamie S. Simpson Martin J. Scanlon 《Biomolecular NMR assignments》2014,8(1):117-121
The α1β1 integrin receptor binds to its main extracellular ligand, collagen, through an inserted domain in its α-subunit called the αI domain (αI). αI contains a metal binding site that allows collagen to coordinate to the domain through a divalent metal ion. Here we report the backbone assignments of the apo and Mg2+ bound state of the isolated human α1I and the chemical shift changes resulting from metal coordination. 相似文献
10.
Phosphoenolpyruvate binding to the C-terminal domain (EIC) of enzyme I of the bacterial phosphotransferase system (PTS) initiates a phosphorylation cascade that results in sugar translocation across the cell membrane and controls a large number of essential pathways in bacterial metabolism. EIC undergoes an expanded to compact conformational equilibrium that is regulated by ligand binding and determines the phosphorylation state of the overall PTS. Here, we report the backbone 1H, 15N and 13C chemical shift assignments of the 70 kDa EIC dimer from the thermophilic bacterium Thermoanaerobacter tengcongensis. Assignments were obtained at 70 °C by heteronuclear multidimensional NMR spectroscopy. In total, 90% of all backbone resonances were assigned, with 264 out of a possible 299 residues assigned in the 1H–15N TROSY spectrum. The secondary structure predicted from the assigned backbone resonance using the program TALOS+ is in good agreement with the X-ray crystal structure of T. tengcongensis EIC. The reported assignments will allow detailed structural and thermodynamic investigations on the coupling between ligand binding and conformational dynamics in EIC. 相似文献
11.
Ca2+ induces a red shift in the absorption spectrum of ferrocytochrome a when added to uncoupled mitochondria, sub-mitochondrial particles or isolated cytochrome aa3. The shift is identical within experimental error to the previously reported energy-linked shift in intact mitochondria (Wikström, M. K. F. (1972), Biochim. Biophys. Acta 283, 385–390). One mol of calcium produces the shift in one mol of cytochrome a, the KD being approx. 20–30 μM. The calcium-induced shift is readily reversed by chelating agents such as EDTA, ethyleneglycol-bis-(μ-aminoethyl ether)N,N′-tetraacetic acid (EGTA) and ATP and is insensitive to uncoupling agents and inhibitors of calcium transport (La3+ and ruthenium red). It is shown that the binding site for calcium that is responsible for the spectral shift is located on the outside of the permeability barrier of the mitochondrial cristae membrane.It is proposed that calcium simulates the energy-linked shift in cytochrome a by binding to a site of cytochrome aa3 that is occupied by protons in energized mitochondria and that is located at the external surface of the mitochondrial membrane. 相似文献
12.
The binding of n-butyl isocyanide to hemoglobin has been investigated by 19F-nuclear magnetic resonance spectroscopy. The 19F-nmr spectrum of hemoglobin trifluoroacetonylated at cysteine β93 exhibits chemical shift changes on binding of ligands to the β chains. Comparison of these changes to the fractional change in the visible spectrum, shows that in the presence of diphosphoglyceric acid initial ligands bind preferentially to α chains. In the absence of DPG, ligation of β chains increases linearly with overall fractional ligation, indicating that binding to α and β chains is random under these conditions. 相似文献
13.
The 13C nuclear magnetic resonance spectra of l-ornithine, of the di- and tripeptide linear derivatives, and of the siderophore-occurring, modified residue δ-N-acetyl-δ-N-hydroxy-l-ornithine (Orn) are reported for 2H2O solutions, at pD 7. The assignment of all the resonances is directly established from the comparative data. This information, together with available literature data, is used to identify the resonances of the metal-free cyclohexapeptides deferriferrichrome, deferriferricrocin, and deferriferrichrysin. The spectra of the analogous peptides at pD 7 in 2H2O are shown to vary in the pattern of the Orn Cβ resonances, suggesting different conformations for deferriferrichrome and the two seryl-containing analogs, in agreement with previously resported 1H nuclear magnetic resonance data. Co-ordination of the Al3+ ion results in extensive changes in both the carbonyl and the aliphatic regions which enhance the overall resolution of the spectra. Most resonances are identified, and many assigned, from the comparative data for the three metal ion co-ordinated analogs. Except for the hydroxamate moiety, directly involved in the complexation event, the drastic chemical shifts induced by metal binding reflect an overall change in the conformational state of the peptides. Differences in the Ca region of the Al3+-bound and metal-free peptides are attributed to strain or environmental effects rather than to inductive effects arising from primary structure. 相似文献
14.
Caroline Smet-Nocca Hélène Launay Jean-Michel Wieruszeski Guy Lippens Isabelle Landrieu 《Journal of biomolecular NMR》2013,55(4):323-337
The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer’s disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the 1H,15N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate. 相似文献
15.
Michael P. Gamcsik Jerry D. Glickson Gerald Zon 《Journal of biomolecular structure & dynamics》2013,31(5):1117-1133
Abstract The interaction of bleomycin A2 and Zn(II)-bleomycin A2 with the oligonucleotide (dC-dG)3 has been monitored by nuclear magnetic resonance spectroscopy. Binding of the drug to the oligonucleotide is indicated by an upfield shift of the bithiazole proton resonances consistent with partial intercalation of this group between base pairs. The effect of temperature and ionic strength on the binding of both free bleomycin and the Zn(II) complex has been studied. Consistent with earlier studies on polynucleotides, the rate of exchange between the free drug and the drug-oligonucleotide complex is rapid on the 1H NMR chemical shift time scale. Binding of the oligonucleotide induced changes in resonances assigned to protons in the metal-binding region of Zn(II)-bleomycin. Intermolecular nuclear Overhauser effect enhancements between bleomycin and the oligonucleotide have not been detected. 相似文献
16.
P.J. Cayley J. Feeney B.J. Kimber 《International journal of biological macromolecules》1980,2(4):251-255
We have measured the 31P n.m.r. spectra of NADP+ and NADPH in their binary complexes with Escherichia coli dihydrofolate reductase and in ternary complexes with the enzyme and folate or methotrexate. The 31P chemical shift of the 2′ phosphate group is the same in all complexes; its value indicates that it is binding in the dianionic state and its pH independence suggests that it is interacting strongly with cationic residue(s) on the enzyme. Similar behaviour has been noted previously for the complexes with the Lactobacillus casei enzyme although the 31P shift is somewhat different in this complex, possibly due to an interaction between the 2′ phosphate group and His 64 which is not conserved in the E. coli enzyme. For the coenzyme complexes with both enzymes 31POC21H2′ spin-spin interactions were detected (7.5–7.8 Hz) on the 2′ phosphate resonances, indicating a POC2H2′ dihedral angle of 30 or 330 : this is in good agreement with the value of 330° measured in crystallographic studies1 (Matthews et al., 1978) on the L. casei enzyme. NADPH-MTX complex. The pyrophosphate resonances are shifted to different extents in the various complexes and there is evidence that there is more OPO bond angle distortion in the E. coli enzyme complexes than in those with the L. casei enzyme. The effects of 31POC51H5′ spin coupling were detected on one pyrophosphate resonance and indicate that the POC5H5′ torsion angle has changed by at least ~30° on binding to the E. coli enzyme: this is considerably less than the distortion (~50°) observed previously in the L. casei enzyme complex. 相似文献
17.
We report the effects of Ca2+ binding on the backbone relaxation rates and chemical shifts of the AD and BD splice variants of the second Ca2+‐binding domain (CBD2) of the sodium–calcium exchanger. Analysis of the Ca2+‐induced chemical shifts perturbations yields similar KD values of 16–24 μM for the two CBD2‐AD Ca2+‐binding sites, and significant effects are observed up to 20 Å away. To quantify the Ca2+‐induced chemical shift changes, we performed a comparative analysis of eight Ca2+‐binding proteins that revealed large differences between different protein folds. The CBD2 15N relaxation data show the CBD2‐AD Ca2+ coordinating loops to be more rigid in the Ca2+‐bound state as well as to affect the FG‐loop located at the opposite site of the domain. The equivalent loops of the CBD2‐BD splice variant do not bind Ca2+ and are much more dynamic relative to both the Ca2+‐bound and apo forms of CBD2‐AD. A more structured FG‐loop in CBD2‐BD is suggested by increased S2 order parameter values relative to both forms of CBD2‐AD. The chemical shift and relaxation data together indicate that, in spite of the small structural changes, the Ca2+‐binding event is felt throughout the molecule. The data suggest that the FG‐loop plays an important role in connecting the Ca2+‐binding event with the other cytosolic domains of the NCX, in line with in vivo and in vitro biochemical data as well as modeling results that connect the CBD2 FG‐loop with the first Ca2+‐binding domain of NCX. Proteins 2010. © 2010 Wiley‐Liss, Inc. 相似文献
18.
J M Friedman D L Rousseau G Navon S Rosenfeld P Glynn K B Lyons 《Archives of biochemistry and biophysics》1979,193(1):14-21
We have observed substantial changes in the resonance Raman spectrum of ruthenium red when it is added to calcium ion binding molecules and organelles, including proteins, phospholipids, chelating agents, and intact mitochondria. The addition of Ca2+ ions can reverse these observed spectral changes. In the case of cytochrome c, ruthenium red binding varies with oxidation state in a manner parallel to that for Ca2+ binding. The resonance Raman spectrum of a ruthenium red-phospholipid complex shows differences from that of a ruthenium red-protein complex, enabling us to distinguish between binding to these different classes of molecules. Our studies suggest that the primary constituent of the low-affinity Ca2+ binding sites in mitochondria is cardiolipin. 相似文献
19.
A pulse sequence that yields three-dimensional 1H chemical shift / 1H-15N heteronuclear dipolar coupling / 15N chemical shift solid-state NMR spectra is demonstrated on a uniformly 15N labeled membrane protein in magnetically aligned phospholipid bilayers. Based on SAMPI4, the pulse sequence yields high
resolution in all three dimensions at a 1H resonance frequency of 900 MHz with the relatively low rf field strength (33 kHz) available for a lossy aqueous sample with
a commercial spectrometer and probe. The 1H chemical shift frequency dimension is shown to select among amide resonances, which will be useful in studies of larger
polytopic membrane proteins where the resonances overlap in two-dimensional spectra. Moreover, the 1H chemical shift, which can be measured from these spectra, provides an additional orientationally dependent frequency as
input for structure calculations.
Both Alexander A. Nevzorov and Sang Ho Park contributed equally to this work. 相似文献
20.
Yeast iso-1 cytochrome c is a naturally occurring protein that possesses an unusually reactive Cysl02 that imbues iso-1 with a complicated solution chemistry which includes spontaneous dimerization and poorly characterized redox reactions. For this reason previous studies of this typical member of the c-type cytochromes have been relegated to variant proteins in which the 102 position has been mutated, with most common changes involving serine and threonine. However, we have determined sequential 1H resonance assignments for the wild-type native protein because it is the actual participant in yeast mitochondrial electron transfer processes and because the wild-type native protein should be the fundamental assignment basis. In addition to 1H resonance assignments for 97 of 106 amino acids, we have also provided an extensive database of long-range NOEs. Comparison of these NOEs and a chemical shift index based upon α-H resonances has lead to identification of solution secondary structural elements that are consistent with the solid-state crystal structure. Although there is currently no efficient expression system that would facilitate isotope labeling of iso-1 cytochrome c, we tried to assess the usefulness of future heteronuclear experiments by using natural-abundance 1H/13C HMQC experiments to unambiguously assign 35 α-C resonances. 相似文献