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1.
We perform a detailed comparison of fast backbone dynamics probed at amide nitrogen versus carbonyl carbon sites for dematin
headpiece C-terminal domain (DHP) and its S74E mutant (DHPS74E). Carbonyl dynamics is probed via auto-correlated longitudinal
rates and transverse C′/C′-Cα CSA/dipolar and C′/C′–N CSA/dipolar cross-correlated rates, while 15N data are taken from a previous study. Resulting values of effective order parameters and internal correlation times support
the conclusion that C′ relaxation reports on a different subset of fast motions compared to those probed at N–H bond vectors
in the same peptide planes. 13C′ order parameters are on the average 0.08 lower than 15N order parameters with the exception of the flexible loop region in DHP. The reduction of mobility in the loop region upon
the S74E mutation can be seen from the 15N order parameters but not from the 13C order parameters. Internal correlation times at 13C′ sites are on the average an order of magnitude longer than those at 15N sites for the well-structured C-terminal subdomains, while the more flexible N-terminal subdomains have more comparable
average internal correlation times. 相似文献
2.
Carbonyl 13C′ relaxation is dominated by the contribution from the 13C′ chemical shift anisotropy (CSA). The relaxation rates provide useful and non-redundant structural information in addition
to dynamic parameters. It is straightforward to acquire, and offers complimentary structural information to the 15N relaxation data. Furthermore, the non-axial nature of the 13C′ CSA tensor results in a T1/T2 value that depends on an additional angular variable even when the diffusion tensor of the protein molecule is axially symmetric.
This dependence on an extra degree of freedom provides new geometrical information that is not available from the NH dipolar
relaxation. A protocol that incorporates such structural restraints into NMR structure calculation was developed within the
program Xplor-NIH. Its application was illustrated with the yeast Fis1 NMR structure. Refinement against the 13C′ T1/T2 improved the overall quality of the structure, as evaluated by cross-validation against the residual dipolar coupling as
well as the 15N relaxation data. In addition, possible variations of the CSA tensor were addressed.
Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
3.
Perazzolo C Wist J Loth K Poggi L Homans S Bodenhausen G 《Journal of biomolecular NMR》2005,33(4):233-242
Major urinary protein (MUP) is a pheromone-carrying protein of the lipocalin family. Previous studies by isothermal titration
calorimetry (ITC) show that the affinity of MUP for the pheromone 2-methoxy-3-isobutylpyrazine (IBMP) is mainly driven by
enthalpy, with a small unfavourable entropic contribution. Entropic terms can be attributed in part to changes in internal motions of the protein upon binding. Slow internal motions can lead to correlated or anti-correlated modulations of the isotropic chemical shifts of carbonyl C′ and amide N nuclei. Correlated chemical shift modulations (CSM/CSM) in MUP
have been determined by measuring differences of the transverse relaxation rates of zero- and double-quantum coherences ZQC{C′N}
and DQC{C′N}, and by accounting for the effects of correlated fluctuations of dipole–dipole couplings (DD/DD) and chemical
shift anisotropies (CSA/CSA). The latter can be predicted from tensor parameters of C′ and N nuclei that have been determined
in earlier work. The effects of complexation on slow time-scale protein dynamics can be determined by comparing the temperature
dependence of the relaxation rates of APO-MUP (i.e., without ligand) and HOLO-MUP (i.e., with IBMP as a ligand).
Electronic supplementary material Electronic supplementary material is available for this article at
and accessible for authorised users. 相似文献
4.
The effects of chemical shift anisotropy (CSA) are evident in line-shapes or side-band analysis in solid-state NMR, in the
observed line positions in partially oriented samples, and in relaxation effects in liquid-state studies. In all of these
cases, the effective shielding tensor is influenced by fast vibrational averaging in addition to larger-amplitude internal
motions and to overall libration or rotation. Here we compute the contributions of vibrational averaging (including zero-point
motions) to the CSA relaxation strengths for the nitrogen and carbonyl carbon in two simple peptide models, and for snapshots
taken from a path-integral simulation of a small protein. Because the 15N shielding tensor is determined by all the atoms of the peptide group, it is less influenced by vibrational motion than (for
example) the N–H dipolar interaction, which is more sensitive to the motion of the light hydrogen atom. Computed order parameters
for CSA averaging are hence much closer to unity than are N–H dipolar order parameters. This leads to a reduction by about
9% in the magnitude of the amide nitrogen CSA that is needed to fit liquid-state relaxation data. Similar considerations apply
to the carbonyl carbon shielding tensor, but in this case the differences between dipolar and CSA averaging are smaller. These
considerations will be important for making comparisons between CSA tensors extracted from various NMR experiments, and for
comparisons to quantum chemical calculations carried out on static conformers. 相似文献
5.
Ming Tang Gemma Comellas Leonard J. Mueller Chad M. Rienstra 《Journal of biomolecular NMR》2010,48(2):103-111
High resolution 13C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been
collected to show that all 15N, 13C′, 13Cα and 13Cβ sites are resolved in 13C–13C and 15N–13C spectra, with significant improvement in T
2 relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T
2 values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that
13Cα T
2′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength,
and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint
of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy
to selectively detect polar residues that are often important for protein function and protein–protein interactions. 相似文献
6.
Dematin is an actin-binding protein abundant in red blood cells and other tissues. It contains a villin-type ‘headpiece’ F-actin-binding
domain at its extreme C-terminus. The isolated dematin headpiece domain (DHP) undergoes a significant conformational change
upon phosphorylation. The mutation of Ser74 to Glu closely mimics the phosphorylation of DHP. We investigated motions in the
backbone of DHP and its mutant DHPS74E using several complementary NMR relaxation techniques: laboratory frame 15N NMR relaxation, which is sensitive primarily to the ps–ns time scale, cross-correlated chemical shift modulation NMR relaxation
detecting correlated μs–ms time scale motions of neighboring 13C′ and 15N nuclei, and cross-correlated relaxation of two 15N–1H dipole–dipole interactions detecting slow motions of backbone NH vectors in successive amino acid residues. The results
indicate a reduction in mobility upon the mutation in several regions of the protein. The additional salt bridge formed in
DHPS74E that links the N- and C-terminal subdomains is likely to be responsible for these changes.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
7.
We developed an NMR pulse sequence, 3D HCA(N)CO, to correlate the chemical shifts of protein backbone 1Hα and 13Cα to those of 13C′ in the preceding residue. By applying 2H decoupling, the experiment was accomplished with high sensitivity comparable to that of HCA(CO)N. When combined with HCACO,
HCAN and HCA(CO)N, the HCA(N)CO sequence allows the sequential assignment using backbone 13C′ and amide 15N chemical shifts without resort to backbone amide protons. This assignment strategy was demonstrated for 13C/15N-labeled GB1 dissolved in 2H2O. The quality of the GB1 structure determined in 2H2O was similar to that determined in H2O in spite of significantly smaller number of NOE correlations. Thus this strategy enables the determination of protein structures
in 2H2O or H2O at high pH values. 相似文献
8.
Tonelli M Masterson LR Hallenga K Veglia G Markley JL 《Journal of biomolecular NMR》2007,39(3):177-185
We present a highly sensitive pulse sequence, carbonyl carbon label selective 1H–15N HSQC (CCLS-HSQC) for the detection of signals from 1H–15N units involved in 13C′–15N linkages. The CCLS-HSQC pulse sequence utilizes a modified 15N CT evolution period equal to 1/(
) (∼33 ms) to select for 13C′–15N pairs. By collecting CCLS-HSQC and HNCO data for two proteins (8 kDa ubiquitin and 20 kDa HscB) at various temperatures
(5–40°C) in order to vary correlation times, we demonstrate the superiority of the CCLS-HSQC pulse sequence for proteins with
long correlation times (i.e. higher molecular weight). We then show that the CCLS-HSQC experiment yields assignments in the
case of a 41 kDa protein incorporating pairs of 15N- and 13C′-labeled amino acids, where a TROSY 2D-HN(CO) had failed. Although the approach requires that the 1H–15N HSQC cross peaks be observable, it does not require deuteration of the protein. The method is suitable for larger proteins
and is less affected by conformational exchange than HNCO experiments, which require a longer period of transverse 15N magnetization. The method also is tolerant to the partial loss of signal from isotopic dilution (scrambling). This approach
will be applicable to families of proteins that have been resistant to NMR structural and dynamic analysis, such as large
enzymes, and partially folded or unfolded proteins. 相似文献
9.
Lundström P Teilum K Carstensen T Bezsonova I Wiesner S Hansen DF Religa TL Akke M Kay LE 《Journal of biomolecular NMR》2007,38(3):199-212
A simple labeling approach is presented based on protein expression in [1-13C]- or [2-13C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone Cα sites, respectively. All of the methyl groups, with the exception of Thr and Ile(δ1) are produced with isolated 13C spins (i.e., no 13C–13C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts
introduced by evolution due to large homonuclear scalar couplings. Carbon-α sites are labeled without concomitant labeling
at Cβ positions for 17 of the common 20 amino acids and there are no cases for which 13Cα−13CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results
obtained complimenting those from more traditional backbone 15N studies. The utility of the labeling is established by recording 13C R
1ρ and CPMG-based experiments on a number of different protein systems. 相似文献
10.
Yin C Aramini JM Ma LC Cort JR Swapna GV Krug RM Montelione GT 《Biomolecular NMR assignments》2011,5(2):215-219
Human interferon-stimulated gene 15 protein (ISG15), also called ubiquitin cross-reactive protein (UCRP), is the first identified
ubiquitin-like protein containing two ubiquitin-like domains fused in tandem. The active form of ISG15 is conjugated to target
proteins via the C-terminal glycine residue through an isopeptide bond in a manner similar to ubiquitin. The biological role
of ISG15 is strongly associated with the modulation of cell immune function, and there is mounting evidence suggesting that
many viral pathogens evade the host innate immune response by interfering with ISG15 conjugation to both host and viral proteins
in a variety of ways. Here we report nearly complete backbone 1HN, 15N, 13C′, and 13Cα, as well as side chain 13Cβ, methyl (Ile-δ1, Leu, Val), amide (Asn, Gln), and indole N–H (Trp) NMR resonance assignments for the 157-residue human ISG15
protein. These resonance assignments provide the basis for future structural and functional solution NMR studies of the biologically
important human ISG15 protein. 相似文献
11.
Heteronuclear NMR spin relaxation studies of conformational dynamics are coming into increasing use to help understand the functions of ribozymes and other RNAs. Due to strong magnetic interactions within the ribose ring, however, these studies have thus far largely been limited to 13C and 15N resonances on the nucleotide base side chains. We report here the application of the alternate-site 13C isotopic labeling scheme, pioneered by LeMaster for relaxation studies of amino acid side chains, to nucleic acid systems. We have used different strains of E. coli to prepare mononucleotides containing 13C label in one of two patterns: Either C1′ or C2′ in addition to C4′, termed (1′/2′,4′) labeling, or nearly complete labeling at the C2′ and C4′ sites only, termed (2′,4′) labeling. These patterns provide isolated H spin systems on the labeled carbon atoms and thus allow spin relaxation studies without interference from scalar or dipolar coupling. Using relaxation studies of AMP dissolved in glycerol at varying temperature to produce systems with correlation times characteristic of different size RNAs, we demonstrate the removal of errors due to interaction in T
1 measurements of larger nucleic acids and in T
1ρ measurements in RNA molecules. By extending the applicability of spin relaxation measurements to backbone ribose groups, this technology should greatly improve the flexibility and completeness of NMR analyses of conformational dynamics in RNA. 相似文献
12.
Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy
We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments
make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration.
We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange
rates negligible within the individual 15N–T
1 timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s−1. Backbone amide 15N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no
systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38,
N35 and for W41ε. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical
exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D2O is employed as a solvent for sample preparation. Due to the intrinsically long 15N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate
regime that is not accessible so far with exchange quench and MEXICO type experiments. 相似文献
13.
The surface dynamics of bacteriorhodopsin was examined by measurements of site-specific 13C–1H dipolar couplings in [3-13C]Ala-labeled bacteriorhodopsin. Motions of slow or intermediate frequency (correlation time <50 µs) scale down 13C–1H dipolar couplings according to the motional amplitude. The two-dimensional dipolar and chemical shift (DIPSHIFT) correlation technique was utilized to obtain the dipolar coupling strength for each resolved peak in the 13C MAS solid-state NMR spectrum, providing the molecular order parameter of the respective site. In addition to the rotation of the Ala methyl group, which scales the dipolar coupling to 1/3 of the rigid limit value, fluctuations of the C–C vector result in additional motional averaging. Typical order parameters measured for mobile sites in bacteriorhodopsin are between 0.25 and 0.29. These can be assigned to Ala103 of the C–D loop and Ala235 at the C-terminal -helix protruded from the membrane surface, and Ala196 of the F–G loop, as well as to Ala228 and Ala233 of the C-terminal -helix and Ala51 from the transmembrane -helix. Such order parameters departing significantly from the value of 0.33 for rotating methyl groups are obviously direct evidence for the presence of fluctuation motions of the Ala C–C vectors of intact preparations of fully hydrated, wild-type bacteriorhodopsin at ambient temperature. The order parameter for Ala160 from the expectantly more flexible E–F loop, however, is unavailable under highest-field NMR conditions, probably because increased chemical shift anisotropy together with intrinsic fluctuation motions result in an unresolved 13C NMR signal. 相似文献
14.
Werner K Lehner I Dhiman HK Richter C Glaubitz C Schwalbe H Klein-Seetharaman J Khorana HG 《Journal of biomolecular NMR》2007,37(4):303-312
Rhodopsin is the visual pigment of the vertebrate rod photoreceptor cell and is the only member of the G protein coupled receptor
family for which a crystal structure is available. Towards the study of dynamics in rhodopsin, we report NMR-spectroscopic
investigations of α,ɛ-15N-tryptophan labeled rhodopsin in detergent micelles and reconstituted in phospholipids. Using a combination of solid state
13C,15N-REDOR and HETCOR experiments of all possible 13C′
i-1 carbonyl/15N
i
-tryptophan isotope labeled amide pairs, and H/D exchange 1H,15N-HSQC experiments conducted in solution, we assigned chemical shifts to all five rhodopsin tryptophan backbone 15N nuclei and partially to their bound protons. 1H,15N chemical shift assignment was achieved for indole side chains of Trp351.30 and Trp1754.65. 15N chemical shifts were found to be similar when comparing those obtained in the native like reconstituted lipid environment
and those obtained in detergent micelles for all tryptophans except Trp1754.65 at the membrane interface. The results suggest that the integrated solution and solid state NMR approach presented provides
highly complementary information in the study of structure and dynamics of large membrane proteins like rhodopsin. 相似文献
15.
Extensive resonance overlap exacerbates assignment of intrinsically disordered proteins (IDPs). This issue can be circumvented
by utilizing 15N, 13C′ and 1HN spins, where the chemical shift dispersion is mainly dictated by the characteristics of consecutive amino acid residues.
Especially 15N and 13C′ spins offer superior chemical shift dispersion in comparison to 13Cα and 13Cβ spins. However, HN-detected experiments suffer from exchange broadening of amide proton signals on IDPs especially under
alkali conditions. To that end, we propose here two novel HA-detected experiments, (HCA)CON(CA)H and (HCA)NCO(CA)H and a new
assignment protocol based on panoply of unidirectional HA-detected experiments that enable robust backbone assignment of IDPs
also at high pH. The new approach was tested at pH 6.5 and pH 8.5 on cancer/testis antigen CT16, a 110-residue IDP, and virtually
complete backbone assignment of CT16 was obtained by employing the novel HA-detected experiments together with the previously
introduced iH(CA)NCO scheme. Remarkably, also those 10 N-terminal residues that remained unassigned in our earlier HN-detection
based assignment approach even at pH 6.5 were now readily assigned. Moreover, theoretical calculations and experimental results
suggest that overall sensitivity of the new experiments is also applicable to small or medium sized globular proteins that
require alkaline conditions. 相似文献
16.
Jehle S Hiller M Rehbein K Diehl A Oschkinat H van Rossum BJ 《Journal of biomolecular NMR》2006,36(3):169-177
A simple spectroscopic filtering technique is presented that may aid the assignment of 13C and 15N resonances of methyl-containing amino-acids in solid-state magic-angle spinning (MAS) NMR. A filtering block that selects methyl resonances is introduced in two-dimensional (2D) 13C-homonuclear and 15N–13C heteronuclear correlation experiments. The 2D 13C–13C correlation spectra are recorded with the methyl filter implemented prior to a 13C–13C mixing step. It is shown that these methyl-filtered 13C-homonuclear correlation spectra are instrumental in the assignment of Cδ resonances of leucines by suppression of Cγ–Cδ cross peaks. Further, a methyl filter is implemented prior to a 15N–13C transferred-echo double resonance (TEDOR) exchange scheme to obtain 2D 15N–13C heteronuclear correlation spectra. These experiments provide correlations between methyl groups and backbone amides. Some of the observed sequential 15N–13C correlations form the basis for initial sequence-specific assignments of backbone signals of the outer-membrane protein G. 相似文献
17.
A set of TROSY-HNCO (tHNCO)-based 3D experiments is presented for measuring 15N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral
overlap. Measurement of backbone 15N R
1, R
1ρ, 15N–{1H} NOE, and 15N CSA/dipolar cross correlation is demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium
channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane
domain, KcsATM, exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, τc = 38 ± 2.5 ns, at 50 °C and a diffusion anisotropy, , corresponding to an aspect ratio a/b ≥ 1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics (S
2 values in the 0.2–0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils.
Relaxation behavior in these domains confirms the position of the C-terminal helix, and indicates that in SDS micelles, this
amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence
of elevated backbone dynamics on the ps–ns time scale for the 5-residue selectivity filter, which selects K+ ions to enter the channel.
Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .
An erratum to this article can be found at 相似文献
18.
19.
Measurement of 15N relaxation in deuterated amide groups in proteins using direct nitrogen detection
15N chemical shielding tensors contain useful structural information, and their knowledge is essential for accurate analysis of protein backbone dynamics. The anisotropic component (CSA) of 15N chemical shielding can be obtained from 15N relaxation measurements in solution. However, the predominant contribution to nitrogen relaxation from 15N-(1)H dipolar coupling in amide groups limits the sensitivity of these measurements to the actual CSA values. Here we present nitrogen-detected NMR experiments for measuring 15N relaxation in deuterated amide groups in proteins, where the dipolar contribution to 15N relaxation is significantly reduced by the deuteration. Under these conditions nitrogen spin relaxation becomes a sensitive probe for variations in 15N chemical shielding tensors. Using the nitrogen direct-detection experiments we measured the rates of longitudinal and transverse 15N relaxation for backbone amides in protein G in D(2)O at 11.7 T. The measured relaxation rates are validated by comparing the overall rotational diffusion tensor obtained from these data with that from the conventional 15N relaxation measurements in H(2)O. This analysis revealed a 17-24 degree angle between the NH-bond and the unique axis of the 15N chemical shielding tensor. 相似文献
20.
The 6-dimensional (6D) APSY-seq-HNCOCANH NMR experiment correlates two sequentially neighboring amide moieties in proteins via the C′ and Cα nuclei, with efficient suppression of the back transfer from Cα to the originating amide moiety. The automatic analysis of two-dimensional (2D) projections of this 6D experiment with the use of GAPRO (Hiller et al., 2005) provides a high-precision 6D peak list, which permits automated sequential assignments of proteins with the assignment software GARANT (Bartels et al., 1997). The procedure was applied to two proteins, the 63-residue 434-repressor(1–63) and the 115-residue TM1290. For both proteins, complete sequential assignments for all NMR-observable backbone resonances were obtained, and the polypeptide segments thus identified could be unambiguously located in the amino acid sequence. These results demonstrate that APSY-NMR spectroscopy in combination with a suitable assignment algorithm can provide fully automated sequence-specific backbone assignments of small proteins.Francesco Fiorito and Sebastian Hiller - Both authors contributed equally to this work 相似文献