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1.
Error estimation and global fitting in transverse-relaxation dispersion experiments to determine chemical-exchange parameters 总被引:2,自引:2,他引:0
Off-resonance effects can introduce significant systematic errors in R2 measurements in constant-time Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation dispersion experiments. For an off-resonance chemical shift of 500 Hz, 15N relaxation dispersion profiles obtained from experiment and computer simulation indicated a systematic error of ca. 3%. This error is three- to five-fold larger than the random error in R2 caused by noise. Good estimates of total R2 uncertainty are critical in order to obtain accurate estimates in optimized chemical exchange parameters and their uncertainties derived from χ2 minimization of a target function. Here, we present a simple empirical approach that provides a good estimate of the total error (systematic + random) in 15N R2 values measured for the HIV protease. The advantage of this empirical error estimate is that it is applicable even when some of the factors that contribute to the off-resonance error are not known. These errors are incorporated into a χ2 minimization protocol, in which the Carver–Richards equation is used fit the observed R2 dispersion profiles, that yields optimized chemical exchange parameters and their confidence limits. Optimized parameters are also derived, using the same protein sample and data-fitting protocol, from 1H R2 measurements in which systematic errors are negligible. Although 1H and 15N relaxation profiles of individual residues were well fit, the optimized exchange parameters had large uncertainties (confidence limits). In contrast, when a single pair of exchange parameters (the exchange lifetime, τex, and the fractional population, pa), were constrained to globally fit all R2 profiles for residues in the dimer interface of the protein, confidence limits were less than 8% for all optimized exchange parameters. In addition, F-tests showed that quality of the fits obtained using τex, pa as global parameters were not improved when these parameters were free to fit the R2 profiles of individual residues. Finally, nearly the same optimized global τex, pa values were obtained, when the 1H and 15N data sets for residues in the dimer interface, were fit independently; the difference in optimized global parameters, ca. 10%, was of marginal significance according to the F-test. 相似文献
2.
Artifacts associated with the measurement of methyl 1H single quantum CPMG-based relaxation dispersion profiles are described. These artifacts arise due to the combination of cross-correlated spin relaxation effects involving intra-methyl 1H–1H dipolar interactions and imperfections in 1H refocusing pulses that are applied during CPMG intervals that quantify the effects of chemical exchange on measured transverse relaxation rates. As a result substantial errors in extracted exchange parameters can be obtained. A simple work-around is presented where the 1H chemical shift difference between the exchanging states is extracted from a combination of 13C single quantum and 13C–1H multiple quantum dispersion profiles. The approach is demonstrated with an application to a folding/unfolding reaction involving a G48M mutant Fyn SH3 domain. 相似文献
3.
Ouwen Zhang Lewis E. Kay J. Paul Olivier Julie D. Forman-Kay 《Journal of biomolecular NMR》1994,4(6):845-858
Summary The backbone 1H and 15N resonances of the N-terminal SH3 domain of the Drosophila signaling adapter protein, drk, have been assigned. This domain is in slow exchange on the NMR timescale between folded and predominantly unfolded states. Data were collected on both states simultaneously, on samples of the SH3 in near physiological buffer exhibiting an approximately 1:1 ratio of the two states. NMR methods which exploit the chemical shift dispersion of the 15N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.Abbreviations 2D, 3D
two-, three-dimensional
- drkN SH3
N-terminal SH3 domain of Drosophila drk
- HSQC
heteronuclear single-quantum spectroscopy
- NOE
nuclear Overhauser enhancement
- SH3
Src homology domain 3
- TOCSY
total correlation spectroscopy 相似文献
4.
Neudecker P Zarrine-Afsar A Choy WY Muhandiram DR Davidson AR Kay LE 《Journal of molecular biology》2006,363(5):958-976
Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central beta-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal beta-strands that exist in the folded state (F). Initial studies focused on mutants where Gly48 is replaced; in an effort to establish whether this intermediate is a general feature of Fyn SH3 folding a series of 15N relaxation experiments monitoring the folding of Fyn SH3 mutants N53P/V55L and A39V/N53P/V55L are reported here. For these mutants as well, folding proceeds through an on-pathway intermediate with similar features to those observed for G48M and G48V Fyn SH3 domains. However, the 15N chemical shifts extracted for the intermediate indicate pronounced non-native contacts between the NH2 and COOH-terminal regions not observed previously. The kinetic parameters extracted for the folding of A39V/N53P/V55L Fyn SH3 from the three-state folding model F<-->I<-->U are in good agreement with folding and unfolding rates extrapolated to zero denaturant obtained from stopped-flow experiments analyzed in terms of a simplified two-state folding reaction. The folding of the triple mutant was studied over a wide range of temperatures, establishing that there is no difference in heat capacities between F and I states. This confirms a compact folding intermediate structure, which is supported by the 15N chemical shifts of the I state extracted from the dispersion data. The temperature-dependent relaxation data simplifies data analysis because at low temperatures (< 25 degrees C) the unfolded state (U) is negligibly populated relative to I and F. A comparison between parameters extracted at low temperatures where the F<-->I exchange model is appropriate with those from the more complex, three-state model at higher temperatures has been used to validate the protocol for analysis of three-site exchange relaxation data. 相似文献
5.
6.
Erik Walinda Daichi Morimoto Masahiro Shirakawa Kenji Sugase 《Journal of biomolecular NMR》2017,67(3):201-209
It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure (“excited states”) has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by 15N R 1ρ relaxation dispersion. The schemes use selective Hartmann–Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713–721, 2005; Hansen et al. in J Am Chem Soc 131:3818–3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-based R 1ρ relaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor. 相似文献
7.
NMR spin relaxation in the rotating frame (R1ρ) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond time scale. Here, we use amide 1H off-resonance R1ρ relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using 15N or 13C relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The 1H off-resonance R1ρ data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 μs. This is in contrast to the case for 15N, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly 15N-enriched samples that were either highly enriched in 2H or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the 1H transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present 1H off-resonance R1ρ experiments are in excellent agreement with those determined previously using a combination of 15N laboratory-frame and off-resonance R1ρ relaxation data, with average values of
and 21 ± 3 μs, respectively. 相似文献
8.
A labeling scheme is introduced that facilitates the measurement of accurate 13Cβ chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use
of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels
of 13C enrichment (30–40%) at Cβ side-chain carbon positions for 15 of the amino acids with little 13C label at positions one bond removed (≈5%). A pair of samples are produced using [1-13C]-glucose/NaH12CO3 or [2-13C]-glucose as carbon sources with isolated and enriched (>30%) 13Cβ positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy.
The utility of such samples for measurement of 13Cβ chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion
studies of a protein–ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles
compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples. 相似文献
9.
Carr–Purcell–Meiboom–Gill (CPMG) type relaxation dispersion experiments are now routinely used to characterise protein conformational dynamics that occurs on the μs to millisecond (ms) timescale between a visible major state and ‘invisible’ minor states. The exchange rate(s) (\( k_{{{\text{ex}}}} \)), population(s) of the minor state(s) and the absolute value of the chemical shift difference \(|{\Delta \varpi }|\) (ppm) between different exchanging states can be extracted from the CPMG data. However the sign of \({\Delta \varpi }\) that is required to reconstruct the spectrum of the ‘invisible’ minor state(s) cannot be obtained from CPMG data alone. Building upon the recently developed triple quantum (TQ) methyl \( ^{1} {\text{H}} \) CPMG experiment (Yuwen in Angew Chem 55:11490–11494, 2016) we have developed pulse sequences that use carbon detection to generate and evolve single quantum (SQ), double quantum (DQ) and TQ coherences from methyl protons in the indirect dimension to measure the chemical exchange-induced shifts of the SQ, DQ and TQ coherences from which the sign of \({\Delta \varpi }\) is readily obtained for two state exchange. Further a combined analysis of the CPMG data and the difference in exchange induced shifts between the SQ and DQ resonances and between the SQ and TQ resonances improves the estimates of exchange parameters like the population of the minor state. We demonstrate the use of these experiments on two proteins undergoing exchange: (1) the ~ 18 kDa cavity mutant of T4 Lysozyme (\( k_{{{\text{ex}}}} \sim\,3500{\text{ s}}^{{ - 1}} \)) and (2) the \(\sim\,4.7\) kDa Peripheral Sub-unit Binding Domain (PSBD) from the acetyl transferase of Bacillus stearothermophilus (\(k_{ex} \sim\,13,000\hbox { s}^{-1}\)). 相似文献
10.
A quantitative analysis of 2D 1H-15N spectra is often complicated by resonance overlap. Here a simple method is presented for resolving overlapped correlations by recording 2D projection planes from HNCO data sets. Applications are presented involving the measurement of 15N T1 relaxation rates in a high molecular weight protein, malate synthase G, and in a system that exchanges between folded and unfolded states, the drkN SH3 domain. By supplementing relaxation data recorded in the conventional way as a series of 2D 1H-15N data sets with a series of a pair of projection planes the number of dynamics probes is increased significantly for both systems studied. 相似文献
11.
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
12.
Guillaume Bouvignies Dmitry M. Korzhnev Philipp Neudecker D. Flemming Hansen Matthew H. J. Cordes Lewis E. Kay 《Journal of biomolecular NMR》2010,47(2):135-141
NMR relaxation dispersion spectroscopy is a powerful method for studying protein conformational dynamics whereby visible,
ground and invisible, excited conformers interconvert on the millisecond time-scale. In addition to providing kinetics and
thermodynamics parameters of the exchange process, the CPMG dispersion experiment also allows extraction of the absolute values
of the chemical shift differences between interconverting states,
| \Updelta [(w)\tilde] | \left| {\Updelta \tilde{\omega }} \right| , opening the way for structure determination of excited state conformers. Central to the goal of structural analysis is the
availability of the chemical shifts of the excited state that can only be obtained once the signs of
\Updelta [(w)\tilde] \Updelta \tilde{\omega } are known. Herein we describe a very simple method for determining the signs of 1HN
\Updelta [(w)\tilde] \Updelta \tilde{\omega } values based on a comparison of peak positions in the directly detected dimensions of a pair of 1HN–15N correlation maps recorded at different static magnetic fields. The utility of the approach is demonstrated for three proteins
that undergo millisecond time-scale conformational rearrangements. Although the method provides fewer signs than previously
published techniques it does have a number of strengths: (1) Data sets needed for analysis are typically available from other
experiments, such as those required for measuring signs of 15N
\Updelta [(w)\tilde] \Updelta \tilde{\omega } values, thus requiring no additional experimental time, (2) acquisition times in the critical detection dimension can be
as long as necessary and (3) the signs obtained can be used to cross-validate those from other approaches. 相似文献
13.
Alexandar L. Hansen Guillaume Bouvignies Lewis E. Kay 《Journal of biomolecular NMR》2013,55(3):279-289
A 13Cα chemical exchange saturation transfer based experiment is presented for the study of protein systems undergoing slow interconversion between an ‘observable’ ground state and one or more ‘invisible’ excited states. Here a labeling strategy whereby [2-13C]-glucose is the sole carbon source is exploited, producing proteins with 13C at the Cα position, while the majority of residues remain unlabeled at CO or Cβ. The new experiment is demonstrated with an application to the folding reaction of the Im7 protein that involves an on-pathway excited state. The obtained excited state 13Cα chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology. 相似文献
14.
Judith N. Haslett Peter B. Kang Mei Han Alvin T. Kho Despina Sanoudou Jay M. Volinski Alan H. Beggs Isaac S. Kohane Louis M. Kunkel 《Mammalian genome》2005,16(10):739-748
The phenotypic differences among Duchenne muscular dystrophy patients, mdx mice, and mdx5cv mice suggest that despite the common etiology of dystrophin deficiency, secondary mechanisms have a substantial influence
on phenotypic severity. The differential response of various skeletal muscles to dystrophin deficiency supports this hypothesis.
To explore these differences, gene expression profiles were generated from duplicate RNA targets extracted from six different
skeletal muscles (diaphragm, soleus, gastrocnemius, quadriceps, tibialis anterior, and extensor digitorum longus) from wild-type,
mdx, and mdx5cv mice, resulting in 36 data sets for 18 muscle samples. The data sets were compared in three different ways: (1) among wild-type
samples only, (2) among all 36 data sets, and (3) between strains for each muscle type. The molecular profiles of soleus and
diaphragm separate significantly from the other four muscle types and from each other. Fiber-type proportions can explain
some of these differences. These variations in wild-type gene expression profiles may also reflect biomechanical differences
known to exist among skeletal muscles. Further exploration of the genes that most distinguish these muscles may help explain
the origins of the biomechanical differences and the reasons why some muscles are more resistant than others to dystrophin
deficiency.
Electronic Supplementary Material Electronic Supplementary material is available for this article at
and accessible for authorised users.
Judith N. Haslett, Peter B. Kang These authors contributed equally to this work. 相似文献
15.
Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3 总被引:1,自引:0,他引:1
Carl Diehl Samuel Genheden Kristofer Modig Ulf Ryde Mikael Akke 《Journal of biomolecular NMR》2009,45(1-2):157-169
The conformational entropy of proteins can make significant contributions to the free energy of ligand binding. NMR spin relaxation enables site-specific investigation of conformational entropy, via order parameters that parameterize local reorientational fluctuations of rank-2 tensors. Here we have probed the conformational entropy of lactose binding to the carbohydrate recognition domain of galectin-3 (Gal3), a protein that plays an important role in cell growth, cell differentiation, cell cycle regulation, and apoptosis, making it a potential target for therapeutic intervention in inflammation and cancer. We used 15N spin relaxation experiments and molecular dynamics simulations to monitor the backbone amides and secondary amines of the tryptophan and arginine side chains in the ligand-free and lactose-bound states of Gal3. Overall, we observe good agreement between the experimental and computed order parameters of the ligand-free and lactose-bound states. Thus, the 15N spin relaxation data indicate that the molecular dynamics simulations provide reliable information on the conformational entropy of the binding process. The molecular dynamics simulations reveal a correlation between the simulated order parameters and residue-specific backbone entropy, re-emphasizing that order parameters provide useful estimates of local conformational entropy. The present results show that the protein backbone exhibits an increase in conformational entropy upon binding lactose, without any accompanying structural changes. 相似文献
16.
Pratibha Kumari Lukas Frey Alexander Sobol Nils-Alexander Lakomek Roland Riek 《Journal of biomolecular NMR》2018,72(3-4):125-137
15N R2 relaxation measurements are key for the elucidation of the dynamics of both folded and intrinsically disordered proteins (IDPs). Here we show, on the example of the intrinsically disordered protein α-synuclein and the folded domain PDZ2, that at physiological pH and near physiological temperatures amide—water exchange can severely skew Hahn-echo based 15N R2 relaxation measurements as well as low frequency data points in CPMG relaxation dispersion experiments. The nature thereof is the solvent exchange with deuterium in the sample buffer, which modulates the 15N chemical shift tensor via the deuterium isotope effect, adding to the apparent relaxation decay which leads to systematic errors in the relaxation data. This results in an artificial increase of the measured apparent 15N R2 rate constants—which should not be mistaken with protein inherent chemical exchange contributions, Rex, to 15N R2. For measurements of 15N R2 rate constants of IDPs and folded proteins at physiological temperatures and pH, we recommend therefore the use of a very low D2O molar fraction in the sample buffer, as low as 1%, or the use of an external D2O reference along with a modified 15N R2 Hahn-echo based experiment. This combination allows for the measurement of Rex contributions to 15N R2 originating from conformational exchange in a time window from µs to ms. 相似文献
17.
Several different factors in the collection and preservation of whale skin and blubber samples were examined to determine their effect on the results obtained by stable nitrogen and carbon isotope (δ15N and δ13C) analysis. Samples of wet killer whale skin retained their original stable isotope values for up to 14 d at 4°C or lower. However, decomposition significantly changed the δ15N value within 3 d at 20°C. Storage at ?20°C was as effective as ?80°C for the preservation of skin and blubber samples for stable isotope analysis for at least a year. By contrast, once a skin sample had been freeze‐dried and lipid extracted, the stable isotope values did not change significantly when it was stored dry at room temperature for at least 12 mo. Preservation of whale skin samples for a month in DMSO‐salt solution, frozen or at room temperature, did not significantly change the δ15N and δ13C values of lipid extracted tissues, although the slight changes seen could influence results of a study if only small changes are expected. 相似文献
18.
Anusha B. Gopalan Tairan Yuwen Lewis E. Kay Pramodh Vallurupalli 《Journal of biomolecular NMR》2018,72(1-2):79-91
Protein conformational changes play crucial roles in enabling function. The Carr–Purcell–Meiboom–Gill (CPMG) experiment forms the basis for studying such dynamics when they involve the interconversion between highly populated and sparsely formed states, the latter having lifetimes ranging from ~?0.5 to ~?5 ms. Among the suite of experiments that have been developed are those that exploit methyl group probes by recording methyl 1H single quantum (Tugarinov and Kay in J Am Chem Soc 129:9514–9521, 2007) and triple quantum (Yuwen et al. in Angew Chem Int Ed Engl 55:11490–11494, 2016) relaxation dispersion profiles. Here we build upon these by developing a third experiment in which methyl 1H double quantum coherences evolve during a CPMG relaxation element. By fitting single, double, and triple quantum datasets, akin to recording the single quantum dataset at static magnetic fields of Bo, 2Bo and 3Bo, we show that accurate exchange values can be obtained even in cases where exchange rates exceed 10,000 s?1. The utility of the double quantum experiment is demonstrated with a pair of cavity mutants of T4 lysozyme (T4L) with ground and excited states interchanged and with exchange rates differing by fourfold (~?900 s?1 and ~?3600 s?1), as well as with a fast-folding domain where the unfolded state lifetime is ~?80 µs. 相似文献
19.
Controls of nitrogen isotope patterns in soil profiles 总被引:5,自引:0,他引:5
To determine the dominant processes controlling nitrogen (N) dynamics in soils and increase insights into soil N cycling from nitrogen isotope (δ15N) data, patterns of 15N enrichment in soil profiles were compiled from studies on tropical, temperate, and boreal systems. The maximum 15N enrichment between litter and deeper soil layers varied strongly with mycorrhizal fungal association, averaging 9.6 ± 0.4‰ in ectomycorrhizal systems and 4.6 ± 0.5‰ in arbuscular mycorrhizal systems. The 15N enrichment varied little with mean annual temperature, precipitation, or nitrification rates. One main factor controlling 15N in soil profiles, fractionation against 15N during N transfer by mycorrhizal fungi to host plants, leads to 15N-depleted plant litter at the soil surface and 15N-enriched nitrogen of fungal origin at depth. The preferential preservation of 15N-enriched compounds during decomposition and stabilization is a second important factor. A third mechanism, N loss during nitrification and denitrification, may account for large 15N enrichments with depth in less N-limited forests and may account for soil profiles where maximum δ15N is at intermediate depths. Mixing among soil horizons should also decrease differences among soil horizons. We suggest that dynamic models of isotope distributions within soil profiles that can incorporate multiple processes could provide additional information about the history of nitrogen movements and transformations at a site. 相似文献
20.
R. Ramachandran C. Sich M. Grüne V. Soskic L. R. Brown 《Journal of biomolecular NMR》1996,7(3):251-255
Summary An approach for the simultaneous acquisition of HCN and HCP as well as HCN-CCH-TOCSY and HCP-CCH-TOCSY triple resonance data sets for 13C-/15N-labelled RNAs is presented. The new HCN-CCH-TOCSY scheme unambiguously links all sugar resonances to the base nitrogen. In addition, simultaneous acquisition of HCN-CCH-TOCSY and HCP-CCH-TOCSY data sets provides sequential and base-type information in a single experiment, thereby saving data acquisition time as well as providing complementary data sets that are useful in clarifying ambiguous assignments. Virtually complete sequence-specific phosphate-ribose 1H, 31P, and base 15N1,9 assignments as well as partial 13C assignments could be obtained in a single experiment for a 0.5-mM sample of a 19-mer ribonucleotide. 相似文献