Temperature dependence of fast carbonyl backbone dynamics in chicken villin headpiece subdomain

Temperature-dependence of protein dynamics can provide information on details of the free energy landscape by probing the characteristics of the potential responsible for the fluctuations. We have investigated the temperature-dependence of picosecond to nanosecond backbone dynamics at carbonyl carbon sites in chicken villin headpiece subdomain protein using a combination of three NMR relaxation rates: ¹³C′ longitudinal rate, and two cross-correlated rates involving dipolar and chemical shift anisotropy (CSA) relaxation mechanisms, ¹³C′/¹³C′-¹³C^α CSA/dipolar and ¹³C′/¹³C′–¹⁵N CSA/dipolar. Order parameters have been extracted using the Lipari-Szabo model-free approach assuming a separation of the time scales of internal and molecular motions in the 2–16°C temperature range. There is a gradual deviation from this assumption from lower to higher temperatures, such that above 16°C the separation of the time scales is inconsistent with the experimental data and, thus, the Lipari-Szabo formalism can not be applied. While there are variations among the residues, on the average the order parameters indicate a markedly steeper temperature dependence at backbone carbonyl carbons compared to that probed at amide nitrogens in an earlier study. This strongly advocates for probing sites other than amide nitrogen for accurate characterization of the potential and other thermodynamics characteristics of protein backbone.     

Phosphorylation-induced changes in backbone dynamics of the dematin headpiece C-terminal domain

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.     

Refinement of protein structure against non-redundant carbonyl <Superscript>13</Superscript>C NMR relaxation

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.     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

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.     

Quantifying Lipari–Szabo modelfree parameters from 13CO NMR relaxation experiments

It is proposed to obtain effective Lipari–Szabo order parameters and local correlation times for relaxation vectors of protein ¹³CO nuclei by carrying out a ¹³CO-R₁ auto relaxation experiment, a transverse CSA/dipolar cross correlation and a transverse ¹³CO CSA/¹³CO–¹⁵N CSA/dipolar cross correlation experiment. Given the global rotational correlation time from ¹⁵N relaxation experiments, a new program COMFORD (CO-Modelfree Fitting Of Relaxation Data) is presented to fit the ¹³CO data to an effective order parameter , an effective local correlation time and the orientation of the CSA tensor with respect to the molecular frame. It is shown that the effective is least sensitive to rotational fluctuations about an imaginary axis and most sensitive to rotational fluctuations about an imaginary axis parallel to the NH bond direction. As such, the information is fully complementary to the ¹⁵N relaxation order parameter, which is least sensitive to fluctuations about the NH axis and most sensitive to fluctuations about the axis. The new paradigm is applied on data of Ca²⁺ saturated Calmodulin, and on available literature data for Ubiquitin. Our data indicate that the order parameters rapport on slower, and sometimes different, motions than the ¹⁵N relaxation order parameters. The CO local correlation times correlate well with the calmodulin’s secondary structure. Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Carbonyl carbon label selective (CCLS) <Superscript>1</Superscript>H–<Superscript>15</Superscript>N HSQC experiment for improved detection of backbone <Superscript>13</Superscript>C–<Superscript>15</Superscript>N cross peaks in larger proteins

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.     

Backbone dynamics of bacteriorhodopsin as studied by <Superscript>13</Superscript>C solid-state NMR spectroscopy

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.     

An intraresidual i(HCA)CO(CA)NH experiment for the assignment of main-chain resonances in <Superscript>15</Superscript>N, <Superscript>13</Superscript>C labeled proteins

An improved pulse sequence, intraresidual i(HCA)CO(CA)NH, is described for establishing solely ¹³C′(i), ¹⁵N(i), ¹H^N(i) connectivities in uniformly ¹⁵N/¹³C-labeled proteins. In comparison to the “out-and-back” style intra-HN(CA)CO experiment, the new pulse sequence offers at least two-fold higher experimental resolution in the ¹³C′ dimension and on average 1.6 times higher sensitivity especially for residues in α-helices. Performance of the new experiment was tested on a small globular protein ubiquitin and an intrinsically unfolded 110-residue cancer/testis antigen CT16/PAGE5. Use of intraresidual i(HCA)CO(CA)NH experiment in combination with the established HNCO experiment was crucial for the assignment of highly disordered CT16.     

Determination of the residue-specific 15N CSA tensor principal components using multiple alignment media

The individual components of the backbone ¹⁵N CSA tensor, σ₁₁, σ₂₂, σ₃₃, and the orientation of σ₁₁ relative to the NH bond described by the angle β have been determined for uniformly labeled ¹⁵N, ¹³C ubiquitin from partial alignment in phospholipid bicelles, Pf1 phage, and poly(ethylene glycol) by measuring the residue-specific residual dipolar couplings and chemical shift deviations. No strong correlation between any of the CSA tensor components is observed with any single structural feature. However, the experimentally determined tensor components agree with the previously determined average CSA principal components [Cornilescu and Bax (2000) J. Am. Chem. Soc. 122, 10143–10154]. Significant deviations from the averages coincide with residues in β-strand or extended regions, while α-helical residue tensor components cluster close to the average values.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

HA-detected experiments for the backbone assignment of intrinsically disordered proteins

We propose a new alpha proton detection based approach for the sequential assignment of natively unfolded proteins. The proposed protocol superimposes on following features: HA-detection (1) enables assignment of natively unfolded proteins at any pH, i.e., it is not sensitive to rapid chemical exchange undergoing in natively unfolded proteins even at moderately high pH. (2) It allows straightforward assignment of proline-rich polypeptides without additional proline-customized experiments. (3) It offers more streamlined and less ambiguous assignment based on solely intraresidual ¹⁵N(i)-¹³C′(i)-H^α(i) (or ¹⁵N(i)-¹³C^α(i)-H^α(i)) and sequential ¹⁵N(i + 1)-¹³C′(i)-H^α(i) (or ¹⁵N(i + 1)-¹³C^α(i)-H^α(i)) correlation experiments together with efficient use of chemical shifts of ¹⁵N and ¹³C′ nuclei, which show smaller dependence on residue type. We have tested the proposed protocol on two proteins, small globular 56-residue GB1, and highly disordered, proline-rich 47-residue fifth repeat of EspF_U. Using the proposed approach, we were able to assign 90% of ¹H^α, ¹³C^α, ¹³C′, ¹⁵N chemical shifts in EspF_U. We reckon that the HA-detection based strategy will be very useful in the assignment of natively unfolded proline-rich proteins or polypeptide chains.     

Calculation of chemical shift anisotropy in proteins

Individual peptide groups in proteins must exhibit some variation in the chemical shift anisotropy (CSA) of their constituent atoms, but not much is known about the extent or origins of this dispersion. Direct spectroscopic measurement of CSA remains technically challenging, and theoretical methods can help to overcome these limitations by estimating shielding tensors for arbitrary structures. Here we use an automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) approach to compute ¹⁵N, ¹³C′ and ¹H chemical shift tensors for human ubiquitin and the GB1 and GB3 fragments of staphylococcal protein G. The average and range of variation of the anisotropies is in good agreement with experimental estimates from solid-state NMR, and the variation among residues is somewhat smaller than that estimated from solution-state measurements. Hydrogen-bond effects account for much of the variation, both between helix and sheet regions, and within elements of secondary structure, but other effects (including variations in torsion angles) may play a role as well.     

HNCO-based measurement of one-bond amide <Superscript>15</Superscript>N-<Superscript>1</Superscript>H couplings with optimized precision

A pair of 3D HNCO-based experiments have been developed with the aim of optimizing the precision of measurement of ¹J_NH couplings. Both pulse sequences record ¹J_NH coupling evolution during the entire constant time interval that ¹⁵N magnetization is dephasing or rephasing with respect to the directly bonded ¹³C′ nucleus, with ¹⁵N¹³C′ multiple quantum coherence maintained during the ¹³C′ evolution period. The first experiment, designed for smaller proteins, produces an apparent doubling of the ¹J_NH coupling without any accompanying increases in line width. The second experiment is a J-scaled TROSY-HNCO experiment in which the ¹J_NH coupling is measured by frequency difference between resonances offset symmetrically about the position of the downfield component of the ¹⁵N doublet (i.e. the TROSY resonance). This experiment delivers significant gains in precision of ¹J_NH coupling measurement compared to existing J-scaled TROSY-HNCO experiments. With the proper choice of acquisition parameters and sufficient sensitivity to acquire a 3D TROSY-HNCO experiment, it is shown that ¹J_NH couplings can be measured with a precision which approaches or exceeds the precision of measurement with which the frequency of the TROSY resonance itself can be determined.     

Alternate-site isotopic labeling of ribonucleotides for NMR studies of ribose conformational dynamics in RNA

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.     

REDOR: an assessment of the efficacy of dipolar recoupling with adiabatic inversion pulses

A numerical assessment of the efficacy of REDOR recoupling of heteronuclear dipolar interactions employing adiabatic dephasing pulses has been carried out by considering an isolated dipolar coupled spin 1/2 I-S system. At moderate magic angle spinning frequencies in the range of 3–6 kHz and when the CSA of the dephased spins is large, it is shown that efficient broadband heteronuclear dipolar recoupling and reliable distance estimates can be achieved even under conditions where a significant fraction of the rotor period is occupied by the adiabatic pulse. The efficacy of REDOR with adiabatic inversion pulses has been demonstrated experimentally in two model ¹⁵N-¹³C spin systems, (¹³C,¹⁵N) Aib-(¹⁵N) Aib-NH₂ (Aib = -aminoisobutyric acid) and (1-¹³C,¹⁵N) glycine.     

Measurement of 15N relaxation in the detergent-solubilized tetrameric KcsA potassium channel

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, KcsA^TM, exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, τ_c = 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     

Backbone assignment and dynamics of human α-synuclein in viscous 2 M glucose solution

We present ¹H^N, ¹⁵N, ¹³C^α, ¹³C^β and ¹³C′ assignments and ¹⁵N transverse relaxation rates (R₂) of a Parkinson’s disease-related intrinsically disordered protein, α-synuclein, in the presence of 2 M (360 g/l) glucose solution.     

Structure determination of proteins in <Superscript>2</Superscript>H<Subscript>2</Subscript>O solution aided by a deuterium-decoupled 3D HCA(N)CO experiment

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.     

Effects of Protein–pheromone Complexation on Correlated Chemical Shift Modulations

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.     

The stable carbon and nitrogen isotopic composition of vegetation in tropical forests of the Amazon Basin, Brazil

Here we present the within-site, seasonal, and interannual variations of the carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope ratios of leaves, wood, bark and litter from four sites in the Amazon region, Brazil. Samples were collected in Manaus (3° 06′07′′ S; 60°01′30′′ W), Ji-Paraná (10°53′07′′ S; 61°57′06′′ W), and Santarém (2°26′35′′ S; 54°42′30′′ W) with mean annual precipitation of 2207, 2040 and 1909 mm respectively. The overall average for all leaf samples was for δ¹³C and for δ¹⁵N (n=756). The leaf δ values at these sites were often but not always statistically distinct from each other. The δ¹³C values varied from to . Pronounced differences in δ¹³C values occurred with height associated with differences in forest structure. The δ¹³C of leaf dry matter showed seasonal variations associated with the length of the dry season, despite the fact that total annual precipitation was similar among the studied sites. Leaf δ¹⁵N values ranged from to a maximum value of , and the Santarém sites showed more enriched values than Manaus and Ji-Paraná sites. No seasonal variation was detected in the δ¹⁵N of leaves, but significant differences were observed among sites and with changes in canopy height. The isotope ratio data are consistent with our current understanding of the roles of light, water availability, and recycling of soil-respired CO₂ influences on δ¹³C and consistent with our understanding that an open nitrogen cycle can lead to high δ¹⁵N values despite a significant number of legumes in the vegetation.     

Broadband <Superscript>15</Superscript>N–<Superscript>13</Superscript>C dipolar recoupling via symmetry-based RF pulse schemes at high MAS frequencies

An approach for generating efficient RN_n^{n_S, n_k} {\rm{RN}}_{n}^{\nu_{\rm{S}}, {\nu_{\rm{k}}}} symmetry-based dual channel RF pulse schemes for γ-encoded broadband ¹⁵N–¹³C dipolar recoupling at high magic angle spinning frequencies is presented. The method involves the numerical optimisation of the RF phase-modulation profile of the basic “R” element so as to obtain heteronuclear double quantum dipolar recoupling sequences with satisfactory magnetisation transfer characteristics. The basic “R” element was implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by a RF phase and amplitude values. The performance characteristics of the sequences were evaluated via numerical simulations and ¹⁵N–¹³C chemical shift correlation experiments. Employing such ¹³C–¹⁵N double-quantum recoupling sequences and the multiple receiver capabilities available in the current generation of NMR spectrometers, the possibility to simultaneously acquire 3D NCC and CNH chemical shift correlation spectra is also demonstrated.