Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance 13C NMR spectroscopy

The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance (13)C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the [(1)H]-(13)C NOE were determined in this study. The C alpha H relaxation measurements were compared to the previously measured (15)N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the chi(1) dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than +/-25 degrees.     

Accessibility of tobacco lipid transfer protein cavity revealed by 15N NMR relaxation studies and molecular dynamics simulations

Plant LTP1 are small helical proteins stabilized by four disulfide bridges and are characterized by the presence of an internal cavity, in which various hydrophobic ligands can be inserted. Recently, we have determined the solution structure of the recombinant tobacco LTP1_1. Unexpectedly, despite a global fold very similar to the structures already known for cereal seed LTP1, its binding properties are different: Tobacco LTP1_1 is able to bind only one monoacylated lipid, whereas cereal LTP1 can bind either one or two. The 3D structure of tobacco LTP1_1 revealed the presence of a hydrophobic cluster, not observed on cereal LTP1 structures, which may hinder one of the two entrances of the cavity defined for wheat LTP1. To better understand the mechanism of lipid entrance for tobacco LTP1_1 and to define the regions of the protein monitoring the accessibility of the cavity, we have complemented our structural data by the study of the internal dynamics of tobacco LTP1_1, using (15)N magnetic relaxation rate data and MD simulations at room and high temperatures. This work allowed us to define two regions of the protein experiencing the largest motions. These two regions delineate a portal that opens up during the simulation constituting a unique entrance of the hydrophobic cavity, in contrast with wheat LTP1 where two routes were detected. The hydrophobic interactions resulting from a few point mutations are strong enough to completely block the second portal so that the accessibility of the cavity is restricted to one entrance, explaining why this particular LTP1 binds only one lipid molecule.     

Internal motional amplitudes and correlated bond rotations in an alpha-helical peptide derived from 13C and 15N NMR relaxation

Peptide GFSKAELAKARAAKRGGY folds in an alpha-helical conformation that is stabilized by formation of a hydrophobic staple motif and an N-terminal capping box (Munoz V. Blanco FJ, Serrano L, 1995, Struct Biol 2:380-385). To investigate backbone and side-chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C- and 15N-enriched and NMR relaxation experiments were performed in water and in water/trifluoroethanol (TFE) solution at four Larmor frequencies (62.5, 125, 150, and 200 MHz for 13C). Relaxation data were analyzed using the model free approach and an anisotropic diffusion model. In water, angular variances of motional vectors range from 10 to 20 degrees and backbone phi,psi bond rotations for helix residues A5, L7, A8, and A10 are correlated indicating the presence of Calpha-H, Calpha-Cbeta, and N-H rocking-type motions along the helix dipole axis. L7 side-chain CbetaH2 and CgammaH motions are also correlated and as motionally restricted as backbone CalphaH, suggesting considerable steric hindrance with neighboring groups. In TFE which stabilizes the fold, internal motional amplitudes are attenuated and rotational correlations are increased. For the side chain of hydrophobic staple residue F2, wobbling-in-a-cone type motions dominate in water, whereas in TFE, the Cbeta-Cgamma bond and phenyl ring fluctuate more simply about the Calpha-Cbeta bond. These data support the Daragan-Mayo model of correlated bond rotations (Daragan VA, Mayo KH, 1996, J Phys Chem 100:8378-8388) and contribute to a general understanding of internal motions in peptides and proteins.     

Internal motions of apo-neocarzinostatin as studied by 13C NMR methine relaxation at natural abundance

Summary Dynamics of the backbone and some side chains of apo-neocarzinostatin, a 10.7 kDa carrier protein, have been studied from ¹³C relaxation rates R1, R2 and steady-state ¹³C-{¹H} NOEs, measured at natural abundance. Relaxation data were obtained for 79 nonoverlapping C resonances and for 11 threonine C single resonances. Except for three C relaxation rates, all data were analysed from a simple two-parameter spectral density function using the model-free approach of Lipari and Szabo. The corresponding C–H fragments exhibit fast (_e < 40 ps) restricted libration motions (S²=0.73 to 0.95). Global examination of the microdynamical parameters S² and _e along the amino acid sequence gives no immediate correlation with structural elements. However, different trends for the three loops involved in the binding site are revealed. The -ribbon comprising residues 37 to 47 is spatially restricted, with relatively large _e values in its hairpin region. The other -ribbon (residues 72 to 87) and the large disordered loop ranging between residues 97–107 experience small-amplitude motions on a much faster (picosecond) time scale. The two N-terminal residues, Ala¹ and Ala², and the C-terminal residue Asn¹¹³, exhibit an additional slow motion on a subnanosecond time scale (400–500 ps). Similarly, the relaxation data for eight threonine side-chain C must be interpreted in terms of a three-parameter spectral density function. They exhibit slower motions, on the nanosecond time scale (500–3000 ps). Three threonine (Thr⁶⁵, Thr⁶⁸, Thr⁸¹) side chains do not display a slow component, but an exchange contribution to the observed transverse relaxation rate R2 could not be excluded at these sites. The microdynamical parameters (S², _e and R2_ex) or (S _infslow ^sup2 , S _inffast ^sup2 and _slow) were obtained from a straightforward solution of the equations describing the relaxation data. They were calculated assuming an overall isotropic rotational correlation time _e for the protein of 5.7 ns, determined using standard procedures from R2/R1 ratios. However, it is shown that the product (1–S²)× _e is nearly independent of _e for residues not exhibiting slow motions on the nanosecond time scale. In addition, this parameter very closely follows the heteronuclear NOEs, which therefore could be good indices for local fast motions on the picosecond time scale.     

Histidine side-chain dynamics and protonation monitored by 13C CPMG NMR relaxation dispersion

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 ¹³C^ε1 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, ¹³C^ε1 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 ¹³C^ε1 dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics of the histidine side chains are discussed.     

Internal dynamics of d(CGCAAATTTGCG)2: a comparison of NMR relaxation measurements with a molecular dynamics simulation

We report the analysis of a 250 ps molecular dynamics simulation of the dodecamer d(CGCAAATTT-GCG)₂ immersed in a rectangular box of 3469 water molecules with 22 Na⁺ counterions. The internal dynamics of the molecule were investigated by studying the relevant autocorrelation functions related to the ¹³C-NMR relaxation parameters of the C1′-H1′ bonds of the sugar rings. The calculated effective correlation times τ _e (∼13 ps) and the order parameter S² (∼0.82) of the Lipari and Szabo formalism (Lipari and Szabo 1982a, b) are in satisfactory agreement with those determined previously by NMR (Gaudin et al. 1995, 1996). ¹H-¹H NOE buildups have also been measured experimentally and agree with those computed from the simulation. These results validate the simulation, and a more detailed analysis of the internal dynamics of the dodecamer was undertaken. Analysis of the distributions and of the autocorrelation functions of the glycosidic angle flucuations χ shows that the rotational motion of the sugar rings about their glycosidic bond conforms to a restricted diffusion mechanism. The amplitude of the motions and the diffusion constant are 20° and 17.10⁹ rad²s^–1 respectively. These values are in good agreement with ¹³C NMR data. Furthermore the simulation allows us to rule out another model also consistent with the experiment, consisting of a two-state jump between a syn and an anti conformation. Received: 19 November 1996 / Accepted: 17 March 1997     

Hydration dependence of backbone and side chain polylysine dynamics: a 13C solid-state NMR and IR spectroscopy study

The molecular dynamics of solid poly-L-lysine has been studied by the following natural abundance (13)C-NMR relaxation methods: measurements of the relaxation times T(1) at two resonance frequencies, off-resonance T(1rho) at two spin-lock frequencies, and proton-decoupled T(1rho). Experiments were performed at different temperatures and hydration levels (up to 17% H(2)O by weight). The natural abundance (13)C-CPMAS spectrum of polylysine provides spectral resolution of all types of backbone and side chain carbons and thus, dynamic parameters could be determined separately for each of them. At the same time, the conformational properties of polylysine were investigated by Fourier transform infrared spectroscopy. The data obtained from the different NMR experiments were simultaneously analyzed using the correlation function formalism and model-free approach. The results indicate that in dry polylysine both backbone and side chains take part in two low amplitude motions with correlation times of the order of 10(-4) s and 10(-9) s. Upon hydration, the dynamic parameters of the backbone remain almost constant except for the amplitude of the slower process that increases moderately. The side chain dynamics reveals a much stronger hydration response: the amplitudes of both slow and fast motions increase significantly and the correlation time of the slow motion shortens by about five orders of magnitude, and at hydration levels of more than 10% H(2)O fast and slow side chain motions are experimentally indistinguishable. These changes in the molecular dynamics cannot be ascribed to any hydration-dependent conformational transitions of polylysine because IR spectra reveal almost no hydration dependence in either backbone or side chain absorption domains. The physical nature of the fast and slow motions, their correlation time distributions, and hydration dependence of microdynamic parameters are discussed.     

Present dynamics of the savanna-forest boundary in the Congolese Mayombe: a pedological,botanical and isotopic (13C and 14C) study

Isolated savannas enclosed by forest are especially abundant in the eastern part of the Congolese Mayombe. They are about 3000 years old, and were more extensive some centuries ago. The boundary between forest and savanna is very abrupt, as a consequence of the numerous savanna fires lit by hunters. Floristic composition and vegetation structure data, organic carbon ratios, ¹⁴C and ¹³C measurements presented here show that forest is spreading over savanna at the present time and suggest that the rate of forest encroachment is is currently between 14 and 75 m per century, and more probably about 20–50 m per century. As most savannas are less than 1 km across, such rates mean, assuming there are no changes in environmental conditions, that enclosed savannas could completely disappear in the Mayombe in about 1000–2000 years.     

1H, 13C, and 15N resonance assignments of a general stress protein GSP13 from Bacillus subtilis

GSP13 encoded by gene yugI is a general stress protein in Bacillus subtilis. The NMR assignments of the protein are essential for its structure determination.     

Structure and dynamic properties of the single disulfide-deficient α-amylase inhibitor [C45A/C73A]tendamistat: An NMR study

Covalent linkages such as disulfide bonds are important for the stabilization of proteins. In the present NMR study we compare the structure and the dynamics of the single disulfide-deficient variant C45A/C73A of the α-amylase inhibitor tendamistat and the wild-type protein, which contains two disulfide bonds (C11-C27 and C45-C73). Complete proton assignment was achieved by standard homonuclear 2D techniques for the variant. Chemical shift differences, intra-strand NOE effects and protected amide proton were used to compare the connectivity of the secondary structure elements of variant and wild-type. Dynamic properties of the wild-type protein were studied by ¹³C_α heteronuclear NOE experiments with carbon in natural abundance. ¹⁵N isotope labeling was necessary to obtain the relaxation parameters of the variant, because of sample degradation. The ¹⁵N resonance assignment was achieved by a ¹⁵N 3D-NOESY-HMQC. Removal of the C45-C73 bond by the C45A/C73A mutation has no influence upon the β-barrel structure of tendamistat beside very local changes at the mutation site. The relaxation data revealed only subtle differences between variant and wild-type on a subnanosecond time scale. Only the N-terminus and G62 in the connecting loop between the anti-parallel β-sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and the secretion efficiency of tendamistat. Proteins 33:285–294, 1998. © 1998 Wiley-Liss, Inc.     

Carbon sequestration under Miscanthus: a study of 13C distribution in soil aggregates

The growing of bioenergy crops has been widely suggested as a key strategy in mitigating anthropogenic CO₂ emissions. However, the full mitigation potential of these crops cannot be assessed without taking into account their effect on soil carbon (C) dynamics. Therefore, we analyzed the C dynamics through four soil depths under a 14‐year‐old Miscanthus plantation, established on former arable land. An adjacent arable field was used as a reference site. Combining soil organic matter (SOM) fractionation with ¹³C natural abundance analyses, we were able to trace the fate of Miscanthus‐derived C in various physically protected soil fractions. Integrated through the whole soil profile, the total amount of soil organic carbon (SOC) was higher under Miscanthus than under arable crop, this difference was largely due to the input of new C. The C stock of the macroaggregates (M) under Miscanthus was significantly higher than those in the arable land. Additionally, the C content of the micro‐within macroaggregates (mM) were higher in the Miscanthus soil as compared with the arable soil. Analysis of the intramicroaggregates particulate organic matter (POM) suggested that the increase C storage in mM under Miscanthus was caused by a decrease in disturbance of M. Thus, the difference in C content between the two land use systems is largely caused by soil C storage in physically protected SOM fractions. We conclude that when Miscanthus is planted on former arable land, the resulting increase in soil C storage contributes considerably to its CO₂ mitigation potential.     

13C NMR relaxation studies of RNA base and ribose nuclei reveal a complex pattern of motions in the RNA binding site for human U1A protein

The widespread importance of induced fit and order-disorder transition in RNA recognition by proteins and small molecules makes it imperative that RNA motional properties are characterized quantitatively. Until now, however, very few studies have been dedicated to the systematic characterization of RNA motion and to their changes upon protein or small-molecule binding. The U1A protein-RNA complexes provide some of the best-studied examples of the role of RNA motional changes upon protein binding. Here, we report (13)C NMR relaxation studies of base and ribose dynamics for the RNA internal loop target of human U1A protein located within the 3'-untranslated region (3'-UTR) of the mRNA coding for U1A itself. We also report the semi-quantitative analysis of both fast (nano- to picosecond) and intermediate (micro- to millisecond) motions for this paradigmatic RNA system. We measure (13)C T(1), T(1rho) and heteronuclear nuclear Overhauser effects (NOEs) for sugar and base nuclei, as well as the power dependence of T(1rho) at 500 MHz and 750 MHz, and analyze these results using the model-free formalism. The results provide a much clearer picture of the type of motions experienced by this RNA in the absence of the protein than was provided by the analysis of the structure based solely on NOEs and scalar couplings. They define a model where the RNA internal loop region "breathes" on a micro- to millisecond timescale with respect to the double-helical regions. Superimposed on this slower motion, the residues at the very tip of the loop undergo faster (nano- to picosecond) motions. We hypothesize that these motions allow the RNA to sample multiple conformations so that the protein can select a structure within the ensemble that optimizes intermolecular contacts.     

Time-saving methods for heteronuclear multidimensional NMR of (13C, 15N) doubly labeled proteins

Summary Heteronuclear 2D (¹³C, ¹H) and (¹⁵N, ¹H) correlation spectra of (¹³C, ¹⁵N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D time-shared or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.Abbreviations HMQC heteronuclear multiple-quantum coherence spectroscopy - HSQC heteronuclear single-quantum coherence spectroscopy - HSMQC heteronuclear single- and multiple-quantum coherence spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy     

On the human CYP2C9*13 variant activity reduction: a molecular dynamics simulation and docking study

Cytochrome P450 2C9 (CYP2C9) plays a key role in the metabolism of clinical drugs. CYP2C9 is a genetically polymorphic enzyme and some of its allelic variants have less activity compared to the wild-type form. Drugs with a narrow therapeutic index may cause serious toxicity to the individuals who carry such allele. CYP2C9*13, firstly identified by some of the present authors in a Chinese poor metabolizer of lornoxicam, is characterized by mutation encoding Leu90Pro substitution. Kinetic experiments show that CYP2C9*13 has less catalytic activity in elimination of diclofenac and lornoxicam in vitro. In order to explore the structure-activity relationship of CYP2C9*13, the three-dimensional structure models of the substrate-free CYP2C9*1 and its variant CYP2C9*13 are constructed on the basis of the X-ray crystal structure of human CYP2C9*1 (PDB code 1R9O) by molecular dynamics simulations. The structure change caused by Leu90Pro replacement is revealed and used to explain the dramatic decrease of the enzymatic activity in clearance of the two CYP2C9 substrates: diclofenac and lornoxicam. The trans configuration of the bond between Pro90 and Asp89 in CYP2C9*13 is firstly identified. The backbone of residues 106-108 in CYP2C9*13 turns over and their side chains block the entrance for substrates accessing so that the entrance of *13 shrinks greatly than that in the wild-type, which is believed to be the dominant mechanism of the catalytic activity reduction. Consequent docking study which is consistent with the results of the kinetic experiments by Guo et al. identifies the most important residues for enzyme-substrate complexes.     

Internal mobility of cyclic RGD hexapeptides studied by 13C NMR relaxation and the model-free approach

Summary The internal mobility of three isomeric cyclic RGD hexapeptides designed to contain two -turns in defined positions, cyclo(Arg-Gly-Asp-Gly-d-Pro-Pro) (I), cyclo(Arg-Gly-Asp-d-Pro-Gly-Pro) (II) and cyclo(Arg-Gly-Asp-d-Pro-Pro-Gly) (III), have been studied by ¹³C NMR longitudinal and transverse relaxation experiments and measurements of steady-state heteronuclear {¹H}-¹³C NOE enhancement with ¹³C at natural abundance. The data were interpreted according to the model-free formalism of Lipari and Szabo, which is usually applied to data from macromolecules or larger sized peptides with overall rotational correlation times exceeding 1 ns, to yield information about internal motions on the 10–100 ps time scale. The applicability of the model-free analysis with acceptable uncertainties to these small peptides, with overall rotational correlation times slightly below 0.3 ns, was demonstrated for this specific instance. Chemical exchange contributions to T₂ from slower motions were also identified in the process. According to the order parameters obtained for its backbone -carbon atoms, II has the most rigid backbone conformation on the 10–100 ps time scale, and I the most flexible. This result coincides with the results of earlier NMR-constrained conformational searches, which indicated greatest uncertainty in the structure of I and least in II.     

Accuracy and precision of NMR relaxation experiments and MD simulations for characterizing protein dynamics

Model-free parameters obtained from nuclear magnetic resonance (NMR) relaxation experiments and molecular dynamics (MD) simulations commonly are used to describe the intramolecular dynamical properties of proteins. To assess the relative accuracy and precision of experimental and simulated model-free parameters, three independent data sets derived from backbone ¹⁵N NMR relaxation experiments and two independent data sets derived from MD simulations of Escherichia coli ribonuclease HI are compared. The widths of the distributions of the differences between the order parameters for pairs of NMR data sets are congruent with the uncertainties derived from statistical analyses of individual data sets; thus, current protocols for analyzing NMR data encapsulate random uncertainties appropriately. Large differences in order parameters for certain residues are attributed to systematic differences between samples for intralaboratory comparisons and unknown, possibly magnetic field-dependent, experimental effects for interlaboratory comparisons. The widths of distributions of the differences between the order parameters for two NMR sets are similar to widths of distributions for an NMR and an MD set or for two MD sets. The linear correlations between the order parameters for an MD set and an NMR set are within the range of correlations observed between pairs of NMR sets. These comparisons suggest that the NMR and MD generalized order parameters for the backbone amide N—H bond vectors are of comparable accuracy for residues exhibiting motions on a fast time scale (<100 ps). Large discrepancies between NMR and MD order parameters for certain residues are attributed to the occurrence of “rare” motional events over the simulation trajectories, the disruption of an element of secondary structure in one of the simulations, and lack of consensus among the experimental data sets. Consequently, (easily detectable) severe distortions of local protein structure and infrequent motional events in MD simulations appear to be the most serious artifacts affecting the accuracy and precision, respectively, of MD order parameters relative to NMR values. In addition, MD order parameters for motions on a fast (<100 ps) timescale are more precisely determined than their NMR counterparts, thereby permitting more detailed dynamic characterization of biologically important residues by MD simulation than is sometimes possible by experimental methods. Proteins 28:481–493, 1997. © 1997 Wiley-Liss, Inc.     

The influence of Mg2+ coordination on 13C and 15N chemical shifts in CKI1RD protein domain from experiment and molecular dynamics/density functional theory calculations

Sequence dependence of ¹³C and ¹⁵N chemical shifts in the receiver domain of CKI1 protein from Arabidopsis thaliana, CKI1_RD, and its complexed form, CKI1_RD?Mg²⁺, was studied by means of MD/DFT calculations. MD simulations of a 20–ns production run length were performed. Nine explicitly hydrated structures of increasing complexity were explored, up to a 40‐amino‐acid structure. The size of the model necessary depended on the type of nucleus, the type of amino acid and its sequence neighbors, other spatially close amino acids, and the orientation of amino acid NH groups and their surface/interior position. Using models covering a 10 and a 15 Å environment of Mg²⁺, a semi‐quantitative agreement has been obtained between experiment and theory for the V67?I73 sequence. The influence of Mg²⁺ binding was described better by the 15 Å as compared to the 10 Å model. Thirteen chemical shifts were analyzed in terms of the effect of Mg²⁺ insertion and geometry preparation. The effect of geometry was significant and opposite in sign to the effect of Mg²⁺ binding. The strongest individual effects were found for ¹⁵N of D70, S74, and V68, where the electrostatics dominated; for ¹³Cβ of D69 and ¹⁵N of K76, where the influences were equal, and for ¹³Cα of F72 and ¹³Cβ of K76, where the geometry adjustment dominated. A partial correlation between dominant geometry influence and torsion angle shifts upon the coordination has been observed. Proteins 2016; 84:686–699. © 2016 Wiley Periodicals, Inc.     

A combined HNCA/HNCO experiment for 15N labeled proteins with 13C at natural abundance

A triple resonance NMR experiment is presented for the simultaneous recording of HNCA and HNCO data sets on ¹⁵N, natural abundance ¹³C samples. The experiment exploits the fact that transfers of magnetization from ¹⁵N to ¹³CO and from ¹⁵N to ¹³C (and back) proceed independently for samples that are not enriched in ¹³C. A factor of 2 in measuring time is gained by recording the two data sets simultaneously with no compromise in spectral quality. An application to a 0.5 mM ¹⁵N labeled sample of protein-L is presented with all expected correlations observed in spectra recorded with a cryogenic probe at 500 MHz.