Evidence of Slow Motions by Cross-Correlated Chemical Shift Modulation in Deuterated and Protonated Proteins

Cross-correlated fluctuations of isotropic chemical shifts can provide evidence for slow motions in biomolecules. Slow side-chain dynamics have been investigated in (15)N and (13)C enriched ubiquitin by monitoring the relaxation of C(alpha)-C(beta) two-spin coherences (Frueh et al., 2001). This method, which had hitherto been demonstrated only for protonated ubiquitin, has now been applied to both protonated and deuterated proteins. Deuteration reduces the dipole-dipole contributions to the DD/DD cross-correlation, thus facilitating the observation of subtle effects due to cross-correlation of the fluctuations of the isotropic (13)C chemical shifts. The decays of double- and zero-quantum coherences are significantly slower in the deuterated protein than in the protonated sample. Slow motions are found both in loops and in secondary structure elements.     

Side chain dynamics monitored by 13C-13C cross-relaxation

A method to measure (13)C-(13)C cross-relaxation rates in a fully (13)C labeled protein has been developed that can give information about the mobility of side chains in proteins. The method makes use of the (H)CCH-NOESY pulse sequence and includes a suppression scheme for zero-quantum (ZQ) coherences that allows the extraction of initial rates from NOE buildup curves.The method has been used to measure (13)C-(13)C cross-relaxation rates in the 269-residue serine-protease PB92. We focused on C(alpha)-C(beta) cross-relaxation rates, which could be extracted for 64% of all residues, discarding serine residues because of imperfect ZQ suppression, and methyl (13)C-(13)C cross-relaxation rates, which could be extracted for 47% of the methyl containing C-C pairs. The C(alpha)-C(beta) cross-relaxation rates are on average larger in secondary structure elements as compared to loop regions, in agreement with the expected higher rigidity in these elements. The cross-relaxation rates for methyl containing C-C pairs show a general decrease of rates further into the side chain, indicating more flexibility with increasing separation from the main chain. In the case of leucine residues also long-range C(beta)-C(delta) cross-peaks are observed. Surprisingly, for most of the leucines a cross-peak with only one of the methyl C(delta) carbons is observed, which correlates well with the chi(2) torsion-angle and can be explained by a difference in mobility for the two methyl groups due to an anisotropic side chain motion.     

Slow backbone dynamics of the C-terminal fragment of human centrin 2 in complex with a target peptide probed by cross-correlated relaxation in multiple-quantum NMR spectroscopy

The C-terminal domain of human centrin 2 (C-HsCen2) strongly binds to P1-XPC, a peptide comprising 17 amino acids with a NWKLLAKGLLIRERLKR sequence. This peptide corresponds to residues N847-R863 of XPC, a protein involved in the recognition of damaged DNA during the initial step of the nucleotide excision repair pathway. The slow internal dynamics of the protein backbone in the C-HsCen-P1-XPC complex was studied by measuring the relaxation rates of zero- and double-quantum coherences involving neighboring pairs of carbonyl 13C and amide 15N nuclei. These relaxation rates, which reflect dynamics on time scales in the range of micro- to milliseconds, vary significantly along the protein backbone. Analysis of the relaxation rates at different CaCl2 concentrations and ionic strengths shows that these slow motions are mainly affected by the binding of a Ca2+ ion to the lower-affinity EF-hand III. Moreover, we discuss the possible functional role of residues that undergo differential exchange in the formation of HsCen homodimers.     

Triple Quantum Decoherence under Multiple Refocusing: Slow Correlated Chemical Shift Modulations of C′ and N Nuclei in Proteins

A new experiment allows the identification of residues that feature slow conformational exchange in macromolecules. Rotations about dihedral angles that are slower than the global correlation time tau(c) cause a modulation of the isotropic chemical shifts of the nuclei. If these fluctuations are correlated they induce a differential line broadening between three-spin single-quantum and triple-quantum coherences involving three nuclei such as the carbonyl C', the neighbouring amide nitrogen N and the amide proton H(N) belonging to a pair of consecutive amino acids. A cross-correlated relaxation rate R (CS/CS)(C'N) can be determined that corresponds to the sum of the isotropic and anisotropic contributions to the chemical shift modulations of the carbonyl carbon and nitrogen nuclei. Only the isotropic contributions depend on the pulse repetition rate of a multiple-refocusing sequence. An attenuation of the relaxation rate with increasing pulse repetition rate can therefore be attributed to slow motions. The asparagine N25 residue of ubiquitin, located in the first alpha-helix, is shown to feature significant slow conformational exchange.     

Factors affecting the use of 13C(alpha) chemical shifts to determine, refine, and validate protein structures

Interest centers here on the analysis of two different, but related, phenomena that affect side-chain conformations and consequently 13C(alpha) chemical shifts and their applications to determine, refine, and validate protein structures. The first is whether 13C(alpha) chemical shifts, computed at the DFT level of approximation with charged residues is a better approximation of observed 13C(alpha) chemical shifts than those computed with neutral residues for proteins in solution. Accurate computation of 13C(alpha) chemical shifts requires a proper representation of the charges, which might not take on integral values. For this analysis, the charges for 139 conformations of the protein ubiquitin were determined by explicit consideration of protein binding equilibria, at a given pH, that is, by exploring the 2(xi) possible ionization states of the whole molecule, with xi being the number of ionizable groups. The results of this analysis, as revealed by the shielding/deshielding of the 13C(alpha) nucleus, indicated that: (i) there is a significant difference in the computed 13C(alpha) chemical shifts, between basic and acidic groups, as a function of the degree of charge of the side chain; (ii) this difference is attributed to the distance between the ionizable groups and the 13C(alpha) nucleus, which is shorter for the acidic Asp and Glu groups as compared with that for the basic Lys and Arg groups; and (iii) the use of neutral, rather than charged, basic and acidic groups is a better approximation of the observed 13C(alpha) chemical shifts of a protein in solution. The second is how side-chain flexibility influences computed 13C(alpha) chemical shifts in an additional set of ubiquitin conformations, in which the side chains are generated from an NMR-derived structure with the backbone conformation assumed to be fixed. The 13C(alpha) chemical shift of a given amino acid residue in a protein is determined, mainly, by its own backbone and side-chain torsional angles, independent of the neighboring residues; the conformation of a given residue itself, however, depends on the environment of this residue and, hence, on the whole protein structure. As a consequence, this analysis reveals the role and impact of an accurate side-chain computation in the determination and refinement of protein conformation. The results of this analysis are: (i) a lower error between computed and observed 13C(alpha) chemical shifts (by up to 3.7 ppm), was found for approximately 68% and approximately 63% of all ionizable residues and all non-Ala/Pro/Gly residues, respectively, in the additional set of conformations, compared with results for the model from which the set was derived; and (ii) all the additional conformations exhibit a lower root-mean-square-deviation (1.97 ppm < or = rmsd < or = 2.13 ppm), between computed and observed 13C(alpha) chemical shifts, than the rmsd (2.32 ppm) computed for the starting conformation from which this additional set was derived. As a validation test, an analysis of the additional set of ubiquitin conformations, comparing computed and observed values of both 13C(alpha) chemical shifts and chi(1) torsional angles (given by the vicinal coupling constants, 3J(N-Cgamma) and 3J(C'-Cgamma), is discussed.     

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.     

Signal assignments and chemical-shift structural analysis of uniformly 13C, 15N-labeled peptide, mastoparan-X, by multidimensional solid-state NMR under magic-angle spinning

Carbon-13 and nitrogen-15 signals of fully isotope-labeled 15-residue peptide, glycinated mastoparan-X, in a solid state were assigned by two- and three-dimensional NMR experiments under magic-angle spinning conditions. Intra-residue spin connectivities were obtained with multidimensional correlation experiments for C'-C(alpha)-C(beta) and N-C(alpha)-C(beta). Sequence specific assignments were performed with inter-residue C(alpha)-C(alpha) and N-C(alpha)C(beta) correlation experiments. Pulse sequences for these experiments have mixing periods under recoupled zero- and double-quantum (13)C-(13)C and (15)N-(13)C dipolar interactions. These correlation spectra allowed the complete assignments of (13)C and (15)N backbone and (13)C(beta) signals. Chemical shift analysis of the (13)C and (15)N signals based on empirical and quantum chemical databases for proteins indicated that the backbone between residues 3 and 14 forms alpha-helix and residue 2 has extended conformation in the solid state. This structure was compared with the G-protein- and membrane-bound structures of mastoparan-X.     

1H, 13C, and 15N backbone assignment and secondary structure of the receptor-binding domain of vascular endothelial growth factor.

Nearly complete sequence-specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor-binding domain of vascular endothelial growth factor (VEGF), a 23-kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple-resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA-(CO)NH] and a 3D 15N-TOCSY-HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45 degrees C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1H alpha, 13C alpha, 13C beta, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N-NOESY-HSQC spectrum. Two helices and a significant amount of beta-sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192-7197].     

Three-dimensional deuterium-carbon correlation experiments for high-resolution solid-state MAS NMR spectroscopy of large proteins

Well-resolved (2)H-(13)C correlation spectra, reminiscent of (1)H-(13)C correlations, are obtained for perdeuterated ubiquitin and for perdeuterated outer-membrane protein G (OmpG) from E. coli by exploiting the favorable lifetime of (2)H double-quantum (DQ) states. Sufficient signal-to-noise was achieved due to the short deuterium T (1), allowing for high repetition rates and enabling 3D experiments with a (2)H-(13)C transfer step in a reasonable time. Well-resolved 3D (2)H(DQ)-(13)C-(13)C correlations of ubiquitin and OmpG were recorded within 3.5?days each. An essentially complete assignment of (2)H(DQα) shifts and of a substantial fraction of (2)H(DQβ) shifts were obtained for ubiquitin. In the case of OmpG, (2)H(DQα) and (2)H(DQβ) chemical shifts of a considerable number of threonine, serine and leucine residues were assigned. This approach provides the basis for a general heteronuclear 3D MAS NMR assignment concept utilizing pulse sequences with (2)H(DQ)-(13)C transfer steps and evolution of deuterium double-quantum chemical shifts.     

Backbone dynamics of reduced plastocyanin from the cyanobacterium Anabaena variabilis: regions involved in electron transfer have enhanced mobility

The dynamics of the backbone of the electron-transfer protein plastocyanin from the cyanobacterium Anabaena variabilis were determined from the (15)N and (13)C(alpha) R(1) and R(2) relaxation rates and steady-state [(1)H]-(15)N and [(1)H]-(13)C nuclear Overhauser effects (NOEs) using the model-free approach. The (13)C relaxation studies were performed using (13)C in natural abundance. Overall, it is found that the protein backbone is rigid. However, the regions that are important for the function of the protein show moderate mobility primarily on the microsecond to millisecond time scale. These regions are the "northern" hydrophobic site close to the metal site, the metal site itself, and the "eastern" face of the molecule. In particular, the mobility of the latter region is interesting in light of recent findings indicating that residues also on the eastern face of plastocyanins from prokaryotes are important for the function of the protein. The study also demonstrates that relaxation rates and NOEs of the (13)C(alpha) nuclei of proteins are valuable supplements to the conventional (15)N relaxation measurements in studies of protein backbone dynamics.     

Structural analysis of silk with 13C NMR chemical shift contour plots.

The polymorphic structures of silk fibroins in the solid state were examined on the basis of a quantitative relationship between the 13C chemical shift and local structure in proteins. To determine this relationship, 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues, and the C alpha chemical shift plot for Gly residues were prepared using atomic co-ordinates from the Protein Data Bank and 13C NMR chemical shift data in aqueous solution reported for 40 proteins. The 13C CP/MAS NMR chemical shifts of Ala, Ser and Gly residues of Bombyx mori silk fibroin in silk I and silk II forms were used along with 13C CP/MAS NMR chemical shifts of Ala residues of Samia cynthia ricini silk fibroin in beta-sheet and alpha-helix forms for the structure analyses of silk fibroins. The allowed regions in the 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues for the structures in silk fibroins, i.e. Silk II, Silk I and alpha-helix, were determined using their 13C isotropic NMR chemical shifts in the solid state. There are two area of the phi,psi map which satisfy the observed Silk I chemical shift data for both the C alpha and C beta carbons of Ala and Ser residues in the 13C chemical shift contour plots.     

Probing multiple effects on 15N, 13C alpha, 13C beta,and 13C' chemical shifts in peptides using density functional theory

We have used density functional calculations on model peptides to study conformational effects on (15)N, (13)C alpha, (13)C beta, and (13)C' chemical shifts, associated with hydrogen bonding, backbone conformation, and side-chain orientation. The results show a significant dependence on the backbone torsion angles of the nearest three residues. Contributions to (15)N chemical shifts from hydrogen bonding (up to 8 ppm), backbone conformation (up to 13 ppm), side-chain orientation and neighborhood residue effects (up to 22 ppm) are significant, and a unified theory will be required to account for their behavior in proteins. In contrast to this, the dependence on sequence and hydrogen bonding is much less for (13)C alpha and (13)C beta chemical shifts (<0.5 ppm), and moderate for carbonyl carbon shifts (<2 ppm). The effects of side-chain orientation are mainly limited to the residue itself for both nitrogen and carbon, but the chi(1) effect is also significant for the nitrogen shift of the following residue and for the (13)C' shift of the preceding residue. The calculated results are used, in conjunction with an additive model of chemical shift contributions, to create an algorithm for prediction of (15)N and (13)C shifts in proteins from their structure; this includes a model to extrapolate results to regions of torsion angle space that have not been explicitly studied by density functional theory (DFT) calculations. Crystal structures of 20 proteins with measured shifts have been used to test the prediction scheme. Root mean square deviations between calculated and experimental shifts 2.71, 1.22, 1.31, and 1.28 ppm for N, C alpha, C beta, and C', respectively. This prediction algorithm should be helpful in NMR assignment, crystal and solution structure comparison, and structure refinement.     

Effector-induced structural fluctuation regulates the ligand affinity of an allosteric protein: binding of inositol hexaphosphate has distinct dynamic consequences for the T and R states of hemoglobin

The present study reports distinct dynamic consequences for the T- and R-states of human normal adult hemoglobin (Hb A) due to the binding of a heterotropic allosteric effector, inositol hexaphosphate (IHP). A nuclear magnetic resonance (NMR) technique based on modified transverse relaxation optimized spectroscopy (TROSY) has been used to investigate the effect of conformational exchange of Hb A in both deoxy and CO forms, in the absence and presence of IHP, at 14.1 and 21.1 T, and at 37 degrees C. Our results show that the majority of the polypeptide backbone amino acid residues of deoxy- and carbonmonoxy-forms of Hb A in the absence of IHP is not mobile on the micros-ms time scale, with the exception of several amino acid residues, that is, beta109Val and beta132Lys in deoxy-Hb A, and alpha40Lys in HbCO A. The mobility of alpha40Lys in HbCO A can be explained by the crystallographic data showing that the H-bond between alpha40Lys and beta146His in deoxy-Hb A is absent in HbCO A. However, the conformational exchange of beta109Val, which is located in the intradimer (alpha 1beta 1 or alpha 2beta 2) interface, is not consistent with the crystallographic observations that show rigid packing at this site. IHP binding appears to rigidify alpha40Lys in HbCO A, but does not significantly affect the flexibility of beta109Val in deoxy-Hb A. In the presence of IHP, several amino acid residues, especially those at the interdimer (alpha 1beta 2 or alpha 2beta 1) interface of HbCO A, exhibit significant conformational exchange. The affected residues include the proximal beta92His in the beta-heme pocket, as well as some other residues located in the flexible joint (betaC helix-alphaFG corner) and switch (alphaC helix-betaFG corner) regions that play an important role in the dimer-dimer rotation of Hb during the oxygenation process. These findings suggest that, upon IHP binding, HbCO A undergoes a conformational fluctuation near the R-state but biased toward the T-state, apparently along the trajectory of its allosteric transition, accompanied by structural fluctuations in the heme pocket of the beta-chain. In contrast, no significant perturbation of the dynamic features on the ms-micros time scale has been observed upon IHP binding to deoxy-Hb A. We propose that the allosteric effector-induced quaternary structural fluctuation may contribute to the reduced ligand affinity of ligated hemoglobin. Conformational exchange mapping of the beta-chain of HbCO A observed at 21.1 T shows significantly increased scatter in the chemical exchange contribution to the transverse relaxation rate ( R ex) values, relative to those at lower fields, due to the enhanced effect of the local chemical shift anisotropy (CSA) fluctuation. A spring-on-scissors model is proposed to interpret the dynamic phenomena induced by the heterotropic effector, IHP.     

Protein backbone chemical shifts predicted from searching a database for torsion angle and sequence homology

Chemical shifts of nuclei in or attached to a protein backbone are exquisitely sensitive to their local environment. A computer program, SPARTA, is described that uses this correlation with local structure to predict protein backbone chemical shifts, given an input three-dimensional structure, by searching a newly generated database for triplets of adjacent residues that provide the best match in phi/psi/chi(1 )torsion angles and sequence similarity to the query triplet of interest. The database contains (15)N, (1)H(N), (1)H(alpha), (13)C(alpha), (13)C(beta) and (13)C' chemical shifts for 200 proteins for which a high resolution X-ray (< or =2.4 A) structure is available. The relative importance of the weighting factors for the phi/psi/chi(1) angles and sequence similarity was optimized empirically. The weighted, average secondary shifts of the central residues in the 20 best-matching triplets, after inclusion of nearest neighbor, ring current, and hydrogen bonding effects, are used to predict chemical shifts for the protein of known structure. Validation shows good agreement between the SPARTA-predicted and experimental shifts, with standard deviations of 2.52, 0.51, 0.27, 0.98, 1.07 and 1.08 ppm for (15)N, (1)H(N), (1)H(alpha), (13)C(alpha), (13)C(beta) and (13)C', respectively, including outliers.     

Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR

The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.     

Carbonyl carbon transverse relaxation dispersion measurements and ms-μs timescale motion in a protein hydrogen bond network

A constant-time, Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation, R(2), dispersion experiment for carbonyl carbons was designed and executed to detect micros-ms time-scale dynamics of protein backbone carbonyl sites. Because of the large (ca. 55 Hz) C(alpha)-C' J-coupling, the carbonyl signal intensity is strongly modulated as the spacing between CPMG pulses is varied, in uniformly (13)C enriched proteins, unless care is taken to minimize the perturbation of the C(alpha) magnetization by the CPMG pulses. CPMG pulse trains consisting of either a band-selective pulse, such as RE-BURP, or rectangular (with an excitation null in the C(alpha) region of the spectrum) pulses were employed in order to minimize C' signal modulation by C(alpha)-C' J-coupling. The performance of these types of CPMG refocusing pulses was assessed by computer simulation, and by comparing dispersion profiles measured for (1) uniformly [(13)C,(15)N, (2)H] ((2)H at non-labile hydrogen sites) labeled, and (2) uniformly (15)N/selectively-(13)C' labeled samples of HIV-1 protease bound to a potent inhibitor, DMP323. In addition, because the uniformly (13)C/(15)N/(2)H labeled sample was well suited to measure (15)N and (1)H R(2) dispersion as well as (13)C' dispersion, conformational exchange in the inter subunit beta-sheet hydrogen-bond network of the inhibitor-bound protease was elucidated using relaxation dispersion data of all three types of nuclei.     

Ubiquitin binding interface mapping on yeast ubiquitin hydrolase by NMR chemical shift perturbation.

The interaction between the 26 kDa yeast ubiquitin hydrolase (YUH1), involved in maintaining the monomeric ubiquitin pool in cells, and the 8.5 kDa yeast ubiquitin protein has been studied by heteronuclear multidimensional NMR spectroscopy. Chemical shift perturbation of backbone (1)H(N), (15)N, and (13)C(alpha) resonances of YUH1, in a YUH1-ubiquitin mixture and in a 35 kDa covalent complex with ubiquitin (a stable analogue of the tetrahedral reaction intermediate), was employed to identify the ubiquitin binding interface of YUH1. This interface mapped on the secondary structure of YUH1 suggests a wide area of contact for ubiquitin, encompassing the N-terminus, alpha1, alpha4, beta2, beta3, and beta6, coincident with the high specificity of YUH1 for ubiquitin. The presence of several hydrophobic clusters in the ubiquitin binding interface of YUH1 suggests that hydrophobic interactions are equally important as ionic interactions in contacting ubiquitin. The residues in the binding interface exhibit a high percentage of homology among the members of the ubiquitin C-terminal hydrolase family, indicating the well-conserved nature of the ubiquitin binding interface reported in this study. The secondary structure of YUH1, from our NMR studies, was similar to the recently determined structure of its human homologue ubiquitin C-terminal hydrolase L3 (UCH-L3), except for the absence of the helix H3 of UCH-L3. This region in YUH1 (helix H3 of UCH-L3) was least perturbed upon ubiquitin binding. Therefore, the binding interface was mapped onto the corresponding residues in the UCH-L3 crystal structure. A model for ubiquitin binding to YUH1 is proposed, in which a good correlation was observed for the lateral binding of ubiquitin to UCH-L3 (YUH1), stabilized by the electrostatic and hydrophobic interactions.     

Prospects for resonance assignments in multidimensional solid-state NMR spectra of uniformly labeled proteins

Summary The feasibility of assigning the backbone ¹⁵N and ¹³C NMR chemical shifts in multidimensional magic angle spinning NMR spectra of uniformly isotopically labeled proteins and peptides in unoriented solid samples is assessed by means of numerical simulations. The goal of these simulations is to examine how the upper limit on the size of a peptide for which unique assignments can be made depends on the spectral resolution, i.e., the NMR line widths. Sets of simulated three-dimensional chemical shift correlation spectra for artificial peptides of varying length are constructed from published liquid-state NMR chemical shift data for ubiquitin, a well-characterized soluble protein. Resonance assignments consistent with these spectra to within the assumed spectral resolution are found by a numerical search algorithm. The dependence of the number of consistent assignments on the assumed spectral resolution and on the length of the peptide is reported. If only three-dimensional chemical shift correlation data for backbone ¹⁵N and ¹³C nuclei are used, and no residue-specific chemical shift information, information from amino acid side-chain signals, and proton chemical shift information are available, a spectral resolution of 1 ppm or less is generally required for a unique assignment of backbone chemical shifts for a peptide of 30 amino acid residues.     

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.     

Assignment of protein backbone resonances using connectivity, torsion angles and 13Cα chemical shifts

A program is presented which will return the most probable sequence location for a short connected set of residues in a protein given just (13)C(alpha) chemical shifts (delta((13)C(alpha))) and data restricting the phi and psi backbone angles. Data taken from both the BioMagResBank and the Protein Data Bank were used to create a probability density function (PDF) using a multivariate normal distribution in delta((13)C(alpha)), phi, and psi space for each amino acid residue. Extracting and combining probabilities for particular amino acid residues in a short proposed sequence yields a score indicative of the correctness of the proposed assignment. The program is illustrated using several proteins for which structure and (13)C(alpha) chemical shift data are available.