NMR: prediction of protein flexibility

We present a protocol for predicting protein flexibility from NMR chemical shifts. The protocol consists of (i) ensuring that the chemical shift assignments are correctly referenced or, if not, performing a reference correction using information derived from the chemical shift index, (ii) calculating the random coil index (RCI), and (iii) predicting the expected root mean square fluctuations (RMSFs) and order parameters (S2) of the protein from the RCI. The key advantages of this protocol over existing methods for studying protein dynamics are that (i) it does not require prior knowledge of a protein's tertiary structure, (ii) it is not sensitive to the protein's overall tumbling and (iii) it does not require additional NMR measurements beyond the standard experiments for backbone assignments. When chemical shift assignments are available, protein flexibility parameters, such as S2 and RMSF, can be calculated within 1-2 h using a spreadsheet program.     

Transient kinetics of ligand binding and role of the C-terminus in the dUTPase from equine infectious anemia virus

Transient kinetics of the equine infectious anemia virus deoxyuridine 5'-triphosphate nucleotide hydrolase were characterized by monitoring the fluorescence of the protein. Rate constants for the association and dissociation of substrate and inhibitors were determined and found to be consistent with a one-step mechanism for substrate binding. A C-terminal part of the enzyme presumed to be flexible was removed by limited trypsinolysis. As a result, the activity of the dUTPase was completely quenched, but the rate constants and fluorescent signal of the truncated enzyme were affected only to a minor degree. We conclude that the flexible C-terminus is not a prerequisite for substrate binding, but indispensable for catalysis.     

Modulation of human dUTPase using small interfering RNA

Analyzing the CYP2A6 gene of subjects who showed a poor metabolic phenotype toward SM-12502, we discovered a novel mutant allele (CYP2A6*4C) lacking the whole CYP2A6 gene. Using genetically engineered Salmonella typhimurium expressing a human CYP, we found that CYP2A6 was involved in the metabolic activation of a variety of nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) contained in tobacco smoke. Taking these results into consideration, we hypothesized that the subjects carrying the CYP2A6*4C allele had lower risk of tobacco-related lung cancer. In accordance with our hypothesis, our epidemiological studies indicated that smokers homozygous for the CYP2A6*4C allele showed much lower odds ratios toward cancer risk. Other mutant alleles reducing the CYP2A6 activity, besides CYP2A6*4C, also reduced the risk of lung cancer in smokers, particularly of squamous-cell carcinoma and small-cell carcinoma, both smoking-related cancers. 8-Methoxypsoralen, an inhibitor of CYP2A6, efficiently prevented the occurrence of adenoma caused by NNK in A/J mice.     

The RXRalpha C-terminus T462 is a NMR sensor for coactivator peptide binding

The C-terminal activation function-2 (AF-2) helix plays a crucial role in retinoid X receptor alpha (RXRα)-mediated gene expression. Here, we report a nuclear magnetic resonance (NMR) study of the RXRα ligand-binding domain complexed with 9-cis-retinoic acid and a glucocorticoid receptor-interacting protein 1 peptide. The AF-2 helix and most of the C-terminal residues were undetectable due to a severe line-broadening effect. Due to its outstanding signal-to-noise ratio, the C-terminus residue, threonine 462 (T462) exhibited two distinct crosspeaks during peptide titration, suggesting that peptide binding was in a slow exchange regime on the chemical shift timescale. Consistently, the K_d derived from T462 intensity decay agreed with that derived from isothermal titration calorimetry. Furthermore, the exchange contribution to the ¹⁵N transverse relaxation rate was measurable in either T462 or the bound peptide. These results suggest that T462 is a sensor for coactivator binding and is a potential probe for AF-2 helix mobility.     

Stochastic searches and NMR experiments on four Lewis A analogues: NMR experiments support some flexibility around the fucosidic bond

We have compared the conformational behavior of three Le^a analogues with that of Le^a using stochastic searches (MOE2005) and selective ROESY experiments. In the analogues either or both the β-d-Gal and α-l-Fuc residues were replaced by β-d-Glc and α-l-Rha units, respectively. All compounds showed similar behavior and even though four conformational families were identified, the calculations and NMR experiments support that the ‘stacked conformation’ known for Le^a is predominant for all analogues. Interestingly, ROESY showed a correlation between H-1 Fuc/Rha and H-3 GlcNAc which, although small, could be seen in all analogues. For two compounds, the corresponding distance was measured and found to be shorter (～3.7 Å) than that found in the global minimum (4.5 Å). While one published study suggests some motion around the fucosidic bond, this constitutes the first experimental evidence supporting such flexibility. Our MD simulation (Amber10/Glycam06) on Le^a was in full agreement with previous studies which described a rigid conformation for this branched trisaccharide. Thus, NMR seems to indicate that these dynamic studies are underestimating flexibility around the fucosidic bond.     

113Cd NMR studies of reconstituted seven-cadmium metallothionein: evidence for structural flexibility

A reproducible method for the reconstitution of rabbit liver metallothionein (MT) containing seven cadmium atoms per mole of protein is described. This protein was studied in detail by 113Cd NMR at 88-, 55-, and 44-MHz frequencies, including the effects of pH, temperature, and ionic strength on the spectra. Our results differ significantly from previous reports of 113Cd NMR on similar samples. Thus, the spectra of both chromatographically distinguishable isoforms MT1 and MT2 were not identical, and neither could be interpreted in terms of a unique static model with the seven Cd ions forming two independent clusters of four and three Cd ions. Large differential shifts of 113Cd resonances were observed with changes in temperature over the range 277-320 K and ionic strength (0.02-0.5 M). At low temperature a slow structural change (half-life of several minutes) was detected. The structure was more rigid at high ionic strength. The frequency dependence and two-dimensional J-resolved spectra revealed that 113Cd resonances were composed of several overlapping peaks, complicating the interpretation of fine structure in one-dimensional spectra. A new flexible model of the Cd cluster in metallothionein is proposed. This model incorporates dynamic thiolate exchange reactions and involves several configurational substates of the protein. The possible relationship of such flexibility to the function of metallothionein is discussed.     

Detection of protein-ligand interaction on the membranes using C-terminus biotin-tagged alamethicin

C-terminal biotin-tagged alamethicin, which has several alpha-aminoisobutyric acid (Aib) residues in its sequence, was synthesized by the preparation of the protected peptide segment using the 2-chlorotrityl resin, followed by conjugation with biotin hydrazide. Suppression of the channel current of the biotin-tagged alamethicin by the addition of streptavidin to the electrolyte was monitorable in real time using the planar lipid-bilayer method. The system was also applicable to the detection of interaction of the biotin-tagged alamethicin with the anti-biotin antibody.     

Phosphorylation and flexibility of cyclic-AMP-dependent protein kinase (PKA) using (31)P NMR spectroscopy

Cell signaling pathways rely on phosphotransfer reactions that are catalyzed by protein kinases. The protein kinases themselves are typically regulated by phosphorylation and concurrent structural rearrangements, both near the catalytic site and elsewhere. Thus, physiological function requires posttranslational modification and deformable structures. A prototypical example is provided by cyclic AMP-dependent protein kinase (PKA). It is activated by phosphorylation, is inhomogeneously phosphorylated when expressed in bacteria, and exhibits a wide range of dynamic properties. Here we use (31)P nuclear magnetic resonance (NMR) spectroscopy to characterize the phosphorylation states and to estimate the flexibility of the phosphorylation sites of 2-, 3-, and 4-fold phosphorylated PKA. The phosphorylation sites Ser10, Ser139, Thr197, and Ser338 are assigned to individual NMR resonances, assisted by complexation with AMP-PNP and dephosphorylation with alkaline phosphatase. Rotational diffusion correlation times estimated from resonance line widths show progressively increasing flexibilities for phosphothreonine 197, phosphoserines 139 and 338, and disorder at phosphoserine 10, consistent with crystal structures of PKA. However, because the apparent rotational diffusion correlation time fitted for phosphothreonine 197 of the activation loop is longer than the overall PKA rotational diffusion time, microsecond to millisecond time scale conformational exchange effects involving motions of phosphothreonine 197 are probable. These may represent "open"-"closed" transitions of the uncomplexed protein in solution. These data represent direct measurements of flexibilities also associated with functional properties, such as ATP binding and membrane association, and illustrate the applicability of (31)P NMR for functional and dynamic characterization of protein kinase phosphorylation sites.     

The effectiveness of 3 stretching techniques on hamstring flexibility using consistent stretching parameters

This study compares the effects of 3 common stretching techniques on the length of the hamstring muscle group during a 4-week training program. Subjects were 19 young adults between the ages of 21 and 35. The criterion for subject inclusion was tight hamstrings as defined by a knee extension angle greater than 20 degrees while supine with the hip flexed 90 degrees . The participants were randomly assigned to 1 of 4 groups. Group 1 (n = 5) was self-stretching, group 2 (n = 5) was static stretching, group 3 (n = 5) was proprioceptive neuromuscular facilitation incorporating the theory of reciprocal inhibition (PNF-R), and group 4 (n = 4) was control. Each group received the same stretching dose of a single 30-second stretch 3 days per week for 4 weeks. Knee extension angle was measured before the start of the stretching program, at 2 weeks, and at 4 weeks. Statistical analysis (p < or = 0.05) revealed a significant interaction of stretching technique and duration of stretch. Post hoc analysis showed that all 3 stretching techniques increase hamstring length from the baseline value during a 4-week training program; however, only group 2 (static stretching) was found to be significantly greater than the control at 4 weeks. These data indicate that static stretching 1 repetition for 30 seconds 3 days per week increased hamstring length in young healthy subjects. These data also suggest that active self-stretching and PNF-R stretching 1 repetition for 30 seconds 3 days per week is not sufficient to significantly increase hamstring length in this population.     

病毒dUTPase研究进展

脱氧尿苷焦磷酸酶（dUTP pyrophosphatase,dUTPasc）广泛存在于真核、原核细胞和病毒等生物有机体中,通过催化水解脱氧尿苷三磷酸（dUTP）,减少尿嘧啶在DNA合成中的错误掺入,降低细胞中的dUTP／dTTP比例,保证DNA复制的正确性和顺利进行。病毒编码的dUTPasc具有种属特异性,且与病毒的毒力和高效复制密切相关。本文就dUTPase的生物学功能、分类特征、表达调控、分布定位及病毒dUTPase功能的研究进展进行了概述,为深入开展dUTPase功能研究提供理论基础。     

噬菌体羧基端展示系统

噬菌体羧基端展示系统在一些方面是优于氨基端展示系统的,它可以用于构建和筛选cDNA噬菌体表面展示库,研究需要有自由羧基端时的蛋白质相互作用以及难以分泌的胞内蛋白的展示,特别是构建和筛选cDNA噬菌体表面展示库,将会成为该类展示系统的最重要的应用领域和进一步发展完善的动力。     

The connexin 43 C-terminus: A tail of many tales

Connexins are chordate gap junction channel proteins that, by enabling direct communication between the cytosols of adjacent cells, create a unique cell signalling network. Gap junctional intercellular communication (GJIC) has important roles in controlling cell growth and differentiation and in tissue development and homeostasis. Moreover, several non-canonical connexin functions unrelated to GJIC have been discovered. Of the 21 members of the human connexin family, connexin 43 (Cx43) is the most widely expressed and studied. The long cytosolic C-terminus (CT) of Cx43 is subject to extensive post-translational modifications that modulate its intracellular trafficking and gap junction channel gating. Moreover, the Cx43 CT contains multiple domains involved in protein interactions that permit crosstalk between Cx43 and cytoskeletal and regulatory proteins. These domains endow Cx43 with the capacity to affect cell growth and differentiation independently of GJIC. Here, we review the current understanding of the regulation and unique functions of the Cx43 CT, both as an essential component of full-length Cx43 and as an independent signalling hub. We highlight the complex regulatory and signalling networks controlled by the Cx43 CT, including the extensive protein interactome that underlies both gap junction channel-dependent and -independent functions. We discuss these data in relation to the recent discovery of the direct translation of specific truncated forms of Cx43. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

13C NMR relaxation and conformational flexibility of the deoxyribose ring.

13C T1's and NOE's have been measured for all protonated carbons of 2'-deoxy-D-ribose (2'-d-ribose), 2'-deoxyadenosine-5'-monophosphate (5'-dAMP), thymidine-3'-monophosphate (3'-TMP) and thymidine-5'-monophosphate (5'-TMP) in D2O solutions. In all of the deoxy sugars examined, NT1 values for C-2' are significantly larger than the values for the remaining carbons. This result is interpreted in terms of rapid puckering motion of C-2'. By contrast, NT1 values measured in ribose are found to be equal, within experimental error. The results are compared with analogous data obtained for the five membered pyrrolidine ring of proline and with results for DNA itself.     

Arginine kinase: joint crystallographic and NMR RDC analyses link substrate-associated motions to intrinsic flexibility

The phosphagen kinase family, including creatine and arginine kinases (AKs), catalyzes the reversible transfer of a “high-energy” phosphate between ATP and a phosphoguanidino substrate. They have become a model for the study of both substrate-induced conformational change and intrinsic protein dynamics. Prior crystallographic studies indicated large substrate-induced domain rotations, but differences among a recent set of AK structures were interpreted as a plastic deformation. Here, the structure of Limulus substrate-free AK is refined against high-resolution crystallographic data and compared quantitatively with NMR chemical shifts and residual dipolar couplings (RDCs). This demonstrates the feasibility of this type of RDC analysis of proteins that are large by NMR standards (42 kDa) and illuminates the solution structure, free from crystal-packing constraints. Detailed comparison of the 1.7 Å resolution substrate-free crystal structure against the 1.7 Å transition-state analog complex shows large substrate-induced domain motions that can be broken down into movements of smaller quasi-rigid bodies. The solution-state structure of substrate-free AK is most consistent with an equilibrium of substrate-free and substrate-bound structures, with the substrate-free form dominating, but with varying displacements of the quasi-rigid groups. Rigid-group rotations evident from the crystal structures are about axes previously associated with intrinsic millisecond dynamics using NMR relaxation dispersion. Thus, “substrate-induced” motions are along modes that are intrinsically flexible in the substrate-free enzyme and likely involve some degree of conformational selection.     

Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme

dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54 degrees C, 23 degrees C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg(2+) binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.     

Plasmodium falciparum dUTPase: studies on protein stability and binding of deoxyuridine derivatives

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme preventing a deleterious incorporation of uracil into DNA, has been thought of as a novel target for anticancer and antiviral drug design. The interaction of Plasmodium falciparum dUTPase (PfdUTPase) with deoxyuridine derivatives (dU, dUMP, dUDP and dUpNHpp) has been studied thermodynamically by both isothermal titration and differential scanning calorimetry. ITC shows no cooperativity for the binding of these derivatives. Dependencies in the binding thermodynamic parameters (enthalpy, entropy and Gibbs energy changes) with the number of phosphate groups in the nucleotide are obtained, and from the heat capacity changes no significant conformational changes upon binding are inferred. DSC shows PfdUTPase trimer is very stable but denatures irreversibly, with a more complex denaturation profile than other homologous trimeric dUTPases. The presence of magnesium ions does not influence the denaturation profile, while the presence of deoxyuridine derivatives increases the stability. The increase depends upon nucleotide concentration and type, with dUDP having the greater effect.     

New genes from old: redeployment of dUTPase by herpesviruses

Published work (D. J. McGeoch, Nucleic Acids Res. 18:4105-4110, 1990; J. E. McGeehan, N. W. Depledge, and D. J. McGeoch, Curr. Protein Peptide Sci. 2:325-333, 2001) has indicated that evolution of dUTPase in the class of herpesviruses that infect mammals and birds involved capture of a host gene followed by a duplication event that resulted in a coding region comprising two fused dUTPase domains. Some of the conserved residues required for enzyme activity were then lost, resulting in a dUTPase containing a single active site with different elements contributed by each half of the protein. Further conserved residues were lost in one subfamily (the Betaherpesvirinae), yielding a protein that is related to herpesvirus dUTPases but has a different and as yet unrecognized function. Evidence from sequence similarities and structural predictions now indicates that several additional genes were derived from the herpesvirus dUTPase gene, probably by duplication. These are UL31, UL82, UL83, and UL84 in human cytomegalovirus (and counterparts in other members of the Betaherpesvirinae) and ORF10 and ORF11 in human herpesvirus 8 (and counterparts in other members of the Gammaherpesvirinae). The findings clarify the evolutionary history of these genes and provide novel insights for structural and functional studies.     

Information-driven protein-DNA docking using HADDOCK: it is a matter of flexibility

Intrinsic flexibility of DNA has hampered the development of efficient protein−DNA docking methods. In this study we extend HADDOCK (High Ambiguity Driven DOCKing) [C. Dominguez, R. Boelens and A. M. J. J. Bonvin (2003) J. Am. Chem. Soc. 125, 1731–1737] to explicitly deal with DNA flexibility. HADDOCK uses non-structural experimental data to drive the docking during a rigid-body energy minimization, and semi-flexible and water refinement stages. The latter allow for flexibility of all DNA nucleotides and the residues of the protein at the predicted interface. We evaluated our approach on the monomeric repressor−DNA complexes formed by bacteriophage 434 Cro, the Escherichia coli Lac headpiece and bacteriophage P22 Arc. Starting from unbound proteins and canonical B-DNA we correctly predict the correct spatial disposition of the complexes and the specific conformation of the DNA in the published complexes. This information is subsequently used to generate a library of pre-bent and twisted DNA structures that served as input for a second docking round. The resulting top ranking solutions exhibit high similarity to the published complexes in terms of root mean square deviations, intermolecular contacts and DNA conformation. Our two-stage docking method is thus able to successfully predict protein−DNA complexes from unbound constituents using non-structural experimental data to drive the docking.     

NMR spectroscopy of phosphorylated wild-type rhodopsin: mobility of the phosphorylated C-terminus of rhodopsin in the dark and upon light activation

Binding of arrestin to light-activated rhodopsin involves recognition of the phosphorylated C-terminus and several residues on the cytoplasmic surface of the receptor. These sites are in close proximity in dark, unphosphorylated rhodopsin. To address the position and mobility of the phosphorylated C-terminus in the active and inactive receptor, we combined high-resolution solution and solid state NMR spectroscopy of the intact mammalian photoreceptor rhodopsin in detergent micelles as a function of temperature. The (31)P NMR resonance of rhodopsin phosphorylated by rhodopsin kinase at the C-terminal tail was observable with single pulse excitation using magic angle spinning until the sample temperature reached -40 degrees C. Below this temperature, the (31)P resonance broadened and was only observable using cross polarization. These results indicate that the phosphorylated C-terminus is highly mobile above -40 degrees C and immobilized at lower temperature. To probe the relative position of the immobilized phosphorylated C-terminus with respect to the cytoplasmic domain of rhodopsin, (19)F labels were introduced at positions 140 and 316 by the reaction of rhodopsin with 2,2,2-trifluoroethanethiol (TET). Solid state rotational-echo double-resonance (REDOR) NMR was used to probe the internuclear distance between the (19)F and the (31)P-labels. The REDOR technique allows (19)F...(31)P distances to be measured out to approximately 12 A with high resolution, but no significant dephasing was observed in the REDOR experiment in the dark or upon light activation. This result indicates that the distances between the phosphorylated sites on the C-terminus and the (19)F sites on helix 8 (Cys 316) and in the second cytoplasmic loop (Cys140) are greater than 12 A in phosphorylated rhodopsin.