Determination of multiple ***φ***-torsion angles in proteins by selective and extensive (13)C labeling and two-dimensional solid-state NMR.

We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive (13)C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective (13)C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-(13)C]glucose preferentially labels the ends of the side chains, while [2-(13)C]glycerol labels the C(alpha) of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles phi; simultaneously, using an isotropic-anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively (13)C labeled protein were performed using (15)N-(13)C 2D correlation spectroscopy. From the time dependence of the (15)N-(13)C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective (13)C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.     

Determination of Multiple φ-Torsion Angles in Proteins by Selective and Extensive C Labeling and Two-Dimensional Solid-State NMR

We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive ¹³C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective ¹³C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-¹³C]glucose preferentially labels the ends of the side chains, while [2-¹³C]glycerol labels the C^α of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles φ simultaneously, using an isotropic–anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively ¹³C labeled protein were performed using ¹⁵N–¹³C 2D correlation spectroscopy. From the time dependence of the ¹⁵N–¹³C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective ¹³C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.     

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

Clean MAS observation of 13C-labeled carbons in membrane-bound HIV-1 and influenza fusion peptides was made by using a rotational-echo double-resonance spectroscopy (REDOR) filter of directly bonded 13C-15N pairs. The clean filtering achieved with the REDOR approach is superior to filtering done with sample difference spectroscopy. In one labeling approach, the peptide had labels at a single 13C carbonyl and its directly bonded 15N. The resulting chemical shift distribution of the filtered signal is used to assess the distribution of local secondary structures at the labeled carbonyl. For the influenza peptide, the Leu-2 carbonyl chemical shift distribution is shown to vary markedly with lipid and detergent composition, as well as peptide:lipid ratio, suggesting that the local peptide structure also has a strong dependence on these factors. Because most carboxylic- and amino-labeled amino acids are commercially available, this REDOR approach should have broad applicability to chemically synthesized peptides as well as bacterially synthesized proteins. In a second labeling approach, the HIV-1 fusion peptide had U-13C, 15N labeling over three sequential residues. When a 1.6 ms REDOR dephasing time is used, only backbone 13C signals are observed. The resulting spectra are used to determine spectral linewidths and to assess feasibility of assignment of uniformly labeled peptide.     

非天然氨基酸在蛋白质动态特性核磁共振研究中的应用

核磁共振（NMR）是蛋白质结构解析的主要方法之一.除可获得蛋白质的高分辨结构外,NMR还可用于观测最接近生理条件的蛋白质动态构象,获得蛋白质行使生物学功能的详细机制.非天然氨基酸定点标记方法可显著减少大分子量蛋白质的信号数目,降低数据采集和分析难度,已广泛运用于蛋白质结构和功能研究.本文介绍了常用的在蛋白质中引入非天然氨基酸的实验方法,包括蛋白质化学合成法、蛋白质化学修饰法、氟代芳香族氨基酸插入和基因密码子编辑的位点特异性插入等方法,并介绍了部分应用非天然氨基酸结合NMR研究大分子量蛋白的成功案例.此外,此篇综述讨论了目前非天然氨基酸标记在蛋白质研究中的局限性及发展方向.     

Assignment of congested NMR spectra: carbonyl backbone enrichment via the Entner-Doudoroff pathway

In NMR spectra of complex proteins, sparse isotope enrichment can be important, in that the removal of many (13)C-(13)C homonuclear J-couplings can narrow the lines and thereby facilitate the process of spectral assignment and structure elucidation. We present a simple scheme for selective yet extensive isotopic enrichment applicable for production of proteins in organisms utilizing the Entner-Doudoroff (ED) metabolic pathway. An enrichment scheme so derived is demonstrated in the context of a magic-angle spinning solid-state NMR (MAS SSNMR) study of Pf1 bacteriophage, the host of which is Pseudomonas aeruginosa, strain K (PAK), an organism that uses the ED pathway for glucose catabolism. The intact and infectious Pf1 phage in this study was produced by infected PAK cells grown on a minimal medium containing 1-(13)C d-glucose ((13)C in position 1) as the sole carbon source, as well as (15)NH(4)Cl as the only nitrogen source. The 37MDa Pf1 phage consists of about 93% major coat protein, 1% minor coat proteins, and 6% single-stranded, circular DNA. As a consequence of this composition and the enrichment scheme, the resonances in the MAS SSNMR spectra of the Pf1 sample were almost exclusively due to carbonyl carbons in the major coat protein. Moreover, 3D heteronuclear NCOCX correlation experiments also show that the amino acids leucine, serine, glycine, and tyrosine were not isotopically enriched in their carbonyl positions (although most other amino acids were), which is as expected based upon considerations of the ED metabolic pathway. 3D NCOCX NMR data and 2D (15)N-(15)N data provided strong verification of many previous assignments of (15)N amide and (13)C carbonyl shifts in this highly congested spectrum; both the semi-selective enrichment patterns and the narrowed linewidths allowed for greater certainty in the assignments as compared with use of uniformly enriched samples alone.     

Rotational resonance in uniformly 13C-labeled solids: effects on high-resolution magic-angle spinning NMR spectra and applications in structural studies of biomolecular systems

We describe investigations of the effects of rotational resonance (R(2)) on solid state (13)C NMR spectra of uniformly (13)C-labeled samples obtained under magic-angle spinning (MAS), and of the utility of R(2) measurements as structural probes of peptides and proteins with multiple uniformly labeled residues. We report results for uniformly (13)C-labeled L-alanine and L-valine in polycrystalline form, and for amyloid fibrils formed by the 15-residue peptide A beta(11-25) with uniform labeling of a four-residue segment. The MAS NMR spectra reveal a novel J-decoupling effect at R(2) conditions that may be useful in spectral assignments for systems with sharp (13)C MAS NMR lines. Pronounced dependences of the apparent isotropic (13)C NMR chemical shifts on MAS frequency near R(2) conditions are also observed. We demonstrate the feasibility of quantitative (13)C-(13)C distance determinations in L-valine, and qualitative determinations of inter-residue (13)C-(13)C contacts in A beta(11-25) fibrils. Finally, we demonstrate a "relayed" R(2) technique that may be useful in structural measurements on systems with poorly resolved (13)C MAS NMR lines.     

3D NMR spectroscopy for resonance assignment and structure elucidation of proteins under MAS: novel pulse schemes and sensitivity considerations

Two types of 3D MAS NMR experiments are introduced, which combine standard (NC,CC) transfer schemes with (1H,1H) mixing to simultaneously detect connectivities and structural constraints of uniformly 15N,13C-labeled proteins with high spectral resolution. The homonuclear CCHHC and CCC experiments are recorded with one double-quantum evolution dimension in order to avoid a cubic diagonal in the spectrum. Depending on the second transfer step, spin systems or proton-proton contacts can be determined with reduced spectral overlap. The heteronuclear NHHCC experiment encodes NH-HC proton-proton interactions, which are indicative for the backbone conformation of the protein. The third dimension facilitates the identification of the amino acid spin system. Experimental results on U-[15N,13C]valine and U-[15N,13C]ubiquitin demonstrate their usefulness for resonance assignments and for the determination of structural constraints. Furthermore, we give a detailed analysis of alternative multidimensional sampling schemes and their effect on sensitivity and resolution.     

Functional group contributions to the auger spectra of methyl cyanide and methyl isocyanide

We have obtained and analyzed the C and N core—valence—valence Auger spectra of methyl cyanide and methyl isocyanide. From both a fingerprint level interpretation, based on nitrogen, acetylene and methane as models, and from detailed spectral assignments, based on one-electron binding energies, we have shown that the spectra can be interpreted as resulting from independent contributions from the methyl group and the cyanide (or isocyanide) group. The contributions from the methyl and cyanide (or isocyanide) groups occur at different energies and exhibit different degrees of hole—hole correlation energy; the latter is shown to be consistent with differing degrees of functional group orbital delocalization. The detailed assignments to experimental spectral features, for the transitions involving the four highest filled molecular orbitais, form a consistent set with only a single exception. Intensity is missing in the region of the C spectrum of methyl cyanide corresponding to transitions involving the cyanide group π and σ orbitals.     

High-Resolution Four-Dimensional HMQC-NOESY-HSQC Spectroscopy

Practical optimization of the 4D [(1)H, (13)C, (13)C, (1)H] HMQC-NOESY-HSQC experiment in terms of distribution of resolution over the indirect dimensions is analyzed in detail. Recommendations for an optimal experiment are based on computer simulations assessing the effective resolution of the experiment, defined as the percentage of all possible NOE cross peaks that can be assigned unambiguously on the basis of the spectral data alone. Using actual (13)C-(1)H spectra of an 18-kDa chaperone protein, the analysis shows that experiments with the best effective resolution are also among the most sensitive ones. When combined with an efficient aliasing scheme that reduces indirect spectral space 124-fold, a 4D experiment that yields unambiguous assignments for 41% of all possible NOE cross peaks can be recorded in 28 h. A high-resolution experiment, which can be recorded in 8 days, yields 61% unambiguous assignments and can be analyzed more easily using standard NMR display software. The predictions are verified with experimental 4D spectra from which 1850 NOEs (914 long-range) were extracted for the 18-kDa chaperone protein.     

Rotational diffusion of membrane proteins in aligned phospholipid bilayers by solid-state NMR spectroscopy

Solid-state NMR experiments on mechanically aligned bilayer and magnetically aligned bicelle samples demonstrate that membrane proteins undergo rapid rotational diffusion about the normal in phospholipid bilayers. Narrow single-line resonances are observed from 15N labeled sites in the trans-membrane helix of the channel-forming domain of the protein Vpu from HIV-1 in phospholipid bilayers with their normals at angles of 0 degrees, 20 degrees, 40 degrees, and 90 degrees, and bicelles with their normals at angles of 0 degrees and 90 degrees with respect to the direction of the applied magnetic field. This could only occur if the entire polypeptide undergoes rotational diffusion about the bilayer normal. Comparisons between experimental and simulated spectra are consistent with a rotational diffusion coefficient (DR) of approximately 10(5)s-1.     

圆果皂甙元的NMR和构型研究

本文利用了多种NMR技术,其中包括¹H ¹³C NMR,DEPT、COSY、C,H-COSY、COLOC和NOE差谱,首次确定了圆果皂甙元的结构及构型。     

Thermodynamically Induced Conformational Changes of the Cyanobacterial Circadian Clock Protein KaiB

Site-directed spin labeling electron spin resonance (ESR) was applied to investigate the local environment of the cyanobacterial circadian clock protein KaiB. We prepared five cysteine residue-substituted mutants of KaiB labeled with maleimide spin label (MSL). By comparing the ESR spectra of KaiBs carrying MSL at different positions (Thr64, Lys67, Tyr94, Gly98, and Ala101), local conformational changes were identified. The ESR spectra of MSL-T64C and MSL-K67C showed the relatively slow motion of MSL characterized by τ?=?79 and 59?ns at 4°C, respectively. The spectra of MSL-Y94C, MSL-G98C and MSL-A101C showed relatively fast motion characterized by τ?=?8.0, 4.1 and 3.1?ns at 4°C, respectively. These differences were explained by the local environments of the position in KaiB. On incubation at 40°C for 24?h, all ESR spectra of the labeled KaiBs changed, which can be explained by the structural relaxation of KaiB.     

Rapid assignment of protein side chain resonances using projection-reconstruction of (4,3)D HC(CCO)NH and intra-HC(C)NH experiments

The reconstruction of higher-dimensional NMR spectra from projections can provide significant savings in instrument time. Here, we demonstrate its application to the (4,3)D HC(CCO)NH and intra-HC(C)NH experiments. The latter experiment contains a novel intra-residue filter element, which selectively correlates the side chain resonances with the corresponding intra-residue amide resonances. Compared to the conventional HC(C)NH experiment, the intra-HC(C)NH experiment reduces the spectral complexity and thus the minimum number of projections required for artifact-free reconstruction by half. The use of the projection-reconstruction technique allows rapid data collection and unambiguous assignment of aliphatic side chain nuclei at high resolution.     

iHADAMAC: a complementary tool for sequential resonance assignment of globular and highly disordered proteins

An experiment, iHADAMAC, is presented that yields information on the amino-acid type of individual residues in a protein by editing the (1)H-(15)N correlations into seven different 2D spectra, each corresponding to a different class of amino-acid types. Amino-acid type discrimination is realized via a Hadamard encoding scheme based on four different spin manipulations as recently introduced in the context of the sequential HADAMAC experiment. Both sequential and intra-residue HADAMAC experiments yield highly complementary information that greatly facilitate resonance assignment of proteins with high frequency degeneracy, as demonstrated here for a 188-residue intrinsically disordered protein fragment of the hepatitis C virus protein NS5A.     

Do topological parameters of amino acids within protein contact networks depend on their physico-chemical properties?

The three dimensional structure of a protein is determined by the interactions of its constituent amino acids. Considering the amino acids as nodes and the non-bonded interactions among them in 3D space as edges, researchers have constructed protein contact networks and analyzed the values of several topological parameters to uncover different important aspects of proteins. Here, we have analyzed some of the topological parameters such as degree, strength, clustering coefficients, betweenness and closeness centrality of each of the twenty amino acids in a set of non-redundant proteins covering all classes and folds. The results show that the values of these topological parameters vary widely with different amino acids. Also, these values differ significantly with different length scales of proteins. Most of the hydrophobic residues along with Cys, Arg and His have larger contributions to the long range connectivities than short range. We have also studied whether the values of topological parameters have any significant dependency on the physico-chemical properties of the amino acids. While the clustering coefficients show a strong negative correlation with residual volumes, surface areas and number of atoms in the side chains of amino acids; the degrees, strengths and betweenness show positive correlations with the mentioned properties. All the topological parameters show high dependency on bulkiness and average area buried of the amino acid residues in all-range residue networks. The average degree shows higher dependency on hydrophobicity, while the average strength is more able to capture the essences of surface area, residual volume and number of atoms of amino acids. The hydrophobicities of the amino acids and their corresponding degrees show a higher positive correlation in long range networks (LRNs) than short range networks (SRNs). The closeness centrality shows high correlation with two hydrophobic scales and no correlation with surface area, residual volume or number of atoms in LRNs. We have further explored the relationship in hydrophobic, hydrophilic and charged residues separately. Interestingly, charged residues show a higher dependency on the number of atoms than their residual volumes and surface areas. Finally, we present a linear regression model relating the network parameters with physico-chemical properties of amino acids.     

Imaging membrane protein helical wheels

Resonance patterns have been observed in 2D solid-state NMR spectra of the transmembrane segment of M2 protein from Influenza A virus in oriented samples reflecting the helical wheel of this alpha-helix. The center of this pattern uniquely defines the helical tilt with respect to the bilayer normal without a need for resonance assignments. The distribution of resonances from amino acid specific labels around the "PISA wheel" defines the rotational orientation of the helix and yields preliminary site-specific assignments. With assignments high-resolution structural detail, such as differences in tilt and rotational orientation along the helical axis leading to an assessment of helical coiling, can be obtained.     

Novel 2D triple-resonance NMR experiments for sequential resonance assignments of proteins

We present 2D versions of the popular triple resonance HN(CO) CACB, HN(COCA)CACB, HN(CO)CAHA, and HN(COCA) CAHA experiments, commonly used for sequential resonance assignments of proteins. These experiments provide information about correlations between amino proton and nitrogen chemical shifts and the alpha- and beta-carbon and alpha-proton chemical shifts within and between amino acid residues. Using these 2D spectra, sequential resonance assignments of H(N), N, C(alpha), C(beta), and H(alpha) nuclei are easily achieved. The resolution of these spectra is identical to the well-resolved 2D (15)N-(1)H HSQC and H(NCO)CA spectra, with slightly reduced sensitivity compared to their 3D and 4D versions. These types of spectra are ideally suited for exploitation in automated assignment procedures and thereby constitute a fast and efficient means for NMR structural determination of small and medium-sized proteins in solution in structural genomics programs.     

The structural analysis of protein sequences based on the quasi-amino acids code

Proteomics is the study of proteins and their interactions in a cell. With the successful completion of the Human Genome Project, it comes the postgenome era when the proteomics technology is emerging. This paper studies protein molecule from the algebraic point of view. The algebraic system (∑,+,*) is introduced, where ∑ is the set of 64 codons. According to the characteristics of (∑,+,*), a novel quasi-amino acids code classification method is introduced and the corresponding algebraic operation table over the set ZU of the 16 kinds of quasi-amino acids is established. The internal relation is revealed about quasi-amino acids. The results show that there exist some very close correlations between the properties of the quasi-amino acids and the codon. All these correlation relationships may play an important part in establishing the logic relationship between codons and the quasi-amino acids during the course of life origination. According to Ma F et al (2003 J. Anhui Agricultural University 30 439), the corresponding relation and the excellent properties about amino acids code are very difficult to observe. The present paper shows that (ZU, ⊕,○x) is a field. Furthermore, the operational results display that the codon tga has different property from other stop codons. In fact, in the mitochondrion from human and ox genomic codon, tga is just tryptophane, is not the stop codon like in other genetic code, it is the case of the Chen W C et al (2002 Acta Biophysica Sinica 18(1) 87). The present theory avoids some inexplicable events of the 20 kinds of amino acids code, in other words it solves the problem of `the 64 codon assignments of mRNA to amino acids is probably completely wrong' proposed by Yang (2006 Progress in Modern Biomedicine 6 3).     

DNP enhanced frequency-selective TEDOR experiments in bacteriorhodopsin

We describe a new approach to multiple ¹³C–¹⁵N distance measurements in uniformly labeled solids, frequency-selective (FS) TEDOR. The method shares features with FS-REDOR and ZF- and BASE-TEDOR, which also provide quantitative ¹⁵N–¹³C spectral assignments and distance measurements in U-[¹³C,¹⁵N] samples. To demonstrate the validity of the FS-TEDOR sequence, we measured distances in [U-¹³C,¹⁵N]-asparagine which are in good agreement with other methods. In addition, we integrate high frequency dynamic nuclear polarization (DNP) into the experimental protocol and use FS-TEDOR to record a resolved correlation spectrum of the Arg-¹³C_γ–¹⁵N_ε region in [U-¹³C,¹⁵N]-bacteriorhodopsin. We resolve six of the seven cross-peaks expected based on the primary sequence of this membrane protein.