Side-chain 13C Resonances Research Articles

Correlation of Backbone Amide and Side-Chain 13C Resonances in Perdeuterated Proteins

Side-chain carbon resonance assignments are difficult to obtain for larger proteins. While standard methods require protons for excitation and detection of magnetization, their presence is often unacceptable and often leads to unacceptable relaxation losses at the directly bound carbon sites. In this paper, pulse sequences are presented which provide connectivities between aliphatic side-chain 13C and amide 1H and 15N chemical shifts in fully deuterated, 13C/15N-enriched proteins. Magnetization either starts off from carbons or from both nitrogens and protons and is passed along the side-chain via 13C–13C isotropic mixing. Direct rather than 13CO-relayed 15N→13Cα or 13Cα→15N transfer steps allow the detection of intraresidual as well as sequential correlations. To avoid ambiguities between these two types in the three-dimensional version of the experiments, a fourth dimension can be introduced to achieve their separation along a 13Cα frequency axis. The novel methods are demonstrated with the uniformly 2H/13C/15N labeled 35-kDa protein diisopropylfluorophosphatase from Loligo vulgaris.

Methods for sequential resonance assignment in solid, uniformly 13C, 15N labelled peptides: quantification and application to antamanide.

The application of adiabatic polarization-transfer experiments to resonance assignment in solid, uniformly 13C-15N-labelled polypeptides is demonstrated for the cyclic decapeptide antamanide. A homonuclear correlation experiment employing the DREAM sequence for adiabatic dipolar transfer yields a complete assignment of the C(alpha) and aliphatic side-chain 13C resonances to amino acid types. The same information can be obtained from a TOBSY experiment using the recently introduced P9(12)1 TOBSY sequence, which employs the J couplings as a transfer mechanism. A comparison of the two methods is presented. Except for some aromatic phenylalanine resonances, a complete sequence-specific assignment of the 13C and 15N resonances in antamanide is achieved by a series of selective or broadband adiabatic triple-resonance experiments. Heteronuclear transfer by adiabatic-passage Hartmann-Hahn cross polarization is combined with adiabatic homonuclear transfer by the DREAM and rotational-resonance tickling sequences into two- and three-dimensional experiments. The performance of these experiments is evaluated quantitatively.

Use of deuterium labeling in NMR: overcoming a sizeable problem

In summary, the use of deuteration in combination with uniformly 13C- and 15N-labeling dramatically improves the quality of NMR spectra of larger proteins. These improvements allow the backbone and side-chain signals of larger proteins to be assigned and aids in the acquisition and analysis of NOE data. Using these methods, three-dimensional structures of proteins up to 30kDa have been determined [[[20]xStructure and ligand recognition of the phosphotyrosine binding domain of Shc. Zhou, M.-M...., and Fesik, S.W. Nature. 1995; 378: 584–592Crossref | PubMedSee all References, [31]xX-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Muchmore, S.W...., and Fesik, S.W. Nature. 1996; 381: 335–341Crossref | PubMedSee all References, [32]xStructure of Bcl-xL–Bak peptide complex reveals how regulators of apoptosis interact. Sattler, M...., and Fesik, S.W. Science. 1996; : in pressSee all References] SWF, unpublished data]. Further developments of NMR methods and variation of labeling techniques promise to push the molecular weight limit even further.

1H, 13C and 15N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b5.

Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain 13C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b5. The assignment of backbone 13C resonances was used to confirm previous 1H and 15N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329-8340]. On the basis of short- and medium-range NOEs and backbone 13C chemical shifts, the solution secondary structure of rat cytochrome b5 has been determined. The striking similarity of backbone 13C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the 13C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins

The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain 13C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D 15N/15N-separated NOESY, as many main-chain and side-chain 1HN/15N resonances as possible must be assigned. Traditionally, only backbone amide 1HN/15N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain 1HN/15N resonances to side-chain 13C resonances with high sensitivity: NH2-filtered 2D 1H-15N HSQC(H2N-HSQC), 3D H2N(CO)C gamma/beta and 3D H2N(COC gamma/beta)C beta/alpha for glutamine and asparagine side-chain amide groups; 2D refocused H(N epsilon/zeta)C delta/epsilon and H(N epsilon/zeta C delta/epsilon)C gamma/delta for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N epsilon)C zeta and nonrefocused H(N epsilon, eta)C zeta for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated 13C-/15N-labeled human carbonic anhydrase II (2H-HCA II). Because more than 95% of all side-chain 13C resonances in 2H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain 1HN/15N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain HN protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only 1HN-1HN NOE restraints. In these studies, the inclusion of NOE restraints to side-chain HN protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592-9593].

Assignment of Side-Chain 13C Resonances in Perdeuterated Proteins

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAssignment of Side-Chain 13C Resonances in Perdeuterated ProteinsBennett T. Farmer II and Ronald A. VentersCite this: J. Am. Chem. Soc. 1995, 117, 14, 4187–4188Publication Date (Print):April 1, 1995Publication History Published online1 May 2002Published inissue 1 April 1995https://doi.org/10.1021/ja00119a042RIGHTS & PERMISSIONSArticle Views155Altmetric-Citations58LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1000 KB) Get e-AlertsSupporting Info (2)»Supporting Information Supporting Information Get e-Alerts

Assignment of the 13C and 13CO resonances for Rhodobacter capsulatus ferrocytochrome c2 using double-resonance and triple-resonance NMR spectroscopy.

Rhodobacter capsulatus cytochrome c2 uniformly labelled with 13C/15N has been prepared. The 13C resonances of the reduced state, including those of the carbonyl and heme 13C, have been assigned using a combination of various two- and three-dimensional correlated NMR experiments. Assignment of the sidechain 13C resonances facilitated correction of a small number of previously misassigned sidechain 1H and led to the additional assignment of 32 1H. It was found that 13C alpha and 13CO secondary shifts were better indicators of secondary structure than 1H alpha and 13C beta secondary shifts. Moreover, it was demonstrated that, despite the significant ring current effects present in heme proteins, 13C alpha and 13CO secondary shifts can be employed to accurately identify secondary structure in heme proteins, independently of NOE experiments.

A systematic approach towards the complete assignment of 13C resonances for horse ferrocytochrome c

The complete 1H-NMR assignments for horse ferrocytochrome c have been reported by Wand and colleagues and by our group at Oxford. Using these 1H assignments, we now report chemical shift assignment for 205 13C resonances arising from horse ferrocytochrome c. This is from a total of 437 13C nuclei with covalently attached protons. These chemical shift assignments have been achieved using 1H-detected two-dimensional heteronuclear 1H-13C correlation techniques. The data have been collected from samples of horse ferrocytochrome c without isotopic enrichment. The complete 13C assignments for all carbons with covalently linked protons are reported for the amino acids Ala, Thr, Val and Gly. Specific assignments are tabulated for all 49 methyl groups, for 52 of 92 alpha-carbon resonances, for 10 resonances associated with the heme group, for all aromatic side-chain 13C resonances which have covalent protons and give rise to observable cross peaks under the experimental conditions used, as well as for a number of other side chains of aliphatic amino acids.