Side Chain Resonance Assignments Research Articles

Resonance assignment strategies for the analysis of NMR spectra of proteins.

Determination of the high resolution solution structure of a protein using nuclear magnetic resonance (NMR) spectroscopy requires that resonances observed in the NMR spectra be unequivocally assigned to individual nuclei of the protein. With the advent of modern, two-dimensional NMR techniques arose methodologies for assigning the 1H resonances based on 2D, homonuclear 1H NMR experiments. These include the sequential assignment strategy and the main chain directed strategy. These basic strategies have been extended to include newer 3D homonuclear experiments and 2D and 3D heteronuclear resolved and edited methods. Most recently a novel, conceptually new approach to the problem has been introduced that relies on heteronuclear, multidimensional so-called triple resonance experiments for both backbone and sidechain resonance assignments in proteins. This article reviews the evolution of strategies for the assignment of resonances of proteins.

Membrane-induced helical conformation of an active candidacidal fragment of salivary histatins

The conformational preference of the candidacidal C-terminal 16 residue fragment (9-24; G-Y-K-R-K-F-H-E-K-H-H-S-H-R-G-Y) of salivary histatin 5 was examined in water, methanol, and dimethyl sulfoxide solutions using 500 MHz two-dimensional-NMR. Fourier transform infrared and CD spectroscopy were used to delineate its membrane-bound conformation in lipid vesicles. The peptide backbone and side-chain proton resonance assignments were accomplished by two-dimensional total correlated and nuclear Overhauser effect (NOE) spectra. The coupling constant (JNH-C alpha H) values determined from the double quantum-filtered correlated spectra, temperature coefficients of NH chemical shifts (d delta/dT), 1H/2H exchange rates on amide resonances, and the set of NOE connectivities were used to delineate backbone conformational features. The high JNH-C alpha H values (> or = 7.4 Hz), absence of any characteristic NH-NH (i, i + 1) or C alpha H-C beta H (i, i + 3) NOE connectivities, high d delta/dT values (> or = 0.004), and the fast 1H/2H amide exchange suggest that the histatin peptide favors unfolded random conformations in aqueous solution at pH 3.8. In contrast, the JNH-C alpha H values (< or = 6.5 Hz), slow 1H/2H exchange, low d delta/dT values (< or = 0.003) observed for amide resonances of residues 5-16, and the characteristic NH-NH (i, i + 1), C alpha H-C beta H (i, i + 3) NOE connectivities, provide evidence for the presence of largely alpha-helical conformations in dimethyl sulfoxide, which mimics the polar aprotic membrane environment. In methanolic solutions, 3(10)-helical conformations could exist as a minor population together with the major alpha-helical conformations. Fourier transform infrared spectroscopy and CD data indicate that lipid environments such as dimyristoylphosphatidylcholine vesicles could induce the peptide to fold into predominantly alpha-helical conformation. The results suggest that in dimethyl sulfoxide and dimyristoylphosphatidylcholine vesicles the candidacidal domain of salivary histatin 5 prefers a largely helical conformation, which could facilitate its interaction with the membrane of Candida albicans. The mechanism of antimicrobial action of this class of polypeptides appears to involve primarily electrostatic and hydrogen-bonding interaction of cationic and polar residues with the head groups of the plasma membranes of target cells.

Unusual helix-containing greek keys in development-specific Ca(2+)-binding protein S. 1H, 15N, and 13C assignments and secondary structure determined with the use of multidimensional double and triple resonance heteronuclear NMR spectroscopy.

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain 1H, 15N, and 13C resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain 1H and 13C assignments have been obtained by analysis of HCCH-TOCSY and 15N-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, 3JNH alpha coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a Greek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca(2+)-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., & Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca(2+)-binding site.

Automated Location of Peak Patterns in Multidimensional NMR Spectra: A Tool for Resonance Assignment in Proteins

Recent developments in protein NMR technology provide spectral data that are highly amenable to analysis by computer software systems. Automated methods of analysis use constraint satisfaction, pseudoenergy minimization, directed search, neural net, simulated annealing, and/or genetic algorithms to establish sequential links and sequence-specific assignments. The most advanced systems provide automated analysis of complete backbone and extensive side-chain resonance assignments for proteins of 50–150 amino acids.

Secondary structure and topology of Acanthamoeba profilin I as determined by heteronuclear nuclear magnetic resonance spectroscopy.

The protein profilin binds to both actin and the head groups of poly)phosphoinositide)s and may regulate both actin assembly and the phosphoinositide signaling pathway. As a first step in understanding the activity of profilin at the molecular level, we have determined the secondary structure of Acanthamoeba profilin I in solution using multidimensional, heteronuclear NMR spectroscopy. Using a combination of triple-resonance (1H, 13C, 15N) experiments, we obtained virtually complete backbone and side-chain resonance assignments based solely on scalar couplings. 3D and 4D NOESY experiments were then used to determine the secondary structure and global fold of Acanthamoeba profilin I. The central feature of the protein structure is a five-stranded antiparallel beta-sheet flanked by three helices and a short two-stranded antiparallel beta-sheet.

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of 13C/15N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (1H and 13C) and backbone (1H, 13Cα and 15N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.

Studies on the Spin-Lattice Relaxation Times of the Carbons of the Investigational Antileukemic Drug and Enzyme Inhibitor MethylglyoxalBis(Amidino-Hydrazone) ['MethylglyoxalBIS(Guanylhydrazone)'] and Its Dialkylglyoxal Analogs

Abstract The first study on the 13C relaxation times of bis(amidinohydrazones) is reported. The spin-lattice relaxation times (T1) of the carbons of the free bases of methylglyoxal bis(amidinohydrazone) (MGBG) and of four dialkylglyoxal analogs thereof were determined with the aid of the inversion recovery method and using dimethyl sulfoxide as the solvent. In the series of compounds studied, one of the side chains was always a methyl group, while the other one was altered (hydrogen, methyl, ethyl, propyl, butyl). Remarkable differences were found to exist between the T1 values of the various carbons within each molecule. The T1 values were in the range 1.5 - 2 s for methyl carbons, 0.16 - 1.9 s for carbons of longer alkyl groups, 4.3 - 7.0 s for unprotonated carbons of the glyoxal moiety, 0.57 s for the protonated glyoxal carbon of MGBG, and 2.6 - 3.1 s for guanidino carbons. The bulk of the differences are explainable by assuming that the major relaxation mechanism for the protonated carbons is dipolar relaxation. In alkyl side chains, the T1 values increased in a very regular fashion down the chain. This effect made possible the assignment of two previously unassigned carbon resonances of the butyl group of BMGBG. T1 studies thus offer a facile and reliable method for the assignment of side-chain carbon resonances of bis(amidinohydrazones). Further, T1 measurements were found to offer a very good method for the individual assignment of the glyoxal carbons of unsymmetrical congeners, whose assignment has so far constituted a problem. The method, based on the finding that the one of the carbons bonded to the shorter alkyl chain has a longer relaxation time than does the other one, made possible the unambiguous assignment of several previously unassigned carbon resonances. The results obtained also offer a reliable method for unambiguously distinguishing between the resonances of glyoxal carbons and guanidino carbons that have been difficult to distinguish from each other because the separation of their chemical shifts is often extremely small. Correlations observed between T1 values and the degree of alkyl substitution in the molecule are discussed, as are also possible relaxation mechanisms. Somewhat unexpectedly, the results obtained suggest that dipolar relaxation through the hydrogens of neighboring carbon atoms may to a significant extent contribute to the relaxation of some unprotonated carbons in bis(amidinohydrazones).

1H and 15N resonance assignments and secondary structure of the human thioredoxin C62A, C69A, C73A mutant.

The complete assignment of 1H and 15N backbone resonances and near-complete 1H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional 15N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay, J.D. et al. (1991) Biochemistry, 30, 2685-2698]. An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.

Secondary structure and side-chain proton and carbon-13 resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy

Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities. The secondary structure of this protein in solution has been elucidated by a qualitative interpretation of nuclear Overhauser effects, hydrogen exchange data, and 3JHNH alpha coupling constants. A clear correlation between the 13C alpha chemical shift and secondary structure is found. The secondary structure in the two globular domains of Drosophila CaM in solution is essentially identical with that of the X-ray crystal structure of mammalian CaM [Babu, Y., Bugg, C. E., & Cook, W.J. (1988) J. Mol. Biol. 204, 191-204], which consists of two pairs of a "helix-loop-helix" motif in each globular domain. The existence of a short antiparallel beta-sheet between the two loops in each domain has been confirmed. The eight alpha-helix segments identified from the NMR data are located at Glu-6 to Phe-19, Thr-29 to Ser-38, Glu-45 to Glu-54, Phe-65 to Lys-77, Glu-82 to Asp-93, Ala-102 to Asn-111, Asp-118 to Glu-127, and Tyr-138 to Thr-146. Although the crystal structure has a long "central helix" from Phe-65 to Phe-92 that connects the two globular domains, NMR data indicate that residues Asp-78 to Ser-81 of this central helix adopt a nonhelical conformation with considerable flexibility.

Assignment of side-chain proton resonances of valine and leucine using two-dimensional relayed coherence transfer spectroscopy

Assignment of side-chain proton resonances of valine and leucine using two-dimensional relayed coherence transfer spectroscopy

Assignment of asparagine-44 side-chain primary amide proton NMR resonances and the peptide amide N1H resonance of glycine-37 in basic pancreatic trypsin inhibitor

New assignments of three previously undetected amide proton NMR resonance lines in bovine pancreatic trypsin inhibitor are reported. These are the peptide amide proton of Gly-37 and the primary amide protons of Asn-44. Specific assignments of Asn-44 and Asn-43 HE and HZ resonances are also reported. The Gly-37 NH and Asn-44 HZ resonances are shifted upfield to 4.3 and 3.4 ppm, respectively, by the ring current of the Tyr-35 aromatic group, while Asn-44 HE resonates at 7.8 ppm. The abnormal chemical shifts of Asn-44 HZ and Gly-37 NH indicate that both NH's interact with the pi-electron cloud of the Tyr-35 ring. This is consistent with their location in the crystal structure. The resonances are resolved by differential labeling techniques and are studied by combined use of NOE and exchange difference spectroscopy.

Assignment of the 1H nuclear magnetic resonance spectrum of the proteinase inhibitor IIA from bull seminal plasma by two-dimensional nuclear magnetic resonance at 500 MHz

The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the protease inhibitor IIA from bull seminal plasma is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional n.m.r. experiments at 500 MHz. Individual assignments were obtained at 18 degrees C and 45 degrees C for the backbone protons of all 57 amino acid residues, with the single exception of the N-terminal pyroglutamate amide proton. The amino acid side-chain resonance assignments are complete, with the exception of 17 long side-chains, i.e. Pro13, Met43 and all the Glu, Gln, Lys and Arg, where only one or two resonances of C beta H2 and in some cases C gamma H2 could be identified. The sequential assignments showed that the order of the two C-terminal residues in the previously established primary structure had to be changed; this was then confirmed by chemical methods. The chemical shifts for the assigned resonances at 18 degrees C and 45 degrees C are listed for an aqueous solution at pH 4.9. A preliminary characterization of the polypeptide secondary structure was obtained from the observed patterns of sequential connectivities.

Assignment of the 1H nuclear magnetic resonance spectrum of the trypsin inhibitor E from Dendroaspis polylepis polylepis: Two-dimensional nuclear magnetic resonance at 500 MHz

The assignment of the 1H nuclear magnetic resonance spectrum of the trypsin inhibitor E from the venom of Dendroaspis polylepis polylepis is described and documented. A sample of 18 mg of the protein was investigated with two-dimensional nuclear magnetic resonance experiments at 500 MHz. The assignments are based entirely on the amino acid sequence and the nuclear magnetic resonance data. Individual assignments were obtained for the backbone and C β protons of all 59 residues of inhibitor E, with the exceptions of the N-terminal amino group, Pro8, Pro13 and the amide proton of Gly37. The amino acid side-chain resonance assignments are complete, with the exception of Pro8, Pro13, C γH 2 of Glu49, all the lysyl and arginyl residues and the three histidyl residues 1, 34 and 53, for which the imidazole ring proton lines have not been assigned individually. The chemical shifts for the assigned resonances are listed for an aqueous solution at 50 °C and pH 3.2.

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra : Basic pancreatic trypsin inhibitor

The assignment of the 1H nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor with the use of two-dimensional 1H nuclear magnetic resonance techniques at 500 MHz is described. The assignments are based entirely on the known amino acid sequence and the nuclear magnetic resonance data. Individual resonance assignments were obtained for all backbone and C β protons, with the exception of those of Arg1, Pro2, Pro13 and the amide proton of Gly37. The side-chain resonance assignments are complete, with the exception of Pro2 and Pro13, the N δ protons of Asn44 and the peripheral protons of the lysine residues and all but two of the arginine residues.