Measurement of homonuclear proton couplings from regular 2D COSY spectra.

An interactive computer procedure is described which determines (1)H--(1)H couplings from fitting the cross-peak multiplets in regular phase-sensitive COSY spectra. The robustness and simplicity of the method rely on the fact that a given cross-peak intensity is not an independent variable in the fitting procedure, making it possible to measure couplings accurately even from individual cross peaks with unresolved multiplet structure.

Measurement of one-bond 15N-13C' dipolar couplings in medium sized proteins.

A simple and accurate method is described for measurement of 1J(C'N) splittings in isotopically enriched proteins. The method is of the quantitative J correlation type, and the 1J(C'N) splitting is derived from the relative intensity in two 3D TROSY-HNCO spectra with 1J(C'N) dephasing intervals of approximately 1/(2 1J(C'N)) (reference intensity) and approximately 1/1J(C'N) (residual intensity). If the two spectra are recorded under identical conditions and with the same number of scans, the random error in the 1J(C'N) value extracted in this manner is inversely related to the signal-to-noise (S/N) in the reference spectrum. A S/N of 30:1 in the reference spectrum yields random errors of less than 0.2 Hz in the extracted 1J(C'N) value. Dipolar couplings obtained from the difference in 1J(C'N) splitting in the isotropic and liquid crystalline phase for the C-terminal domain of calmodulin are in excellent agreement with its 1.68-A crystal structure, but agree considerably less with the 2.2-A structure.

Protein backbone angle restraints from searching a database for chemical shift and sequence homology.

Chemical shifts of backbone atoms in proteins are exquisitely sensitive to local conformation, and homologous proteins show quite similar patterns of secondary chemical shifts. The inverse of this relation is used to search a database for triplets of adjacent residues with secondary chemical shifts and sequence similarity which provide the best match to the query triplet of interest. The database contains 13C alpha, 13C beta, 13C', 1H alpha and 15N chemical shifts for 20 proteins for which a high resolution X-ray structure is available. The computer program TALOS was developed to search this database for strings of residues with chemical shift and residue type homology. The relative importance of the weighting factors attached to the secondary chemical shifts of the five types of resonances relative to that of sequence similarity was optimized empirically. TALOS yields the 10 triplets which have the closest similarity in secondary chemical shift and amino acid sequence to those of the query sequence. If the central residues in these 10 triplets exhibit similar phi and psi backbone angles, their averages can reliably be used as angular restraints for the protein whose structure is being studied. Tests carried out for proteins of known structure indicate that the root-mean-square difference (rmsd) between the output of TALOS and the X-ray derived backbone angles is about 15 degrees. Approximately 3% of the predictions made by TALOS are found to be in error.

Measurement of dipolar couplings for methylene and methyl sites in weakly oriented macromolecules and their use in structure determination.

A simple and effective method is described for simultaneously measuring dipolar couplings for methine, methylene, and methyl groups in weakly oriented macromolecules. The method is a J-modulated 3D version of the well-known [1H-13C] CT-HSQC experiment, from which the J and dipolar information are most accurately extracted by using time-domain fitting in the third, constant-time dimension. For CH2-sites, the method generally yields only the sum of the two individual 13C-1H couplings. Structure calculations are carried out by minimizing the deviation between the measured sum, and the sum predicted for each methylene on the basis of the structure. For rapidly spinning methyl groups the dipolar contribution to the splitting of the outer 13C quartet components can be used directly to constrain the orientation of the C-CH3 bond. Measured sidechain dipolar couplings are in good agreement with an ensemble of NMR structures calculated without use of these couplings.

Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra.

Simple procedures are described for recording complementary in-phase and antiphase J-coupled NMR spectra. The sum and difference of these spectra contain only the upfield and the downfield components of a doublet, making it possible to measure the J splitting directly from these combinations without an increase in resonance overlap relative to the decoupled spectrum. The approach is demonstrated for measurement of 1JNH splittings and 2JHNC splittings in oriented and isotropic ubiquitin. Dipolar couplings obtained from differences in the splittings measured in the oriented and isotropic phases are in excellent agreement with dipolar couplings obtained from direct measurement of the splitting or from a conventional E. COSY-type measurement.

NMRPipe: a multidimensional spectral processing system based on UNIX pipes.

The NMRPipe system is a UNIX software environment of processing, graphics, and analysis tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.

Resonance assignment of methionine methyl groups and chi 3 angular information from long-range proton-carbon and carbon-carbon J correlation in a calmodulin-peptide complex.

Several simple 3D experiments are used to provide J correlations between methionine C epsilon methyl carbons and either the C gamma H2 protons or C beta and C gamma. The intensity of the J correlations provides information on the size of the three-bond J couplings and thereby on the chi 3 torsion angle. In addition, a simple 3D version of the HMBC experiment provides a sensitive link between the C epsilon H3 methyl protons and C gamma. The methods are demonstrated for a 20 kDa complex between calmodulin and a 26-residue peptide fragment of skeletal muscle myosin light chain kinase.

Measurement of HN-H alpha J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods.

Two new methods are described for the measurement of three-bond JHNH alpha couplings in proteins isotopically enriched with 15N. Both methods leave the water magnetization in an unsaturated state, parallel to the z-axis, and therefore offer significant enhancements in sensitivity for rapidly exchanging backbone amide protons. The J couplings can be measured either from a set of constant-time 2D 1H-15N HMQC spectra, which are modulated in intensity by JHNH alpha, or from a water-flip-back version of the 3D HNHA experiment. The method is demonstrated for a sample of calcium-free calmodulin. Residues Lys75-Asp80 have JHNH alpha values in the 6-7 Hz range, suggesting that a break in the 'central helix' occurs at the same position as previously observed in solution NMR studies of Ca(2+)-ligated calmodulin.

The use of 1JC alpha H alpha coupling constants as a probe for protein backbone conformation.

Simple pseudo-3D modifications to the constant-time HSQC and HCACO experiments are described that allow accurate (+/- 0.5 Hz) measurement of one bond JC alpha H alpha coupling constants in proteins that are uniformly enriched with 13C. An empirical phi,psi-surface is calculated which describes the deviation of 1JC alpha H alpha from its random coil value, using 203 1JC alpha H alpha values measured for residues in the proteins calmodulin, staphylococcal nuclease, and basic pancreatic trypsin inhibitor, for which phi and psi are known with good precision from previous X-ray crystallographic studies. Residues in alpha-helical conformation exhibit positive deviations of 4-5 Hz, whereas deviations in beta-sheet are small and, on average, slightly negative. Data indicate that 1JC alpha H alpha depends primarily on psi, and that 1JC alpha H alpha may be useful as a qualitative probe for secondary structure. Comparison of 1JC alpha H alpha coupling constants measured in free calmodulin and in its complex with a 26-amino-acid peptide fragment of myosin light-chain kinase confirm that the calmodulin secondary structure is retained upon complexation but that disruption of the middle part of the 'central helix' is even more extensive than in free calmodulin.

Measurement of 15N-13C J couplings in staphylococcal nuclease.

15N-C alpha and 15N-C' J couplings were measured for the backbone of staphylococcal nuclease, uniformly enriched with 15N and 13C. It is found that the 1JC'N coupling is similar for beta-sheet, J = 14.8 +/- 0.5 and for alpha-helix, J = 14.8 +/- 0.4 but tends to be larger for the unstructured N- and C-terminal ends of the protein (J = 15.6 +/- 0.5). On average, 1JNC alpha are smaller for alpha-helical residues (J = 9.6 +/- 0.3 Hz) compared to beta-sheet (J = 10.9 +/- 0.8 Hz) and a substantial difference is observed for 2JNC alpha in alpha-helices (J = 6.4 +/- 0.4 Hz) and beta-sheets (J = 8.3 +/- 0.8 Hz).

Toward a computer assisted analysis of NOESY spectra: a multivariate data analysis of an RNA NOESY spectrum.

A multivariate data-representation of a portion of the H-NOESY spectrum of an RNA octamer duplex was used to explore the possibility of using Principal Component Analysis and Partial Least Squares Discrimination for pattern recognition. In this case, it is found that the methods can: (i) distinguish slices containing signal from those containing only noise, (ii) locate slices containing overlapping signals, and (iii) in some cases to segregate slices with unique aspects such as those from terminal nucleotides, overlapping signals, purine-H8, pyrimidine-H6 and adenine-H2 containing slices. These properties can easily be included in a scheme to automate spectral analysis. The formulation described here does not distinguish patterns needed to automate sequential assignment of resonances in NOESY spectra of RNA.