Application of automated NOE assignment to three-dimensional structure refinement of a 28 kDa single-chain T cell receptor

An automated procedure for NOE assignment and three-dimensional structure refinement is presented. The input to the procedure consists of (1) an ensemble of preliminary protein NMR structures, (2) partial sequence-specific assignments for the protein and (3) the positions and volumes of unassigned NOESY cross peaks. Chemical shifts for unassigned side chain protons are predicted from the preliminary structures. The chemical shifts and unassigned NOESY cross peaks are input to an automated procedure for NOE assignment and structure calculation (ARIA) [Nilges et al. (1997) J. Mol. Biol., 269, 408–422]. ARIA is optimized for the task of structure refinement of larger proteins. Errors are filtered to ensure that sequence-specific assignments are reliable. The procedure is applied to the 27.8 kDa single-chain T cell receptor (scTCR). Preliminary NMR structures, nearly complete backbone assignments, partial assignments of side chain protons and more than 1300 unassigned NOESY cross peaks are input. Using the procedure, the resonant frequencies of more than 40 additional side chain protons are assigned. Over 400 new NOE cross peaks are assigned unambiguously. Distances derived from the automatically assigned NOEs improve the precision and quality of calculated scTCR structures. In the refined structures, a hydrophobic cluster of side chains on the scTCR surface that binds major histocompatibility complex (MHC)/antigen is revealed. It is composed of the side chains of residues from three loops and stabilizes the conformation of residues that interact with MHC.     

The effect of selective deuteration on magnetization transfer in larger proteins

Summary The effects of selective deuteration on calculated NOESY intensities have been analyzed for the structure of theE. coli trp aporepressor, a 25 kDa protein. It is shown that selectively deuteratedtrp aporepressor proteins display larger calculated NOESY intensities than those for the same interproton distances in the natural abundance protein. The relatively larger magnetization transfer is demonstrated by a comparison of the NOE build-up curves for specific proton pairs, and for the calculated NOE intensities of short-range NOEs to backbone amide protons. This increase in intensity is especially pronounced for the NH¹–NH¹⁺¹ cross peaks in the -helical regions, and particularly for amide protons of two sequential deuterated residues. The effect is shown to be further intensified for longer mixing times. It is also shown that in all cases, each amide proton exhibits stronger NOEs to its own side chain, with an enhanced effect for deuterated derivatives. This theoretical analysis demonstrates that an evaluation of the relative NOE intensities for different selectively deuterated analogs may be an important tool in assigning NMR spectra of large proteins. These results also serve as a guide for the interpretation of NOEs in terms of distances for structure calculations based on data using selectively deuterated proteins.     

A simple spectral-driven procedure for the refinement of DNA structures by NMR spectroscopy.

We have developed a simple and quantitative procedure (SPEDREF) for the refinement of DNA structures using experimental two-dimensional nuclear Overhauser effect (2D NOE) data. The procedure calculates the simulated 2D NOE spectrum using the full matrix relaxation method on the basis of a molecular model. The volume of all NOE peaks is measured and compared between the experimental and the calculated spectra. The difference of the experimental and simulated volumes is minimized by a conjugated gradient procedure to adjust the interproton distances in the model. An agreement factor (analogous to the crystallographic R-factor) is used to monitor the progress of the refinement. The procedure is an The agreement is considered to be complete when several parameters, including the R-factor, the energy associated with the molecule, the local conformation (as judged by the sugar pseudorotation), and the global conformation (as judged by the helical x-displacement), are refined to their respective convergence. With the B-DNA structure of d(CGATCG) as an example, we show that DNA structure may be refined to produce calculated NOE spectra that are in excellent agreement with the experimental 2D NOE spectra. This is judged to be effective by the low R-factor of approximately 15%. Moreover, we demonstrate that not only are NOE data very powerful in providing details of the local structure but, with appropriate weighting of the NOE constraints, the global structure of the DNA double helix can also be determined, even when starting with a grossly different model. The reliability and limitations of a DNA structure as determined by NMR spectroscopy are discussed.     

Sequence-specific solution structure of d-GGTACGCGTACC

Complete resonance assignments of nonexchangeable base protons and sugar protons in d-GGTACGCGTACC at 500 MHz have been obtained by two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The characteristic phase-sensitive multiplet patterns of the ntrasugar cross peaks in the omega 1-scaled COSY spectrum have been used to estimate several scalar coupling constants (J). These coupling constants combined with the intranucleotide COSY cross peak intensities have been used to identify the sugar pucker of individual nucleotide units. In most cases, the deoxyribose rings adopt a conformation close to O4'-endo. Spin-diffusion has been monitored from the buildup of the normalized volumes of NOE cross peaks in NOESY spectra as a function of mixing time. A set of 55 intranucleotide and internucleotide interproton distances have been estimated from the low mixing time NOESY spectrum (tau m = 75 ms). The estimated intranucleotide proton-proton distances have been used to determine the individual glycosidic dihedral angles of the nucleotide units which lie in the anti domain. It is observed that the molecule adopts an overall conformation close to that of the B-form although there are differences in the intricate details.     

Influence of the completeness of chemical shift assignments on NMR structures obtained with automated NOE assignment

Reliable automated NOE assignment and structure calculation on the basis of a largely complete, assigned input chemical shift list and a list of unassigned NOESY cross peaks has recently become feasible for routine NMR protein structure calculation and has been shown to yield results that are equivalent to those of the conventional, manual approach. However, these algorithms rely on the availability of a virtually complete list of the chemical shifts. This paper investigates the influence of incomplete chemical shift assignments on the reliability of NMR structures obtained with automated NOESY cross peak assignment. The program CYANA was used for combined automated NOESY assignment with the CANDID algorithm and structure calculations with torsion angle dynamics at various degrees of completeness of the chemical shift assignment which was simulated by random omission of entries in the experimental ¹H chemical shift lists that had been used for the earlier, conventional structure determinations of two proteins. Sets of structure calculations were performed choosing the omitted chemical shifts randomly among all assigned hydrogen atoms, or among aromatic hydrogen atoms. For comparison, automated NOESY assignment and structure calculations were performed with the complete experimental chemical shift but under random omission of NOESY cross peaks. When heteronuclear-resolved three-dimensional NOESY spectra are available the current CANDID algorithm yields in the absence of up to about 10% of the experimental ¹H chemical shifts reliable NOE assignments and three-dimensional structures that deviate by less than 2 Å from the reference structure obtained using all experimental chemical shift assignments. In contrast, the algorithm can accommodate the omission of up to 50% of the cross peaks in heteronuclear- resolved NOESY spectra without producing structures with a RMSD of more than 2 Å to the reference structure. When only homonuclear NOESY spectra are available, the algorithm is slightly more susceptible to missing data and can tolerate the absence of up to about 7% of the experimental ¹H chemical shifts or of up to 30% of the NOESY peaks.Abbreviations: BmPBP^A – Bombyx mori pheromone binding protein form A; CYANA – combined assignment and dynamics algorithm for NMR applications; NMR – nuclear magnetic resonance; NOE – nuclear Overhauser effect; NOESY – NOE spectroscopy; RMSD – root-mean-square deviation; WmKT – Williopsis mrakii killer toxin     

Selective excitation of intense solvent signals in the presence of radiation damping

Summary Selective water excitation schemes are provided which rely on the radiation damping effect in probeheads characterized by high quality factors. The schemes are implemented in homonuclear NOE and ROE experiments, designed for the selective observation of water-protein cross peaks and their assignment using standard probeheads. The one-dimensional NOE and ROE experiments selectively record the cross section through the water signal usually measured in two-dimensional NOESY and ROESY spectra, and the two-dimensional NOE-NOESY and ROE-NOESY experiments selectively measure the cross section through the water line from 3D NOESY-NOESY and ROESY-NOESY spectra, respectively.     

Protein NMR structure determination with automated NOE-identification in the NOESY spectra using the new software ATNOS

Novel algorithms are presented for automated NOESY peak picking and NOE signal identification in homonuclear 2D and heteronuclear-resolved 3D [¹H,¹H]-NOESY spectra during de novoprotein structure determination by NMR, which have been implemented in the new software ATNOS (automated NOESY peak picking). The input for ATNOS consists of the amino acid sequence of the protein, chemical shift lists from the sequence-specific resonance assignment, and one or several 2D or 3D NOESY spectra. In the present implementation, ATNOS performs multiple cycles of NOE peak identification in concert with automated NOE assignment with the software CANDID and protein structure calculation with the program DYANA. In the second and subsequent cycles, the intermediate protein structures are used as an additional guide for the interpretation of the NOESY spectra. By incorporating the analysis of the raw NMR data into the process of automated de novoprotein NMR structure determination, ATNOS enables direct feedback between the protein structure, the NOE assignments and the experimental NOESY spectra. The main elements of the algorithms for NOESY spectral analysis are techniques for local baseline correction and evaluation of local noise level amplitudes, automated determination of spectrum-specific threshold parameters, the use of symmetry relations, and the inclusion of the chemical shift information and the intermediate protein structures in the process of distinguishing between NOE peaks and artifacts. The ATNOS procedure has been validated with experimental NMR data sets of three proteins, for which high-quality NMR structures had previously been obtained by interactive interpretation of the NOESY spectra. The ATNOS-based structures coincide closely with those obtained with interactive peak picking. Overall, we present the algorithms used in this paper as a further important step towards objective and efficient de novoprotein structure determination by NMR.     

A two-dimensional NMR study of the solution structure of a DNA dodecamer comprising the concensus sequence for the specific DNA-binding sites of the glucocorticoid receptor protein

A two-dimensional 500-MHz 1H-NMR study on the non-self-complementary double-stranded DNA dodecamer 5'd(C-C-A-G-A-A-C-A-G-T-G-G)5'd(C-C-A-C-T-G-T-T-C-T-G-G), is presented. This oligonucleotide contains the consensus octanucleotide sequence 5'd(A-G-A-A-C-A-G-T) for the specific DNA-binding sites of the glucocorticoid receptor protein [Payvar, F. et al. (1984) Cell 35, 381-392]. Using a combination of two-dimensional pure phase absorption nuclear Overhauser enhancement (NOESY) and homonuclear J-correlated (COSY) spectroscopy all non-exchangeable base (with the exception fo the adenine H2 protons), methyl and deoxyribose H1', H2', H2", H3' and H4' resonances are assigned unambiguously using a sequential resonance assignment strategy. From the relative intensities of the cross peaks in the pure phase absorption NOESY spectra at two mixing times it is shown that the dodecamer adopts a B-type conformation in solution.     

A T1 rho-filtered two-dimensional transferred NOE spectrum for studying antibody interactions with peptide antigens.

Transferred nuclear Overhauser effect (TRNOE) spectroscopy can be used to study intra- and intermolecular interactions of bound ligands complexed with large proteins. However, the 2D NOE (NOESY) spectra of large proteins are very poorly resolved and it is very difficult to discriminate the TRNOE cross peaks, especially those due to intermolecular interactions, from the numerous cross peaks due to intramolecular interactions in the protein. In previous studies we measured two-dimensional difference spectra that show exclusively TRNOE and exchange cross-peaks (Anglister, J., 1990. Quart. Rev. Biophys. 23:175-203). Here we show that a filtering method based on the difference between the T1rho values of the ligand and the protein protons can be used to directly obtain a two-dimensional transferred NOE spectrum in which the background cross-peaks due to intramolecular interactions in the protein are very effectively removed. The usefulness of this technique to study protein ligand interactions is demonstrated for two different antibodies complexed with a peptide of cholera toxin (CTP3). It is shown that the T1 rho-filtering alleviates t problems encountered in our previous measurements of TRNOE by the difference method. These problems were due to imperfections in the subtraction of two spectra measured for two different samples.     

Determination of the conformation of Lewis blood group oligosaccharides by simulation of two-dimensional nuclear Overhauser data

Through control of both the nmr probe temperature and of the solvent viscosity, phase-sensitive two-dimensional 1H nuclear Overhauser data (NOESY) at 300 and 500 MHz are obtained with excellent signal-to-noise ratios for Lewis blood group penta- and hexasaccharides isolated from human milk. Relatively long mixing times are required to produce measurable NOE intensities in these oligosaccharides, which makes a full relaxation matrix analysis necessary. By measurements of selective T1 for a few isolated 1H resonances, it was possible to generate a simulation of the complete NOESY spectrum at arbitrary mixing time for comparison with the experimental data. From an exhaustive search of the conformational space, it was found that only a small range of glycosidic dihedral angles of the nonreducing terminal Lewis blood group determinant fragments of the milk oligosaccharides LNF-2 and LND-1 produce simulated spectra agreeing within experimental error to the data. Conformational energy calculations reveal that each of these conformations is also one of minimum energy. It is concluded that the Lewis(a) and Lewis(b) oligosaccharides adopt relatively compact rigid structures in solution, as shown by the observation of cross peaks between protons in nonadjacent residues. Like the blood group A and H oligosaccharides, there exists only a small dependence of the conformation for Lewis(a) and Lewis(b) oligosaccharides on solvent. The apparent lack of dependence of conformation of these oligosaccharides on DMSO in D2O suggests that modification of solvent viscosity with mixtures of DMSO:D2O may provide a useful general strategy of NOESY studies of oligosaccharides.     

Refinement of the spatial structure of the gramicidin A ion channel]

The spatial structure of the gramicidin A (GA) transmembrane ion-channel was refined on the base of cross-peak volumes measured in NOESY spectra (mixing time tau m = 100 and 200 ms). The refinement methods included the comparison of experimental cross-peak volumes with those calculated for low-energy GA conformations, dynamic averaging of the low-energy conformation set and restrained energy minimization. Accuracy of the spatial structure determination was estimated by the penalty function Fr defined as a root mean square deviation of interproton distances corresponding to the calculated and experimental cross-peak volumes. As the initial conformation we used the right-handed pi 6,3 LD pi 6,3 LD helix established on the base of NMR data regardless of the cross-peak volumes. The conformation is in a good agreement with NOE cross-peak volumes (Fr 0.2 to 0.5 A depending on NOESY spectrum). For a number of NOEs formed by the side chain protons, distances errors were found as much as 0.5-2.0 A. Restrained energy minimization procedure had little further success. However some of these errors were eliminated by the change in torsional angle chi 2 of D-Leu12 and dynamic averaging of the Val7 side chain conformations. Apparently, majority of deviations of the calculated and experimental cross-peak volumes are due to the intramolecular mobility of GA and cannot be eliminated within the framework of rigid globule model. In summary the spatial structure of GA ion-channel can be thought as a set of low-energy conformations, differing by the side chain torsion angles chi 1 Val7 and chi 2 D-Leu4 and D-Leu10 and the orientation of the C-terminal ethanolamine group. Root mean square differences between the atomic coordinates of conformations are in the range of 0.3-0.8 A.     

Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA

Combined automated NOE assignment and structure determination module (CANDID) is a new software for efficient NMR structure determination of proteins by automated assignment of the NOESY spectra. CANDID uses an iterative approach with multiple cycles of NOE cross-peak assignment and protein structure calculation using the fast DYANA torsion angle dynamics algorithm, so that the result from each CANDID cycle consists of exhaustive, possibly ambiguous NOE cross-peak assignments in all available spectra and a three-dimensional protein structure represented by a bundle of conformers. The input for the first CANDID cycle consists of the amino acid sequence, the chemical shift list from the sequence-specific resonance assignment, and listings of the cross-peak positions and volumes in one or several two, three or four-dimensional NOESY spectra. The input for the second and subsequent CANDID cycles contains the three-dimensional protein structure from the previous cycle, in addition to the complete input used for the first cycle. CANDID includes two new elements that make it robust with respect to the presence of artifacts in the input data, i.e. network-anchoring and constraint-combination, which have a key role in de novo protein structure determinations for the successful generation of the correct polypeptide fold by the first CANDID cycle. Network-anchoring makes use of the fact that any network of correct NOE cross-peak assignments forms a self-consistent set; the initial, chemical shift-based assignments for each individual NOE cross-peak are therefore weighted by the extent to which they can be embedded into the network formed by all other NOE cross-peak assignments. Constraint-combination reduces the deleterious impact of artifact NOE upper distance constraints in the input for a protein structure calculation by combining the assignments for two or several peaks into a single upper limit distance constraint, which lowers the probability that the presence of an artifact peak will influence the outcome of the structure calculation. CANDID test calculations were performed with NMR data sets of four proteins for which high-quality structures had previously been solved by interactive protocols, and they yielded comparable results to these reference structure determinations with regard to both the residual constraint violations, and the precision and accuracy of the atomic coordinates. The CANDID approach has further been validated by de novo NMR structure determinations of four additional proteins. The experience gained in these calculations shows that once nearly complete sequence-specific resonance assignments are available, the automated CANDID approach results in greatly enhanced efficiency of the NOESY spectral analysis. The fact that the correct fold is obtained in cycle 1 of a de novo structure calculation is the single most important advance achieved with CANDID, when compared with previously proposed automated NOESY assignment methods that do not use network-anchoring and constraint-combination.     

Sequential resonance assignments in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy

A sequential assignment procedure is outlined, based on two-dimensional NOE ( NOESY ) and two-dimensional J-correlated spectroscopy ( COSY ), for assigning the nonexchangeable proton resonances in NMR spectra of oligonucleotides. As presented here the method is generally applicable to right-handed helical oligonucleotides of intermediate size. We applied it to a lac operator DNA fragment consisting of d( TGAGCGG ) and d( CCGCTCA ) and obtained complete assignments for the adenine H8, guanine H8, cytosine H6 and H5, thymine H6 and 5-methyl, and the deoxyribose H1', H2', H2", H3', and H4' resonances, as well as some H5', H5" (pairwise) assignments. These assignments are required for the analysis of two-dimensional NOE and J-coupling data in terms of the solution structure of oligonucleotides.     

Determination of local conformation of proteins from 1H-NMR spectroscopy data

A method is proposed to determine conformations of amino acid residues of the protein and effective correlation time tau c from cross-peak intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra. The method consists in fitting complete relaxation matrix of dipeptide unit protons to experimental cross-peak intensities by varying phi, psi, chi torsional angles and tau c. To verify the method, NOESY spectra of basic pancreatic trypsin inhibitor (BPTI) were theoretically generated at mixing times tau m = 25-300 ms and tau c = 4 ns and used for local structure determination. The method works well with optimum for measurement of NOE intensities tau m 100-200 ms. As a result, the backbone phi, psi torsion angles were unambiguously determined at tau m = 100 ms for all but Gly residues of BPTI, and chi 1 angles were determined for the majority of side chains. The obtained dipeptide unit conformations are very close to the BPTI crystallographic structure: root mean square deviation (RMSD) of interproton distances within dipeptide units, on the average, is 0.08 A (maximal deviation 0.44 A), and RMSD of phi and psi angles are 18 and 9 degrees, respectively (maximal deviations are 44 and 22 degrees).     

Solution conformation of purine-pyrimidine DNA octamers using nuclear magnetic resonance, restrained molecular dynamics and NOE-based refinement

The solution structures of two alternating purine-pyrimidine octamers, [d(G-T-A-C-G-T-A-C)]2 and the reverse sequence [d(C-A-T-G-C-A-T-G)]2, are investigated by using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Chemical shift assignments are obtained for non-exchangeable protons by a combination of two-dimensional correlation and nuclear Overhauser enhancement (NOE) spectroscopy experiments. Distances between protons are estimated by extrapolating distances derived from time-dependent NOE measurements to zero mixing time. Approximate dihedral angles are determined within the deoxyribose ring from coupling constants observed in one and two-dimensional spectra. Sets of distance and dihedral determinations for each of the duplexes form the bases for structure determination. Molecular dynamics is then used to generate structures that satisfy the experimental restraints incorporated as effective potentials into the total energy. Separate runs start from classical A and B-form DNA and converge to essentially identical structures. To circumvent the problems of spin diffusion and differential motion associated with distance measurements within molecules, models are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated using a full matrix analysis procedure. The refined structures generally have the global features of B-type DNA. Some, but not all, variations in dihedral angles and in the spatial relationships of adjacent base-pairs are observed to be in synchrony with the alternating purine-pyrimidine sequence.     

Characterization by 1H NMR of glycosidic conformations in the tetramolecular complex formed by d(GGTTTTTGG).

We have conducted two dimensional NOESY studies on the molecule d(G2T5G2) to characterize the structure of the tetramolecular complex previously identified by calorimetric and spectroscopic studies (1). Analysis of the NOE and exchange cross peaks observed in the NOESY spectra establishes the formation of structured conformations at low temperature (5 degrees C). Significantly, within each strand of these structured conformations, the G1 and G8 residues adopt syn glycosidic torsion angles, while the G2 and G9 residues adopt anti glycosidic torsion angles. Consequently, any structure proposed for the tetramolecular complex of d(G2T5G2) must have alternating G(syn) and G(anti) glycosidic torsion angles within each strand. The implications of this observation for potential structures of the tetramolecular complex of d(G2T5G2) are discussed.     

Structure determination of [d(ATATATAUAT)]2 via two-dimensional NOE spectroscopy and molecular dynamics calculations

Proton homonuclear two-dimensional (2D) NOE spectra were obtained for the decamer [d(ATATATAUAT)]2 as a function of mixing time, and proton resonance assignments were made. Quantitative assessment of the 2D NOE cross-peak intensities was used in conjunction with the program MARDIGRAS, which entails a complete relaxation matrix analysis of the 2D NOE peak intensities, to obtain a set of upper and lower bound interproton distance constraints. The analysis with MARDIGRAS was carried out using three initial models: A-DNA, B-DNA and Z-DNA. The distance constraints determined were essentially the same regardless of initial structure. These experimental structural constraints were used with restrained molecular dynamics calculations to determine the solution structure of the decamer. The molecular dynamics program AMBER was run using A-DNA or B-DNA as starting model. The root-mean-square (rms) difference between these two starting models is 0.504 nm. The two starting models were subjected to 22.5 ps of restrained molecular dynamics calculations. The coordinates of the last 10.5 ps of the molecular dynamics runs were averaged to give two final structures. MDA and MDB. The rms difference between these two structures is 0.09 nm, implying convergence of the two molecular dynamics runs. The 2D NOE spectral intensities calculated for the derived structures are in good agreement with experimental spectra, based on sixth-root residual index analysis of intensities. A detailed examination of the structural features suggests that while the decamer is in the B-family of DNA structures, many torsion angle and helical parameters alternate from purine to pyrimidine, with kinks occurring at the U-A steps.     

Solution structure of the EcoRI DNA sequence: refinement of NMR-derived distance geometry structures by NOESY spectrum back-calculations

The solution structure of the self-complementary DNA duplex [d(CGCGAATTCGCG)]2, which contains the EcoRI restriction site sequence GAATTC at the center, has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Time-dependent nuclear Overhauser effect spectra were used to obtain the initial cross-relaxation rates between 155 pairs of protons. These initial cross-relaxation rates were converted into interproton distances and entered into a distance (bounds) matrix. A distance geometry algorithm (DSPACE) was used to create embedded starting structures and to refine these structures until they showed good agreement with the distance matrix; symmetry constraints were included in the refinement procedure, making the two strands in the refined distance geometry structures virtually identical and significantly improving the agreement with the distance matrix. The NOESY spectrum for one of these distance geometry structures was then calculated from the explicit coordinates by numerically integrating all the z-magnetization transfer pathways among neighboring protons within a specified radius. Distances in this distance geometry structure that did not agree with the experimental NOESY time course were then adjusted accordingly. This process was iterated until a good agreement between calculated and experimental NOESY spectra was reached. The final structure, which generates good agreement with the experimental NOESY spectrum, displays kinks at the C3-G4 base step and at the A6-T7 base step that appear to be similar to those reported for the EcoRI restriction site DNA bound to its endonuclease. The solution structure is not the same as the crystal structure of this DNA duplex.     

Interproton distance bounds from 2D NOE intensities: Effect of experimental noise and peak integration errors

Summary The effect of experimental and integration errors on the calculations in interproton distances from NOE intensities is examined. It is shown that NOE intensity errors can have a large impact on the distances determined. When multiple spin (spin diffusion) effects are significant, the calculated distances are often underestimated, even when using a complete relaxation matrix analysis. In this case, the bias of distances to smaller values is due to the random errors in the NOE intensities. We show here that accurate upper and lower bounds of the distances can be obtained if the intensity errors are properly accounted for in the complete relaxation matrix calculations, specifically the MARDIGRAS algorithm. The basic MARDIGRAS algorithm has been previously described [Borgias, B.A. and James, T.L. (1990) J. Magn. Reson., 87, 475–487]. It has been shown to provide reasonably good interproton distance bounds, but experimental errors can compromise the quality of the resulting restraints, especially for weak cross peaks. In a new approach introduced here, termed RANDMARDI (random error MARDIGRAS), errors due to random noise and integration errors are mimicked by the addition of random numbers from within a specified range to each input intensity. Interproton distances are then calculated for the modified intensity set using MARDIGRAS. The distribution of distances that define the upper and lower distance bounds is obtained by using N randomly modified intensity sets. RANDMARDI has been used in the solution structure determination of the interstrand cross-link (XL) formed between 4-hydroxymethyl-4,5,8-trimethylpsoralen (HMT) and the DNA oligomer d(5-GCGTACGC-3)₂ [Spielmann, H.P. et al. (1995) Biochemistry, 34, 12937–12953]. RANDMARDI generates accurate distance bounds from the experimental NOESY cross-peak intensities for the fixed (known) interproton distances in XL. This provides an independent internal check for the ability of RANDMARDI to accurately fit the experimental data. The XL structure determined using RANDMARDI-generated restrains is in good agreement with other biophysical data that indicate that there is no bend introduced into the DNA by the cross-link. In contrast, isolated spin-pair approximation calculations give distance restraints that, when applied in a restrained molecular dynamics protocol, produce a bent structure.Abbreviations NOE nuclear Overhauser effect - SD standard deviation - HMT 4-hydroxymethyl-4,5,8-trimethylpsoralen - XL psoralen-DNA interstrand cross-link     

Intermolecular relaxation has little effect on intra-peptide exchange-transferred NOE intensities

Exchange-transferred nuclear Overhauser enhancement (etNOE) provides a useful method for determining the 3-dimensional structure of a ligand bound to a high-molecular-weight complex. Some concern about the accuracy of such structures has arisen because indirect relaxation can occur between the ligand and macromolecule. Such indirect relaxation, or spin diffusion, would lead to errors in interproton distances used as restraints in structure determination. We address this concern by assessing the extent of intermolecular spin diffusion in nineteen peptide-protein complexes of known structure. Transferred NOE intensities were simulated with the program CORONA (Calculated OR Observed NOESY Analysis) using the rate-matrix approach to include contributions from indirect relaxation between protein-ligand and intraligand proton pairs. Intermolecular spin diffusion contributions were determined by comparing intensities calculated with protonated protein to those calculated with fully deuterated protein. The differences were found to be insignificant overall, and to diminish at short mixing times and high mole ratios of peptide to protein. Spin diffusion between the peptide ligand and the protein contributes less to the etNOE intensities and alters fewer cross peaks than the well-studied intramolecular spin diffusion effects. Errors in intraligand interproton distances due to intermolecular relaxation effects were small on average and can be accounted for with the restraint functions commonly used in NMR structure determination methods. In addition, a rate-matrix approach to calculate distances from etNOESY intensities using a volume matrix comprising only intraligand intensities was found to give accurate values. Based on these results, we conclude that structures determined from etNOESY data are no less accurate due to spin diffusion than structures determined from conventional NOE intensities.