S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 distances could be determined with an accuracy of fact, it is our experience that it is of prime importance about +0.3 A for short distances (1.8-3.0 A), of for simulated annealing to derive distance constraints t 0. a for distances between 3 and 4A, and which are assured to be correct. How to identify the of +0.5 A for distances between 4.0 and 50A [77]. types of errors? One simple approach, which we have o When considering the relaxation matrix refined not seen suggested in the literature, however, is to stances obtained by van de ven et al. [74], it is compare the relaxation matrix estimates with ISPA sconcerting to find that, in addition to the majority or 'modified Ispa derived distances since the latter of correctly determined distances, a number are cal- are assuredly correct, within albeit somewhat larger culated to be too large, with errors ranging up to 5 A, error bounds, large deviations directly pinpoint and in the extreme case they are said to have suspect'distance estimates exploded. These ' exploded distances not only In summary, the relaxation matrix approa distances as small as 2.6 A. These wrong distances distances <3.0 A)in better estimates than probably result from the difficulties in estimating the obtained with a 'modified ISPA oac spin diffusion contribution to the total NOE intensity. other hand, the relaxation matrix approaches may his may be due to errors in the NOE intensities or also give distances that are wrong by a large amount rrors in the model distances or a combination of these These erroneous distances are difficult to pinpoint. In effects. To account in the calculations for the first contrast, the simulations by van de ven et al. [74]or source of error the following method has been imple- Borgias and James [67] indicate that the simple ISPA mented in MARDIGRAS. The calculations are (or a modified ISPA approach when applied)gives epeated at least 30 times while randomly varying distance estimates that are assuredly correct, albeit NOE intensities with a certain noise level for each with slightly larger error bounds. The advantage of NOE 2D dataset(e.g. 0.002-0.003 for absolute inten- the IsPa method is its simplicity, which allows one sity and 5-10% integration error)[68]. The final to easily identify those distances that are likely to be distances are taken as the average, for the error one affected most by spin diffusion. In addition, one can can conservatively take the maximum and minimum experimentally minimize spin diffusion effects by distance values or, as Schmitz and James suggest [68] choosing relatively short mixing times when record- some intermediate range, leading to error ranges of ing the NOESY spectra. Furthermore, several factors +0.25-0.4 A. This procedure does not however that may affect the distance calculations are not well identify the erroneous distances with certainty and accounted for in the relaxation matrix equations errors resulting from a wrong starting model may Libration motions may lead to S values varying still evolve. Different starting models could be from values of 0.8 to 0.6, thereby affecting NOEs employed for estimating unknown fixed distances. proportionally. When considering that spin diffusion Schmitz and James [68] note that a starting model leads to a lower effective distance dependence of the closer to the true model improves the estimates. The NOEs, e.g. from the sixth to the fourth inverse power advantage in this respect of MORASS and IRMA is this results in relative errors of 5-8% in the distances that restrained MD structure calculations are done in Nucleic acids do not behave as isotropic tumbling each iteration step, thus improving the model molecules, but are rather asymmetric. This also estimates in each iteration step, thus reducing this affects the NOE calculations(see Section 8). Thus, possible source of error On the other hand, we have minimal error bounds of at least 10% corresponding found that errors in the distances are very difficult to to +0.2 A at 2 A are called for. These issues provide detect in the restrained MD calculations (XPLOR, another incentive for sticking to somewhat conserva- 78 The structure is often adjusted to compensate tive distance constraints, which may as well be for the erroneous distance constraint in such a way derived from the less precise 'modified ISPA that distance violations show up not at the site of the approach. We suggest therefore the use of conserva- erroneous constraint but elsewhere in the structure tive error bounds for the non-exchanging protons of Thus, simulated annealing does not provide a certain +0.2-0.3 A up to 2.6A, + 0.3 A from 2.6 A to 3.3 means to identify erroneous distance constraints. In A, +0.4 A from 3.2 A to 4.0A, +0.5 A from 4.0A
distances could be determined with an accuracy of about 6 0.3 A˚ for short distances (1.8–3.0 A˚ ), of 6 0.4 A˚ for distances between 3 and 4 A˚ , and of 6 0:5 A˚ for distances between 4.0 and 5.0 A˚ [77]. When considering the relaxation matrix refined distances obtained by van de Ven et al. [74], it is disconcerting to find that, in addition to the majority of correctly determined distances, a number are calculated to be too large, with errors ranging up to 5 A˚ , and in the extreme case they are said to have ‘exploded’. These ‘exploded’ distances not only occur for large true distances, but also for true distances as small as 2.6 A˚ . These wrong distances probably result from the difficulties in estimating the spin diffusion contribution to the total NOE intensity. This may be due to errors in the NOE intensities or errors in the model distances or a combination of these effects. To account in the calculations for the first source of error the following method has been implemented in MARDIGRAS. The calculations are repeated at least 30 times while randomly varying NOE intensities with a certain noise level for each NOE 2D dataset (e.g. 0.002–0.003 for absolute intensity and 5–10% integration error) [68]. The final distances are taken as the average; for the error one can conservatively take the maximum and minimum distance values or, as Schmitz and James suggest [68], some intermediate range, leading to error ranges of 6 0:25–0.4 A˚ . This procedure does not however identify the erroneous distances with certainty and errors resulting from a wrong starting model may still evolve. Different starting models could be employed for estimating unknown fixed distances. Schmitz and James [68] note that a starting model closer to the true model improves the estimates. The advantage in this respect of MORASS and IRMA is that restrained MD structure calculations are done in each iteration step, thus improving the model estimates in each iteration step, thus reducing this possible source of error. On the other hand, we have found that errors in the distances are very difficult to detect in the restrained MD calculations (XPLOR, [78]). The structure is often adjusted to compensate for the erroneous distance constraint in such a way that distance violations show up not at the site of the erroneous constraint but elsewhere in the structure. Thus, simulated annealing does not provide a certain means to identify erroneous distance constraints. In fact, it is our experience that it is of prime importance for simulated annealing to derive distance constraints which are assured to be correct. How to identify the types of errors? One simple approach, which we have not seen suggested in the literature, however, is to compare the relaxation matrix estimates with ISPA or ‘modified’ ISPA derived distances. Since the latter are assuredly correct, within albeit somewhat larger error bounds, large deviations directly pinpoint ‘suspect’ distance estimates. In summary, the relaxation matrix approaches result for most distances (especially the short distances , 3.0 A˚ ) in better estimates than those obtained with a ‘modified’ ISPA approach. On the other hand, the relaxation matrix approaches may also give distances that are wrong by a large amount. These erroneous distances are difficult to pinpoint. In contrast, the simulations by van de Ven et al. [74] or Borgias and James [67] indicate that the simple ISPA (or a modified ISPA approach when applied) gives distance estimates that are assuredly correct, albeit with slightly larger error bounds. The advantage of the ISPA method is its simplicity, which allows one to easily identify those distances that are likely to be affected most by spin diffusion. In addition, one can experimentally minimize spin diffusion effects by choosing relatively short mixing times when recording the NOESY spectra. Furthermore, several factors that may affect the distance calculations are not well accounted for in the relaxation matrix equations. Libration motions may lead to S2 values varying from values of 0.8 to 0.6, thereby affecting NOEs proportionally. When considering that spin diffusion leads to a lower effective distance dependence of the NOEs, e.g. from the sixth to the fourth inverse power, this results in relative errors of 5–8% in the distances. Nucleic acids do not behave as isotropic tumbling molecules, but are rather asymmetric. This also affects the NOE calculations (see Section 8). Thus, minimal error bounds of at least 10% corresponding to 6 0.2 A˚ at 2 A˚ are called for. These issues provide another incentive for sticking to somewhat conservative distance constraints, which may as well be derived from the less precise ‘modified’ ISPA approach. We suggest therefore the use of conservative error bounds for the non-exchanging protons of: 6 0.2–0.3 A˚ up to 2.6 A˚ , 6 0.3 A˚ from 2.6 A˚ to 3.3 A˚ , 6 0.4 A˚ from 3.2 A˚ to 4.0 A˚ , 6 0.5 A˚ from 4.0 A˚ 302 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387
S.S. Wijmenga, B N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 to 5.0 A and +0.6 A for distances >50A. For torsion angles and also allows the determination of exchanging protons, one could use 0 to 5A, but see the fraction of N-or S-pucker of the sugar pucker Schmidt and James [68]. Deriving distance constraints while the distance d (6 3") by itself does not determine via a ' ISPA with conservative error bounds well the fraction of N-or S-pucker. As pointed out in as described above prevents on the one hand putting Ref [62] uncertainties>+0.5 A make it virtually too tight constraints on the long distances and still impossible to determine the fraction of N-or providing reasonable estimates for short distances S-pucker. The glycosidic torsion angle remains well while on the other hand it also prevents choosing determined by the distance d(6/8: 2")even with an unduly loose error bounds uncertainty as large as +0.7A. with an uncertainty Are conservative or ' loose' distance constraints of +0. 2 A in the distance d 3, 5 /5")the fraction of detrimental to the structure determination compared y rotamer is defined, but rather loosely On the other to'tight distance constraints? The answer seems to be hand, a combination of d (6; 5/5) is sufficient to that they are not. This has recently been elegantly define this fraction rather well [62]. Uncertainties demonstrated by Allain and Varani [79] using NO >+0.5 a make it virtually impossible to determine data from the RNa hammerhead as a model system the fraction of y rotamer(see Fig. 3.1 in Ref. [62]) They classified distances loosely into three categories The same error bounds for isPa estimates derived i.e. 0-2. A(corresponding to dest t 0.55A if one above start from about =0. 2 A for distances around considers that the shortest distance is 1. 8 A any- 2.0 A and broaden to about +0.5a at 5. A: the how), 2.9-3.5 A(dest + 0.3 A), and 3.5-5.0A(dst+ error in the "loose as well as the tight constraint 0.75 A). These distance ranges correspond in fact sets used by Allain and Varani also increases from quite closely to the ones mentioned above. These roughly 0.3-0.5 A for short distances between ranges are used for all protons except the exchange- 2.0 and 3.0 A to +0.7 A for distances between 3.5 able or strongly overlapping protons, for which the and 5.0 A. These error bounds correspond to uncer- bounds were set to 0-50A. When tightening the tainties which fall somewhere in between the +0.2A ind + 0.5A range. Thus. for both sets of constraints 1. 8-2. A(dest +0.55 A), 2.5-3.5 A(dest +0.5 A), the error bounds on the intra-residue distances are and 3.5-5.0A(dest +0.75 A), the precision of the sufficiently narrow that the glycosidic torsion angle structures does not significantly improve, i.e. the and sugar pucker, as well as the y rotamer, can be rmsd of the final structures improves by 0. 2 A at determined reasonably well. The conclusion that one best (Table 2 of Allain and Varani [79]). Van de is forced to draw from these investigations is that Ven and Hilbers [80](see also Hilbers et al. [81] rather conservative distance constraints(see above) have also investigated how the precision of the dis- can be employed without detrimental effect on the tance data affects the precision of the structures ultimate precision of the structure Employing all ds(6/8/1721273 6/8/5)distances and How does the number of constraints and their assuming that they are determined with a precision of spread through the molecular structure affect the pre + 0.25 A, they find that in a g to g dinucleotide step cision of the derived structure? As can be seen from the twist is determined with a range of about 42 and Table 1, the spread in space of the NMR accessible the rise with a range of roughly 1.5A. For the distances in a helix is rather uneven. A large propor- unrealistically high precision of +0. 1 A, they find tion of the total number of short distances are intra- similar values, namely, 40 for the twist and 1.2 A for residue(48%), a considerable percentage of which do the rise. The increased tightness of the distance ranges not confer structural information. In addition, the does not considerably improve the precision of the most easily measured sequential distances, the group helix parameters. Van de ven and Hilbers [80] and of H8/H6 to sugar proton distances, constitute a large Wijmenga et al. [62] have investigated how precisely part of the sequential distances(20%). Unfortunately, torsion angles within a nucleotide unit are determined these sequential sugar-to-base distances only define by distances given a +0.2 A uncertainty in the dis- one side of the base plane. The same applies for the tances. It is found that a combination of d (6 2/23) base-to-base distances(6%). A large number of the distances quite accurately determine the glycosidic sequential distances involve sequential sugar-to-sugar
to 5.0 A˚ , and . 6 0.6 A˚ for distances . 5.0 A˚ . For exchanging protons, one could use 0 to 5 A˚ , but see Schmidt and James [68]. Deriving distance constraints via a ‘modified’ ISPA with conservative error bounds as described above prevents on the one hand putting too tight constraints on the long distances and still providing reasonable estimates for short distances, while on the other hand it also prevents choosing unduly loose error bounds. Are conservative or ‘loose’ distance constraints detrimental to the structure determination compared to ‘tight’ distance constraints? The answer seems to be that they are not. This has recently been elegantly demonstrated by Allain and Varani [79] using NOE data from the RNA hammerhead as a model system. They classified distances loosely into three categories, i.e. 0–2.9 A˚ (corresponding to dest 6 0.55 A˚ if one considers that the shortest distance is 1.8 A˚ anyhow), 2.9–3.5 A˚ (dest 6 0.3 A˚ ), and 3.5–5.0 A˚ (dest 6 0.75 A˚ ). These distance ranges correspond in fact quite closely to the ones mentioned above. These ranges are used for all protons except the exchangeable or strongly overlapping protons, for which the bounds were set to 0–5.0 A˚ . When tightening the constraints to ranges of 1.8–2.4 A˚ (dest 6 0.3 A˚ ), 1.8–2.9 A˚ (dest 6 0.55 A˚ ), 2.5–3.5 A˚ (dest 6 0.5 A˚ ), and 3.5–5.0 A˚ (dest 6 0.75 A˚ ), the precision of the structures does not significantly improve, i.e. the rmsd of the final structures improves by 0.2 A˚ at best (Table 2 of Allain and Varani [79]). Van de Ven and Hilbers [80] (see also Hilbers et al. [81]) have also investigated how the precision of the distance data affects the precision of the structures. Employing all ds(6/8/19/29/20/39;6/8/5) distances and assuming that they are determined with a precision of 6 0.25 A˚ , they find that in a G to G dinucleotide step the twist is determined with a range of about 428 and the rise with a range of roughly 1.5 A˚ . For the unrealistically high precision of 6 0.1 A˚ , they find similar values, namely, 408 for the twist and 1.2 A˚ for the rise. The increased tightness of the distance ranges does not considerably improve the precision of the helix parameters. Van de Ven and Hilbers [80] and Wijmenga et al. [62] have investigated how precisely torsion angles within a nucleotide unit are determined by distances given a 6 0.2 A˚ uncertainty in the distances. It is found that a combination of di(6;29/20/39) distances quite accurately determine the glycosidic torsion angles and also allows the determination of the fraction of N- or S-pucker of the sugar pucker, while the distance di(6;39) by itself does not determine well the fraction of N- or S-pucker. As pointed out in Ref. [62] uncertainties . 6 0.5 A˚ make it virtually impossible to determine the fraction of N- or S-pucker. The glycosidic torsion angle remains well determined by the distance di(6/8;29) even with an uncertainty as large as 6 0.7 A˚ . With an uncertainty of 6 0.2 A˚ in the distance di(39;59/50) the fraction of gþ rotamer is defined, but rather loosely. On the other hand, a combination of di(6;59/50) is sufficient to define this fraction rather well [62]. Uncertainties . 6 0.5 A˚ make it virtually impossible to determine the fraction of gþ rotamer (see Fig. 3.1 in Ref. [62]). The same error bounds for ISPA estimates derived above start from about 6 0.2 A˚ for distances around 2.0 A˚ and broaden to about 6 0.5 A˚ at 5.0 A˚ ; the error in the ‘loose’ as well as the ‘tight’ constraint sets used by Allain and Varani also increases from roughly 6 0.3–0.5 A˚ for short distances between 2.0 and 3.0 A˚ to 6 0.7 A˚ for distances between 3.5 and 5.0 A˚ . These error bounds correspond to uncertainties which fall somewhere in between the 6 0.2 A˚ and 6 0.5 A˚ range. Thus, for both sets of constraints the error bounds on the intra-residue distances are sufficiently narrow that the glycosidic torsion angle and sugar pucker, as well as the gþ rotamer, can be determined reasonably well. The conclusion that one is forced to draw from these investigations is that rather conservative distance constraints (see above) can be employed without detrimental effect on the ultimate precision of the structure. How does the number of constraints and their spread through the molecular structure affect the precision of the derived structure? As can be seen from Table 1, the spread in space of the NMR accessible distances in a helix is rather uneven. A large proportion of the total number of short distances are intraresidue (48%), a considerable percentage of which do not confer structural information. In addition, the most easily measured sequential distances, the group of H8/H6 to sugar proton distances, constitute a large part of the sequential distances (20%). Unfortunately, these sequential sugar-to-base distances only define one side of the base plane. The same applies for the base-to-base distances (6%). A large number of the sequential distances involve sequential sugar-to-sugar S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387 303
S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 distances(20%). They define the backbone quite well, compared to that in the GG step. In the Cg dinucleo- but are difficult to measure since the protons involved tide one finds distances involving H6 and H5 protons reside in very crowded regions of the NMR spectra. whereas in the gg dinucleotide only distances involv Very few detectable cross-strand distances exist, ing H8 protons are found. Similarly, the systematic except for those invol ving imino and amino protons study of Allain and Varani [791, using the hammer- and H2 to HI' protons in an A-helix. Furthermore, in head as a model system, shows that inclusion of loose contrast to a-helices in proteins where one finds NH(i) constraints(see above) involving exchangeable pro- to NH(i+3)distances, in nucleic acids no such long- tons greatly increases the definition, while adding range distances are found. This rather uneven spread sugar-to-sugar constraints and torsion angles gives through the chemical structure of the nMr detectable further improvement but to a lesser extent. For the distances, together with the lack of long-range so-called realistic loose' constraint set they find distances, is expected to detrimentally affect the approximate ranges of 10 for the twist and 0.4 A ision by which the helical structure can be deter- for the rise( see above for the distance ranges, this mined and thereby the precision of torsion angles and/ set contains no sugar-to-sugar constraints but includes or stacking and helical parameters such as twist and constraints involving exchangeable protons apart rise. How extensive should the constraint set be in from order to be able to define structural elements These ranges are narrower than those found by van reasonably well? de ven and Hilbers which again demonstrates the We find in the case of a 12-mer RNA duplex with a advantageous effect of a larger number of constraints tandem ga base pair that very high precision is on the precision. Note that van de ven and Hilbe chieved when about 30 'loose distance constraints considered isolated g to g orc to g dinucleotide per residue are employed; the constraint set included, steps, while in the study of Allain and Varani the besides the easily accessible constraints, sequential bases are part of a helix, which limits the allowed sugar-to-sugar distances and torsion angle constraints conformational space. This also demonstrates that for d and y, but not for a, B, e and s[76, 77]. The pair- the number of constraints required to obtain higl wise rmsd of the center part of the duplex consisting structural definition is quite context dependent. In of four base pairs which included the tandem ga base loop regions, and bulge regions where no base pair pairs was found to be 0.6 A. This highly precise struc- constraints are present, either the precision will be ture allowed the determination of the base pairing of lower with the same number of constraints or a larger the tandem ga base pair(no base pair constraints number of constraints is required to achieve the same were applied for the tandem ga part of the structure). high level of precision as in a helix. Finally, we note This suggests that the inclusion of the sequential that the in-depth studies of James and coworkers sugar-to-sugar distances is quite important. 68, 69] and of Luxon and gorenstein [72] on DNA Gorenstein and co-workers [71] also found that duplexes show that using a large number(> 10/resi- inclusion of sequential sugar-to-sugar distances due)of rather precise constraints(see above ), which gives considerable improvement in the definition of are well spread through the molecular structure, leads the derived structure. Van de Ven and Hilbers [80] to highly defined NMr derived structures. It appears (see also Ref [81 have investigated how the spread to be possible to distinguish sequence dependent f the distance constraints through the molecule structural effects affects the precision of the derived structure When using distance constraints with a precision 4.5. Conclusion of +0.25 A they find that in a g to g dinucleotide step the twist is determined with a range of 42 and the For the precision of NMr derived structures, the rise with a range of roughly 1.5 A (see above). Better number of (structurally relevant) distance constraints results are obtained when the dinucleotide is C to G; is more important than the precision of constraints the range for the twist is then 22 and for the rise The number of structurally relevant constraints 0.8 A. This improved definition results from a better should be around 15 to 30 per residue. They should spatial spread of the protons in the CG step as far as possible be uniformly spread through
distances (20%). They define the backbone quite well, but are difficult to measure since the protons involved reside in very crowded regions of the NMR spectra. Very few detectable cross-strand distances exist, except for those involving imino and amino protons and H2 to H19 protons in an A-helix. Furthermore, in contrast to a-helices in proteins where one finds NH(i) to NH(i þ 3) distances, in nucleic acids no such longrange distances are found. This rather uneven spread through the chemical structure of the NMR detectable distances, together with the lack of long-range distances, is expected to detrimentally affect the precision by which the helical structure can be determined and thereby the precision of torsion angles and/ or stacking and helical parameters such as twist and rise. How extensive should the constraint set be in order to be able to define structural elements reasonably well? We find in the case of a 12-mer RNA duplex with a tandem GA base pair that very high precision is achieved when about 30 ‘loose’ distance constraints per residue are employed; the constraint set included, besides the easily accessible constraints, sequential sugar-to-sugar distances and torsion angle constraints for d and g, but not for a, b, « and z [76,77]. The pairwise rmsd of the center part of the duplex consisting of four base pairs which included the tandem GA base pairs was found to be 0.6 A˚ . This highly precise structure allowed the determination of the base pairing of the tandem GA base pair (no base pair constraints were applied for the tandem GA part of the structure). This suggests that the inclusion of the sequential sugar-to-sugar distances is quite important. Gorenstein and co-workers [71] also found that inclusion of sequential sugar-to-sugar distances gives considerable improvement in the definition of the derived structure. Van de Ven and Hilbers [80] (see also Ref. [81]) have investigated how the spread of the distance constraints through the molecule affects the precision of the derived structure. When using distance constraints with a precision of 6 0.25 A˚ they find that in a G to G dinucleotide step the twist is determined with a range of 428 and the rise with a range of roughly 1.5 A˚ (see above). Better results are obtained when the dinucleotide is C to G; the range for the twist is then 228 and for the rise 0.8 A˚ . This improved definition results from a better spatial spread of the protons in the CG step as compared to that in the GG step. In the CG dinucleotide one finds distances involving H6 and H5 protons, whereas in the GG dinucleotide only distances involving H8 protons are found. Similarly, the systematic study of Allain and Varani [79], using the hammerhead as a model system, shows that inclusion of loose constraints (see above) involving exchangeable protons greatly increases the definition, while adding sugar-to-sugar constraints and torsion angles gives further improvement but to a lesser extent. For the so-called realistic ‘loose’ constraint set they find approximate ranges of 108 for the twist and 0.4 A˚ for the rise (see above for the distance ranges; this set contains no sugar-to-sugar constraints but includes constraints involving exchangeable protons apart from sugar-to-base and base-to-base constraints). These ranges are narrower than those found by van de Ven and Hilbers, which again demonstrates the advantageous effect of a larger number of constraints on the precision. Note that van de Ven and Hilbers considered isolated G to G or C to G dinucleotide steps, while in the study of Allain and Varani the bases are part of a helix, which limits the allowed conformational space. This also demonstrates that the number of constraints required to obtain high structural definition is quite context dependent. In loop regions, and bulge regions where no base pair constraints are present, either the precision will be lower with the same number of constraints or a larger number of constraints is required to achieve the same high level of precision as in a helix. Finally, we note that the in-depth studies of James and coworkers [68,69] and of Luxon and Gorenstein [72] on DNA duplexes show that using a large number ( . 10/residue) of rather precise constraints (see above), which are well spread through the molecular structure, leads to highly defined NMR derived structures. It appears to be possible to distinguish sequence dependent structural effects. 4.5. Conclusion For the precision of NMR derived structures, the number of (structurally relevant) distance constraints is more important than the precision of constraints. The number of structurally relevant constraints should be around 15 to 30 per residue. They should as far as possible be uniformly spread through the 304 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387
S.S. Wijmenga, B.M.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 Table 2 One-bond coupling constants Juc(Hz)in the base and sugar moieties of C/ N labeled 5-AMP, 5'-GMP, 5'-UMP and 5'-CMP 5′-AMPa 5′-GMP 5′-UMP 5′CMPb 216.0 203.2 H5 178.7 1758 HI 166.5 1514 H3′c 1520 HB 150.9 151.1 146 450 a average values derived from ID 'H and C spectra recorded at 500 and 700 MHz. b average values derived from ID C spectrum recorded at 600 MHz. in cyclic nucleotides, which have a defined sugar conformation, one finds for N- and S-type sugars JH?'c2 values of 158.4 Hz and 149.2 Hz, respectively, and for JHcy values of 149.6 Hz and 156.8 Hz, respectively. The error in the coupling constant values is given to be about ±0.3Hz[49 for 5-AMP, 5'-GMP, 5'-UMP and 5'-CMP the y torsion angle is a mixture of rotamers [49] 2209); in the cyclic nucleotides, rpApA rpGp(dG)), the y torsion angle is in a defined conformation, gauche + the JH5'cs and JHscs are then 151 Hz and 141 Hz, respectively e near isochronous resonances H5 and H5" value given is from proton-coupled IDC spectrum. derived from ID H spectrum at 400 MHz. Table adapted from Ref [49] chemical structure of the molecule, i.e. rather than new heteronuclear J-couplings have become including a large number from one category (e.g. accessible. Knowledge of the values of J-coupling intra-nucleotide), constraints should be derived in constants is essential for the rational design and such a way that each category is represented (intra- application of resonance assignment techniques nucleotide and its subdivisions or sequential and based on through-bond coherence transfer. Further cross-strand inter-nucleotide and its subdivisions, more, these couplings provide important additional see Table 1). In view of the uncertainties in the dis- parameters for the determination of torsion angles tance determination from NOE data, precise distances In this section we present an overview of these are prone to error and rather conservative distance heteronuclear J-couplings as well as ranges should be used for structure refinement proton J-coupling constants, their torsion angles, and describe NMR tech elr alues. The J-couplings 5.J discussed according to their torsion angle dependence, except for the J-couplings in the bases With the development of C and N labeling and the JcH-couplings which are discussed techniques for nucleic acids a large number of separately One-bond coupling constants Jcc(Hz) in the base and sugar moieties ofC/N labeled 5-AMP, 5'-GMP, 5'-UMP and 5'-CMP 5′-AMP 5′GMP UMP 5-CM 42.6 37 42.9 average values derived from iD C spectra recorded at 500 and 700 MHz(5-AMP and 5'-UMP) from ID C spectrum recorded at 600 MHz (5'-GMP and 5'-CMP). The error in the coupling constant values is given to be about + 0.4 Hz 49). Table adapted from Ref. [49]
chemical structure of the molecule, i.e. rather than including a large number from one category (e.g. intra-nucleotide), constraints should be derived in such a way that each category is represented (intranucleotide and its subdivisions or sequential and cross-strand inter-nucleotide and its subdivisions, see Table 1). In view of the uncertainties in the distance determination from NOE data, precise distances are prone to error and rather conservative distance ranges should be used for structure refinement. 5. J-couplings With the development of 13C and 15N labeling techniques for nucleic acids a large number of new heteronuclear J-couplings have become accessible. Knowledge of the values of J-coupling constants is essential for the rational design and application of resonance assignment techniques based on through-bond coherence transfer. Furthermore, these couplings provide important additional parameters for the determination of torsion angles. In this section we present an overview of these heteronuclear J-couplings as well as of proton– proton J-coupling constants, their relation to torsion angles, and describe NMR techniques for determining their values. The J-couplings are discussed according to their torsion angle dependence, except for the J-couplings in the bases and the 1 JCH-couplings which are discussed separately. Table 2 One-bond coupling constants 1 JHC (Hz) in the base and sugar moieties of 13C/ 15N labeled 59-AMP, 59-GMP, 59-UMP and 59-CMP 1 JCH 59-AMPa 59-GMPb 59-UMPa 59-CMPb H8 215.9 216.0 H2 203.2 H6 184.9 184.1 H5 178.7 175.8 H19 166.5 166.1 170.2 169.5 H29 c 150.9 151.4 151.7 151.2 H39 c 152.6 152.0 152.2 151.3 H49 150.9 151.1 150.3 148.9 H59 d 148.3 146.9e 148.3 147.2f H50 d 145.2 146.9e 144.9 145.0f a average values derived from 1D 1 H and 13C spectra recorded at 500 and 700 MHz. b average values derived from 1D 13C spectrum recorded at 600 MHz. c in cyclic nucleotides, which have a defined sugar conformation, one finds for N- and S-type sugars 1 J H29C29 values of 158.4 Hz and 149.2 Hz, respectively, and for 1 J H39C39 values of 149.6 Hz and 156.8 Hz, respectively. The error in the coupling constant values is given to be about 6 0:3 Hz [49]. d for 59-AMP, 59-GMP, 59-UMP and 59-CMP the g torsion angle is a mixture of rotamers [49] 2209}; in the cyclic nucleotides, rhpApAi, rhpGp(dG)i, the g torsion angle is in a defined conformation, gauche þ ; the 1 J H59C59 and 1 J H50C59 are then 151 Hz and 141 Hz, respectively. e near isochronous resonances H59 and H50; value given is from proton-coupled 1D 13C spectrum. f derived from 1D 1 H spectrum at 400 MHz. Table adapted from Ref. [49]. Table 3 One-bond coupling constants 1 JCC (Hz) in the base and sugar moieties of 13C/ 15N labeled 59-AMP, 59-GMP, 59-UMP and 59-CMP 1 JCC 59-AMPa 59-GMPa 59-UMPa 59-CMPa C19-C29 42.2 42.6 43.0 43.4 C29-C39 38.1 37.8 37.8 37.4 C39-C49 38.0 38.3 38.5 38.7 C49-C59 42.3 42.9 42.9 43.0 a average values derived from 1D 13C spectra recorded at 500 and 700 MHz (59-AMP and 59-UMP) from 1D 13C spectrum recorded at 600 MHz (59-GMP and 59-CMP). The error in the coupling constant values is given to be about 6 0.4 Hz [49]. Table adapted from Ref. [49]. S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387 305
S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 NH2 4 H1’-c=4Hz AMP C4=< 3H2 H GMP 59 UMP H1 H1-C6-3.7Hz (ray NH213 12N126Hs MP
Fig. 3. 306 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387