清华大学：《生物核磁共振波谱学》课程教学资源（文献资料，英文版）The use of nmr methods for conformational

292 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 Cytosine Thymine Uracil Guanine i+1 Fig 1. Structure and atom numbering in nucleic acids, according to the IUPAC/UB guidelines 61, of the five common bases(pyrimidines C,T and U; purines G and A)(A), and of the B-D-deoxy )riboses(B and C).(B)also shows the torsion angles in the sugar-phosphate backbone(a, B, 6, e and $)and the glycosidic torsion angle x( the exact definition is given in the text), a designation of the chain 5 to 3 direction and the unit numbering in a polynucleotide chain. (C) shows the puckering of the two most common B-D-(deoxy )ribose sugar ring conformations, the C2'-endo(or S-type )and the C3'-endo(or N-type)conformati subsequently going into more detail. Finally, we 4.1.Overview of short distances and their general iscuss their derivation from NOESY spectra and characteristics their use as constraints in simulated annealing protocols In Table 1 we have summarized the short distances

subsequently going into more detail. Finally, we discuss their derivation from NOESY spectra and their use as constraints in simulated annealing protocols. 4.1. Overview of short distances and their general characteristics In Table 1 we have summarized the short distances Fig. 1. Structure and atom numbering in nucleic acids, according to the IUPAC/IUB guidelines [61], of the five common bases (pyrimidines C, T and U; purines G and A) (A), and of the b-D-(deoxy)riboses (B and C). (B) also shows the torsion angles in the sugar–phosphate backbone (a, b, g, d, « and z) and the glycosidic torsion angle x (the exact definition is given in the text), a designation of the chain 59 to 39 direction and the unit numbering in a polynucleotide chain. (C) shows the puckering of the two most common b-D-(deoxy)ribose sugar ring conformations, the C29-endo (or S-type) and the C39-endo (or N-type) conformations. 292 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)287-387 293 Overview of short distances per residue L. constant 4. base-sugar 632 3000 0841 Inter-nucleotide 1. non-exchangeable I sequential sugar-base 2. sequential base-base 3. sequential sugar-sugar I. within base pair M: measurable distances <5 to 6 A[62]:A: tely conformation independent distances; B: distances that are conformation in within approximately +0.2 A; C: 'structural distances', i.e. confomation dependent distances with variation >0.2 A(see text):C:NMR accessible structural' distances: D: NMR accessible 'structural distances that are different in A- and B-helices. The intra-nucleotide distances: 1. The constant distances d 2: 2), d,(5, 5), dA(6, 5)and d(6, M); 2. the sugar-to-sugar distances, dA1'-4: 1-4); they all fall into group B, except for d, (2: 4)and d,(1, 4), which fall into groups C and C, while group d only contains d( 2: 43. the distances d(2-4, 5/ 5); group B contains the distances dA( 2/2.575); group C contains d(374, 575): none of them fall into groups C or D, 4. sugar-to-base distances d,(6/8: 1-5); they are subdivided according to: group B, d,(6/8: 4), group C, d( 6/8; 1-3, 575), group C, excluded d 6/8; 575), group D, dA6/8, 1-3). The distances d( S/M: 1-5)are not taken into account since they are larger than 5 A[62]. The inter-nucleotide distance (considered are the distances to and from Cytosine in a GCG trinucleotide sequence (see Fig. 2). I. Non-exchangeable protons: 1. sequential sugar-to-base distances, d(1-3; 8)and ds(1-3: 6/5); all of them fall into categories C, C and D; 2. base-to-base distances, ds(6/5; 8)and d, (8: 6/ 5); all of them fall into categories C, C and D; 3. sequential sugar-to-sugar distances, ds(1-2, 4, 5), ds(5",1-2,4), ds(2: 3), ds(3, 2), ds(2, 2)and ds (2, 2); all of them are conformation dependent (category C), but only dx2: 3)and d33, 2) are easily accessible tween A-type and B-type helices. Il. Inter-nucleotide distances involving exchangeable protons: 1. The distances within a bas d(NH2: NH); this distance depends on conformation(category C), is NMR accessible (C ), but does not differ between A-type helices; 2. the sequential distances are ds(NH2: NH)and ds NH; NH2); they both fall into category C and C, but not into category D: strand distances are d(NH2: NH2)s, and d(NH2: NH2)3, they fall into category C, Cand D(however, note that the NH2 resonances of G may be broadened making them inaccessible for NMR) ( 5-6 A)and categorized them into two main dependence on conformation is indicated(a to D). groups, intra-nucleotide and inter-nucleotide dis- with category A referring to conformation indepen- tances, with further subdivision to reflect more dent distances, category B to distances that can vary detailed conformational characteristics. The inter- by less than +0. 2 A, and category C to structural nucleotide distances fall into the two broad groups distances, i.e. distances that convey structural infor- of sequential and cross-strand distances involving mation since they can vary by more than +0.2 A non-exchanging protons and exchanging protons, Thestructural distances, category C, are subdivided respectively. The sequential distances involving into two further categories to indicate their usefulness non-exchanging protons are again subdivided into category C contains those structuraldistances that sugar-to-sugar distances, base-to-base distances and are reasonably well accessible by NMR, and category ugar-to-base distances. Within each category their D refers to NMR accessiblestructural'distances that

( , 5–6 A˚ ) and categorized them into two main groups, intra-nucleotide and inter-nucleotide dis￾tances, with further subdivision to reflect more detailed conformational characteristics. The inter￾nucleotide distances fall into the two broad groups of sequential and cross-strand distances involving non-exchanging protons and exchanging protons, respectively. The sequential distances involving non-exchanging protons are again subdivided into sugar-to-sugar distances, base-to-base distances and sugar-to-base distances. Within each category their dependence on conformation is indicated (A to D), with category A referring to conformation indepen￾dent distances, category B to distances that can vary by less than 6 0.2 A˚ , and category C to ‘structural’ distances, i.e. distances that convey structural infor￾mation since they can vary by more than 6 0.2 A˚ . The ‘structural’ distances, category C, are subdivided into two further categories to indicate their usefulness; category C9 contains those ‘structural’ distances that are reasonably well accessible by NMR, and category D refers to NMR accessible ‘structural’ distances that Table 1 Overview of short distances per residue Type % M A B C C9 D Intra-nucleotide 1. constant 5 3 3 0 0 0 0 2. sugar–sugar 16 10 0 8 2 2 1 3. sugar–59/50 13 8 0 4 4 0 0 4. base–sugar 12 7 0 1 6 4 4 sum 46 28 3 13 12 6 5 Inter-nucleotide I. non-exchangeable 1. sequential sugar–base 20 12 0 0 12 12 12 2. sequential base–base 6 4 0 0 4 4 0 3. sequential sugar–sugar 20 12 0 0 12 2 2 4. cross-strand (3%) (2) 0 0 (2) (2) (2) sum 46 28 0 0 28 18 14 II. exchangeable (imino/amino) 1. within base pair 2 1 0 0 1 1 0 2. sequential 3 2 0 0 2 2 0 3. cross-strand 3 2 0 0 2 2 2 sum 8 5 0 0 5 5 2 Total 100 61 3 13 45 29 20 % 100 5 21 74 48 33 M: measurable distances , 5 to 6 A˚ [62]; A: completely conformation independent distances; B: distances that are conformation independent within approximately 6 0.2 A˚ ; C: ‘structural distances’, i.e. conformation dependent distances with variation . 0.2 A˚ (see text); C9: NMR accessible ‘structural’ distances; D: NMR accessible ‘structural’ distances that are different in A- and B-helices. The intra-nucleotide distances: 1. The constant distances di(29;20), di(59;50), di(6;5) and di(6;M); 2. the sugar-to-sugar distances, di(19-49;19-49); they all fall into group B, except for di(20;49) and di(19;49), which fall into groups C and C9, while group D only contains di(20;49); 3. the distances di(29-49;59/ 50); group B contains the distances di(29/20;59/50); group C contains di(39/49;59/50); none of them fall into groups C9 or D; 4. sugar-to-base distances di(6/8;19-50); they are subdivided according to: group B, di(6/8;49), group C, di(6/8;19-39, 59/50), group C9, excluded di(6/8;59/50), group D, di(6/8;19-39). The distances di(5/M;19-50) are not taken into account since they are larger than 5 A˚ [62]. The inter-nucleotide distances (considered are the distances to and from Cytosine in a GCG trinucleotide sequence (see Fig. 2). I. Non-exchangeable protons: 1. sequential sugar-to-base distances, ds(19-39;8) and ds(19-39;6/5); all of them fall into categories C, C9 and D; 2. base-to-base distances, ds(6/5;8) and ds(8;6/ 5); all of them fall into categories C, C9 and D; 3. sequential sugar-to-sugar distances, ds(19-20,49;50), ds(50;19-20,49), ds(29;39), ds(39;29), ds(29;20) and ds(29;20); all of them are conformation dependent (category C), but only ds(29;39) and ds(39;29) are easily accessible and differ between A-type and B-type helices. II. Inter-nucleotide distances involving exchangeable protons: 1. The distances within a base pair are dc(NH2; NH); this distance depends on conformation (category C), is NMR accessible (C9), but does not differ between A-type and B-type helices; 2. the sequential distances are ds(NH2;NH) and ds(NH;NH2); they both fall into category C and C9, but not into category D; 2. the cross￾strand distances are dc(NH2;NH2) 59 and dc(NH2;NH2) 39; they fall into category C, C9 and D (however, note that the NH2 resonances of G may be broadened making them inaccessible for NMR). S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387 293

S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 also show differences depending on whether they are di(1: 4). Only d(2; 4)differs significantly present in an A- or B-type helix between S-type and N-type conformers, with As can be seen from Table 1 approximately 60 d(2;4 4. 2A for the s-type conformer distances per residue can in principle be measured (pseudorotation angle P= 160)and dH(2: 4)= The number of distances that are constant within 2.8A for the N-type conformer (P=10 +0.2 A is rather high. They represent about 26% Although it is in principle possible to determine of the total number of measurable distances Their the sugar conformation from the di(2": 4")distance percentage is even higher for the intra-nucleotide dis- the accuracy of the determination is limited. The tances, of which they represent about 57%(16 out of di(2: 4) distance is difficult to determine from 28). The distances that convey relevant structural NOE intensities because of spin diffusion effects information (in helices) and are reasonably well due to the close proximity of the H2"and H2 accessible by NMR represent less then half (48%)of protons. Also note that in RNA the H2 proton is the total number of distances, while only 20 are dif- bsent, so that these sugar distances cannot be used ferent between A-and B-type helices(33%). Note at all to determine the puckering. The distance also the small number of structurally very important di(1: 4)is almost identical for N-type and s cross-strand and sequential distances involving pe sugars 3.4 A), but has a lower value for exchanging protons which establish base pairing sugar rings with an intermediate pseudorotation (8%), and the small number of cross-strand distances angle, di(1: 4)=2.6A for P=90. Here again involving non-exchanging protons (3%) On the other spin diffusion can adversely affect the accuracy hand, sequential sugar-to-base and sugar-to-sugar dis- distance of the determination tances, which are so important for establishing base 3. The distances dA(3: 575)depend only weakly on stacking and defining the phosphate backbone, are the sugar ring conformation, but significantly on both relatively large in number(20%). The former the y torsion angle, while the distances di 44; 57 e mostly reasonably well accessible by NMR 6) only depend on the y torsion angle. The whereas the latter are extremely difficult to establish distances di(374; 5/5")therefore allow the deter- Thus, a rather uneven spread in the short distances is mination of the torsion angle [62, 63]. This can be found through the chemical structure. As a conse- done in conjunction with relevant J-couplings(see quence, important structural features such as base Section 5). Given an uncertainty in these distances ing often hinge on the presence of a particular of +0.2 A, they do not discriminate well between NOE contact reflecting one short distance the different ranges of the y torsion angle, in ticular when an equilibrium between g* and g 4.2. Overview of structurally important intra tamers exists. The distances di(2/2 575) nucleotide distances depend on both the sugar puckering and the torsion angle y, but their dependence is weak, and they are The intra-nucleotide distances in dna and rna of the order of 5 to 6 A [62] can conveniently be subdivided according to the 4. The distance between HI and H8/6, d(1: 6/8) categories indicated in Table 1, i.e. (1)conformation depends only on the glycosidic torsion angle x. It independent distances,(2)distances between sugar thus provides a means for determining this torsion protons, (3)distances between H2/2/3/4 and H5/ angle. However, the maximum difference in the 5"(4)distances between HI'through H575"and base values of di(1: 6/8)for x in the syn domain (x= 60°) and in the anti domain(x=240°) is only about 1.2 A. Given that in practice the uncertainty 1. The conformation independent distances are: the in the distance determination from noe data is of geminal proton distances, d 2, 2 )and d 5, 5) the order of±0.2Ato±0.5A. it is to be of 1.8A, d( 5: 6)(= 2.45 A)in Cytosine and expected that the use of d; (1: 6/8)is a rather impre Uracyl, and d 6, M)in Thymidine cise means to determine the x torsion angle. The 2. The distances within the sugar ring are all indepen- other sugar proton to base proton distances, dH(2/ dent of its conformation, except for di(2: 4)and 21314: 6/8), depend on both the sugar puckering

also show differences depending on whether they are present in an A- or B-type helix. As can be seen from Table 1 approximately 60 distances per residue can in principle be measured. The number of distances that are constant within 6 0:2 A˚ is rather high. They represent about 26% of the total number of measurable distances. Their percentage is even higher for the intra-nucleotide dis￾tances, of which they represent about 57% (16 out of 28). The distances that convey relevant structural information (in helices) and are reasonably well accessible by NMR represent less then half (48%) of the total number of distances, while only 20 are dif￾ferent between A- and B-type helices (33%). Note also the small number of structurally very important cross-strand and sequential distances involving exchanging protons which establish base pairing (8%), and the small number of cross-strand distances involving non-exchanging protons (3%). On the other hand, sequential sugar-to-base and sugar-to-sugar dis￾tances, which are so important for establishing base stacking and defining the phosphate backbone, are both relatively large in number (20%). The former are mostly reasonably well accessible by NMR, whereas the latter are extremely difficult to establish. Thus, a rather uneven spread in the short distances is found through the chemical structure. As a conse￾quence, important structural features such as base pairing often hinge on the presence of a particular NOE contact reflecting one short distance. 4.2. Overview of structurally important intra￾nucleotide distances The intra-nucleotide distances in DNA and RNA can conveniently be subdivided according to the categories indicated in Table 1, i.e. (1) conformation independent distances, (2) distances between sugar protons, (3) distances between H29/20/39/49 and H59/ 50, (4) distances between H19 through H59/50 and base protons. 1. The conformation independent distances are: the geminal proton distances, di(29;20) and di(59;50), of 1.8 A˚ , di(5;6) ( ¼ 2.45 A˚ ) in Cytosine and Uracyl, and di(6,M) in Thymidine. 2. The distances within the sugar ring are all indepen￾dent of its conformation, except for di(20;49) and di(19;49). Only di(20;49) differs significantly between S-type and N-type conformers, with di(20;49) ¼ 4.2 A˚ for the S-type conformer (pseudorotation angle P ¼ 1608) and di(20;49) ¼ 2.8 A˚ for the N-type conformer (P ¼ 108). Although it is in principle possible to determine the sugar conformation from the di(20;49) distance, the accuracy of the determination is limited. The di(20;49) distance is difficult to determine from NOE intensities because of spin diffusion effects, due to the close proximity of the H29 and H20 protons. Also note that in RNA the H20 proton is absent, so that these sugar distances cannot be used at all to determine the puckering. The distance di(19;49) is almost identical for N-type and S￾type sugars (3.4 A˚ ), but has a lower value for sugar rings with an intermediate pseudorotation angle, di(19;49) ¼ 2.6 A˚ for P ¼ 908. Here again spin diffusion can adversely affect the accuracy distance of the determination. 3. The distances di(39; 59/50) depend only weakly on the sugar ring conformation, but significantly on the g torsion angle, while the distances di(49; 59/ 50) only depend on the g torsion angle. The distances di(39/49; 59/50) therefore allow the deter￾mination of the torsion angle [62,63]. This can be done in conjunction with relevant J-couplings (see Section 5). Given an uncertainty in these distances of 6 0.2 A˚ , they do not discriminate well between the different ranges of the g torsion angle, in par￾ticular when an equilibrium between gþ and gt rotamers exists. The distances di(29/20; 59/50) depend on both the sugar puckering and the torsion angle g, but their dependence is weak, and they are of the order of 5 to 6 A˚ [62]. 4. The distance between H19 and H8/6, di(19;6/8), depends only on the glycosidic torsion angle x. It thus provides a means for determining this torsion angle. However, the maximum difference in the values of di(19;6/8) for x in the syn domain (x ¼ 608) and in the anti domain (x ¼ 2408) is only about 1.2 A˚ . Given that in practice the uncertainty in the distance determination from NOE data is of the order of 6 0.2 A˚ to 6 0.5 A˚ , it is to be expected that the use of di(19;6/8) is a rather impre￾cise means to determine the x torsion angle. The other sugar proton to base proton distances, di(29/ 20/39/49;6/8), depend on both the sugar puckering 294 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387

S.S. Wimenga, B NM van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 295 and the x torsion angle. The distance d(4: 6/8) reproduced from Wijmenga et al. [62] gives the does not convey useful structural information sequential distances, ds(; r) cross-strand since its dependence on these parameters is weak distances, dc r)and d(l; r), found in A-DNA [62]. The distances di (2/2/3: 6/8)are on the other B-DNA and rna helices hand quite useful. Each of these distances defines The cross-strand distances, dcdl, r) and desc; r). the x torsion angle quite well, because of their involving exchangeable protons are indicative of quite strong dependence on the torsion angle x base pair formation. The sequential distances involv [62]. Their dependence on the sugar puckering is ing either exchanging or non-exchanging protons are rather weak, in particular for the distances di(2/ indicative of base stacking. However, only a limited 2; 6/8)[62]. Despite this weak dependence on the number depend on the type of helix conformation. In sugar puckering, a concerted use of d(2273: 6/8) both A- and B-type helices, short base-to-base dis- makes it possible to determine the percentage N- tances, d, (6/8/5/M: 6/8/5/M), are present, depending type or S-type pucker, but to achieve a reasonable on the sequence. Similarly, all distances involving level of precision requires that the uncertainty in exchanging pro otons are very sin their values should be less than +0.5A [62]. w B-type helices. The differences occur for the cross- finally note that Lane and co-workers[64] have shown strand and sequential distances involving H2 protons the improved reliability of sugar pucker determination d&(2; 1/2)3 and ds(2; 1), the sequential sugar-to- using these distances together with J-couplings base distances, ds(2/273: 6/8/5), and for a number The H5/H5"to base proton distances, di(/5: 6/8) of stances,d(2n2”;5 depend on three torsion angles, y, 8 and x. Their 5),d, (2 3),d, (22)and d(1: 5). Short cross- dependence on the sugar pucker(o), and on th strand, as well as sequential H2 to HI'distances glycosidic torsion angle (x) in the usual anti are present in A-type helices, but absent in B-type domain (180-240)is weak, but they depen helices. Short sequential H2 to H6/8 distances and quite strongly on the y torsion angle. In particular long h2" to H6/8 distances are seen in A-helices. ;6/8) while in B-helices the reverse is found. The sugar- to 4.5 A), while for y' the distance d,(5: 6/8) to-sugar distances show the following pattern: Short becomes short(2.5 to 2.9 A). As has been s o8) sequential H2/H2"to H5 /5"distances in A-helices, ith uncertainties in the distance estimates in the while in B-helices these distances are long, rather order of +0.2 A, they determine quite well the long, but measurable, sequential H2 to H3 distances torsion angle y[62]. The distances d (5 /5"; 6/8) In A-helices, which are over 7 A and thus not measur- can be quite useful in NOESY spectra of dNa able in B-helices; finally, long(>7 A)sequential H2 since the related NoE cross pea to h2 and hi to h5" distances in a-helices. which in a crowded spectral region. This does not hold are relatively short in B-helices. While the distances true for RNa where these cross peaks overlap with involving H2 and the h2 /2"to base distances are he other H6/8 to H2 /3'NOE cross peaks. On the quite accessible from NMR spectra, the sugar-to- other hand, the distances d;(3/4; 5/5) all sugar distances are difficult to determine since the relate to cross peaks in crowded spectral sugar proton resonances reside in quite crowded regions for both DNA and RNA and are thus spectral regions difficult to establish 4.4. Derivation of distances from NOESY spectra and structure characterization using distances 4.3. Overview of structurally important sequential and cross-strand distances We will discuss here the three aspects of NMr accessible distances that are of particular relevance Helical conformations form an important part of for structure determination. First, how precisely can nucleic acid structures. We therefore present an over- distances be derived from NOE data? Secondly, how iew of the distances in the two most commonly does this on affect the precision of the deter- found helix types, A-helices and B-helices. Fig. 2, mined structure? Thirdly, how does the spread and

and the x torsion angle. The distance di(49;6/8) does not convey useful structural information since its dependence on these parameters is weak [62]. The distances di(29/20/39;6/8) are on the other hand quite useful. Each of these distances defines the x torsion angle quite well, because of their quite strong dependence on the torsion angle x [62]. Their dependence on the sugar puckering is rather weak, in particular for the distances di(29/ 20;6/8) [62]. Despite this weak dependence on the sugar puckering, a concerted use of di(29/20/39;6/8) makes it possible to determine the percentage N￾type or S-type pucker, but to achieve a reasonable level of precision requires that the uncertainty in their values should be less than 6 0.5 A˚ [62]. We finally note that Lane and co-workers [64] have shown the improved reliability of sugar pucker determination using these distances together with J-couplings. The H59/H50 to base proton distances, di (59/50;6/8), depend on three torsion angles, g, d and x. Their dependence on the sugar pucker (d), and on the glycosidic torsion angle (x) in the usual anti domain (180–2408) is weak, but they depend quite strongly on the g torsion angle. In particular, for gþ both di(59; 6/8) and di(50;6/8) are long (3.7 to 4.5 A˚ ), while for gt the distance di(50;6/8) becomes short (2.5 to 2.9 A˚ ). As has been shown, with uncertainties in the distance estimates in the order of 6 0.2 A˚ , they determine quite well the torsion angle g [62]. The distances di(59/50;6/8) can be quite useful in NOESY spectra of DNA, since the related NOE cross peaks do not reside in a crowded spectral region. This does not hold true for RNA where these cross peaks overlap with the other H6/8 to H29/39 NOE cross peaks. On the other hand, the distances di(39/49;59/50) all relate to cross peaks in crowded spectral regions for both DNA and RNA and are thus difficult to establish. 4.3. Overview of structurally important sequential and cross-strand distances Helical conformations form an important part of nucleic acid structures. We therefore present an over￾view of the distances in the two most commonly found helix types, A-helices and B-helices. Fig. 2, reproduced from Wijmenga et al. [62], gives the sequential distances, ds(l;r), and cross-strand distances, dci(l;r) and dcs(l;r), found in A-DNA, B-DNA and RNA helices. The cross-strand distances, dci(l;r) and dcs(l;r), involving exchangeable protons are indicative of base pair formation. The sequential distances involv￾ing either exchanging or non-exchanging protons are indicative of base stacking. However, only a limited number depend on the type of helix conformation. In both A- and B-type helices, short base-to-base dis￾tances, ds(6/8/5/M;6/8/5/M), are present, depending on the sequence. Similarly, all distances involving exchanging protons are very similar in A- and B-type helices. The differences occur for the cross￾strand and sequential distances involving H2 protons, dcs(2;19/2)39 and ds(2;19), the sequential sugar-to￾base distances, ds(29/20/39;6/8/5), and for a number of sequential sugar-to-sugar distances, ds(29/20;59/ 50), ds(29;39), ds(20;20) and ds(19;50). Short cross￾strand, as well as sequential H2 to H19 distances, are present in A-type helices, but absent in B-type helices. Short sequential H29 to H6/8 distances and long H20 to H6/8 distances are seen in A-helices, while in B-helices the reverse is found. The sugar￾to-sugar distances show the following pattern: Short sequential H29/H20 to H59/50 distances in A-helices, while in B-helices these distances are long; rather long, but measurable, sequential H29 to H39 distances in A-helices, which are over 7 A˚ and thus not measur￾able in B-helices; finally, long ( . 7 A˚ ) sequential H20 to H29 and H19 to H50 distances in A-helices, which are relatively short in B-helices. While the distances involving H2 and the H29/20 to base distances are quite accessible from NMR spectra, the sugar-to￾sugar distances are difficult to determine since the sugar proton resonances reside in quite crowded spectral regions. 4.4. Derivation of distances from NOESY spectra and structure characterization using distances We will discuss here the three aspects of NMR accessible distances that are of particular relevance for structure determination. First, how precisely can distances be derived from NOE data? Secondly, how does this precision affect the precision of the deter￾mined structure? Thirdly, how does the spread and S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387 295

S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 A HS HS"H4H3'H2 H2"HI H8 NH2 NH [ NH2 HS H6 HI H2" F H3'H4'H5"H5' HS"" H3 H2H2" H6 CH3 NH NH2 H2 H8 8HEB2田HB H5H5”H4H3H2"H2 CH3NH NH2 H2 H8 HI' H2"H2H H3" H4 H5" HSA HSHS"H4'H3'H2'H2 中中 H2'H3'H4'HS"H5 HSHS"H4'H3'H2'H2" 6 HS NH2JNH NH2 H8 HI H2"H2 H3 H4 HS"HS HSH5H4H3H2'H2HI H6 CH3 NHNH2 H2 H8 HI H2"H2 2" H3 H4 HS"HSA HS'H5"H4 H2"HI HS H2 NH2]NH CH3 H6 HI"" H3"H4 H5"H5 H5 HS"H4 H3 H2 H2"HI H6 HS NH2NH NH2 H8 HI H2"H2 H4 HS"HS HS HS" H4' H3 H2H2"HI H8 H2 NH2 NH CH3 H6 HI H2"H2 H3 H4 HS"HS" 田FHHH田HNH2 H CH H6 HI H2H2 H3 H4H5H5 EHm业正mcm[N正正EE时3 HS HS" H4 H3" H2 H2" HI HB NH2 NH NH2 HS H6 HI H2"H2 H3" H4 HS"H5 IHS HS" H4 H3 HZ H2 HI H8 NH2 NH NH2 H5 H6 HI' H2H2. H3' H4' H5" H5' HS H5"H4 H3 H2 H2"HI'H8 H2 NH2 NH CH3 H6 HI H2"H2 H3 H4 HS"H H5 H5 H4 H3 H2H2"HI H8 NH2 NH NH2 HS H6 HI H2" H2 H3" H4 H5"H5 H5’H5”H4H3′H2′H2"H′H8NH2NH 12 H5 H6 HI'H2H2'H3'H4'H5"H5 5 3 base stacking in A-DNA(A), B-DNA(B)and RNA(C). The meaning of the symbols is: 0-2.5 A( thick solid line), 2.5-3.0 A(solid line ), 3.0-4.0 A(dashed line), 4.0-50A (dotted line)

Fig. 2. Overview of short sequential and inter-strand proton–proton distances for all possible combinations of base stacking in A-DNA (A), B-DNA (B) and RNA (C). The meaning of the symbols is: 0–2.5 A˚ (thick solid line), 2.5–3.0 A˚ (solid line), 3.0–4.0 A˚ (dashed line), 4.0–5.0 A˚ (dotted line). 296 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387