Blommers M J, van de Ven F J, van der Marel G A, van Boom J H, Hilbers C W
N.S.R. Center for Molecular Design, Structure and Synthesis, University of Nijmegen, The Netherlands.
Eur J Biochem. 1991 Oct 1;201(1):33-51. doi: 10.1111/j.1432-1033.1991.tb16253.x.
The hairpin formed by d(ATCCTATTTATAGGAT) was studied by means of two-dimensional NMR spectroscopy and conformational analysis. Almost all 1H resonances of the stem region could be assigned, while the 1H and 31P spectra of the loop region were interpreted completely; this includes the stereospecific assignment of the H5' and H5" resonances. The derivation of the detailed loop structure was carried out in a stepwise fashion including some improved and new methods for structure determination from NMR data. In the first step, the mononucleotide structures were examined. The conformational space available to the mononucleotide was scanned systematically by varying the glycosidic torsion angle and pseudorotational parameters. Each generated conformer was tested against the experimental J coupling constants and NOE parameters. In the following stage, the structures of dinucleotides and longer fragments were derived. Inter-residue distances between protons were calculated by means of a procedure in which the simulated NOEs, obtained via a relaxation-matrix approach, were fitted to the experimental NOEs without the introduction of a molecular model. In addition, the backbone torsion angles beta, gamma and epsilon were deduced from homocoupling and heterocoupling constants. These data served as constraints in the next step, in which the loop sequence was subjected to a multi-conformer generation procedure. The resulting structures were tested against the mentioned constraints and disregarded if these constraints were violated. This yielded a family of structures for the loop region, confined to a relatively narrow conformational space. A representative conformation was subsequently docked on a B-type stem which fulfilled the structural constraints (derived from the NMR experiments for the stem region) to yield the hairpin structure. Results obtained from subsequent restrained-molecular-mechanics as well as free-molecular-mechanics calculations are in accordance with those obtained by means of the analysis described above. The structure of the hairpin loop is a compactly folded conformation and the first base of the central TTTA region forms a Hoogsteen T-A pair with the fourth base. This Hoogsteen base pair is stacked upon the sixth base pair of the B-type double-helical stem. The second base of the loop is folded into the minor groove, whereas the third base of the loop is partly stacked on the first and fourth bases. The phosphate backbone exhibits a sharp turn between the third and fourth nucleotides of the loop. The peculiar structure of this hairpin loop is discussed in relation to loop folding in DNA and RNA hairpins and in relation to a general model for loop folding.
通过二维核磁共振光谱和构象分析研究了由d(ATCCTATTTATAGGAT)形成的发夹结构。茎区几乎所有的1H共振峰都能被归属，而环区的1H和31P谱也得到了完整的解释；这包括对H5'和H5"共振峰的立体专一性归属。环区详细结构的推导是逐步进行的，包括一些改进的和新的从核磁共振数据确定结构的方法。第一步，研究单核苷酸结构。通过改变糖苷扭转角和假旋转参数，系统地扫描单核苷酸可利用的构象空间。每个生成的构象异构体都根据实验测得的J耦合常数和NOE参数进行检验。在接下来的阶段，推导二核苷酸和更长片段的结构。质子间的残基间距离通过一种程序计算得出，在该程序中，通过弛豫矩阵方法获得的模拟NOE与实验测得的NOE进行拟合，而无需引入分子模型。此外，主链扭转角β、γ和ε由同核耦合和异核耦合常数推导得出。这些数据在下一步中作为约束条件，在此步骤中，环序列经过多构象异构体生成程序。所得结构根据上述约束条件进行检验，如果违反这些约束条件则予以舍弃。这产生了一系列环区结构，局限于相对狭窄的构象空间。随后将一个代表性构象对接在一个满足结构约束条件（从茎区的核磁共振实验推导得出）的B型茎上，以得到发夹结构。后续的受限分子力学以及自由分子力学计算所得结果与通过上述分析得到的结果一致。发夹环的结构是一种紧密折叠的构象，中央TTTA区域的第一个碱基与第四个碱基形成一个Hoogsteen T-A对。这个Hoogsteen碱基对堆积在B型双螺旋茎的第六个碱基对上。环的第二个碱基折叠进入小沟，而环的第三个碱基部分堆积在第一个和第四个碱基上。磷酸主链在环的第三个和第四个核苷酸之间呈现出一个急转弯。结合DNA和RNA发夹中环的折叠以及环折叠的一般模型，讨论了这个发夹环的特殊结构。
