Cornish Peter V, Giedroc David P, Hennig Mirko
Department of Biochemistry and Biophysics, 2128 TAMU, Texas A&M University, College Station, TX 77843-2128, USA.
J Biomol NMR. 2006 Jul;35(3):209-23. doi: 10.1007/s10858-006-9033-x.
A variety of powerful NMR experiments have been introduced over the last few years that allow for the direct identification of different combinations of donor and acceptor atoms involved in hydrogen bonds in biomolecules. This ability to directly observe tertiary structural hydrogen bonds in solution tremendously facilitates structural studies of nucleic acids. We show here that an adiabatic HNN-COSY pulse scheme permits observation and measurement of J(N,N) couplings for nitrogen sites that are separated by up to 140 ppm in a single experiment at a proton resonance frequency of 500 MHz. Crucial hydrogen bond acceptor sites in nucleic acids, such as cytidine N3 nitrogens, can be unambiguously identified even in the absence of detectable H41 and H42 amino protons using a novel triple-resonance two-dimensional experiment, denoted H5(C5C4)N3. The unambiguous identification of amino nitrogen donor and aromatic nitrogen acceptor sites associated with both major groove as well as minor groove triple base pairs reveal the details of hydrogen bonding networks that stabilize the complex architecture of frameshift-stimulating mRNA pseudoknots. Another key tertiary interaction involving a 2'-OH hydroxyl proton that donates a hydrogen bond to an aromatic nitrogen acceptor in a cis Watson-Crick/sugar edge interaction can also be directly detected using a quantitative J(H,N) 1H,15N-HSQC experiment.
在过去几年中，已经引入了各种强大的核磁共振实验，这些实验能够直接识别生物分子中参与氢键的供体和受体原子的不同组合。这种在溶液中直接观察三级结构氢键的能力极大地促进了核酸的结构研究。我们在此表明，一种绝热的HNN-COSY脉冲序列允许在500 MHz的质子共振频率下，在单个实验中观察和测量相隔高达140 ppm的氮位点的J(N,N)耦合。即使在没有可检测到的H41和H42氨基质子的情况下，使用一种新型的三共振二维实验，即H5(C5C4)N3，也能够明确识别核酸中关键的氢键受体位点，如胞苷N3氮。与大沟以及小沟三碱基对相关的氨基氮供体位点和芳香氮受体位点的明确识别，揭示了稳定移码刺激mRNA假结复杂结构的氢键网络的细节。另一种关键的三级相互作用涉及一个2'-OH羟基质子，它在顺式沃森-克里克/糖边缘相互作用中向一个芳香氮受体提供氢键，也可以使用定量J(H,N) 1H,15N-HSQC实验直接检测到。
