Study of nucleic acid structure and dynamics by novel NMR methods

通过新型 NMR 方法研究核酸结构和动力学

基本信息

项目摘要

Ribonucleic acid structure determination by NMR spectroscopy relies primarily on local structural restraints provided by 1H-1H NOEs and J-couplings. When employed loosely, these restraints are broadly compatible with A- and B-like helical geometries and give rise to calculated structures that are highly sensitive to the force fields employed during refinement. A survey of recently reported NMR structures reveals significant variations in helical parameters, particularly the major groove width. Although helical parameters observed in high-resolution X-ray crystal structures of isolated A-form RNA helices are sensitive to crystal packing effects, variations among the published X-ray structures are significantly smaller than those observed in NMR structures. We have shown that restraints derived from aromatic 1H-13C residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) can overcome NMR restraint and force field deficiencies and afford structures with helical properties similar to those observed in high-resolution X-ray structures. Multiple new methods for accurate measurement of residual dipolar couplings in larger nucleic acids have also been developed. In particular, analogous to the recently introduced ARTSY method for measurement of one-bond 1H-15N residual dipolar couplings (RDCs) in large perdeuterated proteins, we have developed new methods for measurement of base 13C-1H and 15N-1H RDCs in protonated nucleic acids. Measurements are based on quantitative analysis of intensities in 1H-15N and 13C-1H TROSY-HSQC spectra, and have been demonstrated for a 71-nucleotide adenine riboswitch. Results compare favorably with those of conventional frequency-based measurements in terms of completeness and convenience of use. The ARTSY method derives the size of the coupling from the ratio of intensities observed in two TROSY-HSQC spectra recorded with different dephasing delays, thereby minimizing potential resonance overlap problems. Precision of the RDC measurements is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC reference spectrum, and is approximately given by 30/(S/N) Hz for 15N-1H and 65/(S/N) Hz for 13C-1H. The signal-to-noise ratio of both 1H-15N and 1H-13C spectra greatly benefits when water magnetization during the experiments is not perturbed, such that rapid magnetization transfer from bulk water to the nucleic acid, mediated by rapid amino and hydroxyl hydrogen exchange coupled with 1H-1H NOE transfer, allows for fast repetition of the experiment. RDCs in the mutated helix 1 of the riboswitch are compatible with nucleotide-specifically modeled, idealized A-form geometry and a static orientation relative to the helix 2/3 pair, which differs by ca 6 relative to the X-ray structure of the native riboswitch.
核磁共振谱确定核糖核酸结构主要依赖于1H-1HNOE和J-偶联提供的局部结构限制。当松散使用时,这些约束与A和B类螺旋几何形状广泛兼容,并产生对精化过程中采用的力场高度敏感的计算结构。对最近报道的核磁共振结构的调查显示,螺旋参数,特别是主槽宽度有显著的变化。虽然在分离的A-型RNA螺旋的高分辨X射线晶体结构中观察到的螺旋参数对晶体堆积效应很敏感,但已发表的X射线结构之间的变化明显小于在核磁共振结构中观察到的变化。结果表明,由芳香族1H-13C剩余偶极耦合(RDC)和剩余化学位移各向异性(RCSA)产生的约束可以克服核磁共振约束和力场缺陷,并提供类似于高分辨X射线结构中观察到的螺旋性质的结构。 还开发了多种准确测量较大核酸中剩余偶极耦合的新方法。特别是,类似于最近引进的ARTSY方法来测量大的全氚蛋白质中的单键1H-15N剩余偶极耦合(RDC),我们发展了测量质子化核酸中碱基13C-1H和15N-1H RDC的新方法。测量是基于对1H-15N和13C-1H TROSY-HSQC谱强度的定量分析,并已证明是针对71个核苷酸的腺嘌呤核糖开关。在完备性和使用便利性方面,结果与传统的基于频率的测量结果相比是有利的。ARTSY方法从两个TROSY-HSQC谱中观察到的强度之比得出耦合的大小,以不同的去相延迟记录,从而最小化潜在的共振重叠问题。RDC测量的精度受可在2D TROSY-HSQC参考谱中获得的信噪比S/N的限制,对于15N-1H,其测量精度约为30/(S/N)赫兹,对于13C-1H,约为65/(S/N)赫兹。当实验过程中的水磁化不受干扰时,1H-15N和1H-13C谱的信噪比都很大,这样,通过快速的氨基和羟基氢交换以及1H-1H NOE转移,从体水到核酸的快速磁化转移允许快速重复实验。核糖开关突变螺旋1中的RDC与核苷酸特异建模的理想化A型几何形状和相对于螺旋2/3对的静态取向兼容,这与天然核糖开关的X射线结构相差约6。

项目成果

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Ad Bax其他文献

Ad Bax的其他文献

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{{ truncateString('Ad Bax', 18)}}的其他基金

Structure and membrane binding of alpha-synuclein
α-突触核蛋白的结构和膜结合
  • 批准号:
    7967275
  • 财政年份:
  • 资助金额:
    $ 10.02万
  • 项目类别:
Structural study of the HIV1 gp41 coat protein
HIV1 gp41外壳蛋白的结构研究
  • 批准号:
    7967823
  • 财政年份:
  • 资助金额:
    $ 10.02万
  • 项目类别:
Study of hemagglutinin membrane fusion domain
血凝素膜融合结构域的研究
  • 批准号:
    8741545
  • 财政年份:
  • 资助金额:
    $ 10.02万
  • 项目类别:
Sructural study of the M4 Immune Evasion Protein
M4免疫逃避蛋白的结构研究
  • 批准号:
    9148956
  • 财政年份:
  • 资助金额:
    $ 10.02万
  • 项目类别:
Structural study of the HIV1 gp41 coat protein
HIV1 gp41外壳蛋白的结构研究
  • 批准号:
    8939688
  • 财政年份:
  • 资助金额:
    $ 10.02万
  • 项目类别:
Study of hemagglutinin membrane fusion domain
血凝素膜融合结构域的研究
  • 批准号:
    8349890
  • 财政年份:
  • 资助金额:
    $ 10.02万
  • 项目类别:
Protein structure and dynamics from residual dipolar couplings
残余偶极耦合的蛋白质结构和动力学
  • 批准号:
    8148713
  • 财政年份:
  • 资助金额:
    $ 10.02万
  • 项目类别:
Sructural study of immuno regulatory proteins by NMR spectroscopy
通过核磁共振波谱研究免疫调节蛋白的结构
  • 批准号:
    9357217
  • 财政年份:
  • 资助金额:
    $ 10.02万
  • 项目类别:
Protein structure and dynamics from residual dipolar couplings
残余偶极耦合的蛋白质结构和动力学
  • 批准号:
    7967277
  • 财政年份:
  • 资助金额:
    $ 10.02万
  • 项目类别:
Protein and nucleic acid structure and dynamics from residual dipolar couplings
残余偶极耦合的蛋白质和核酸结构和动力学
  • 批准号:
    8349704
  • 财政年份:
  • 资助金额:
    $ 10.02万
  • 项目类别:

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