ANALYSIS OF THE BACKBONE DYNAMICS OF INTERLEUKIN-1-BETA USING 2-DIMENSIONAL INVERSE DETECTED HETERONUCLEAR N-15-H-1 NMR-SPECTROSCOPY
ANALYSIS OF THE BACKBONE DYNAMICS OF INTERLEUKIN-1-BETA USING 2-DIMENSIONAL INVERSE DETECTED HETERONUCLEAR N-15-H-1 NMR-SPECTROSCOPY
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DOI：
10.1021/bi00484a006
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发表时间：
1990-08-14
期刊：
影响因子：
2.9
通讯作者：
GRONENBORN, AM
中科院分区：
文献类型：
--
作者：
CLORE, GM;DRISCOLL, PC;GRONENBORN, AM
15N均一标记白介素1β的骨架动力学用二维逆探测异核15N-1H核磁共振波谱对其进行了研究。获得了90%主链酰胺基团在600 MHz光谱仪频率下的15N T1、T2和NOE数据。这些数据为三个时间尺度上的运动提供了证据。所有残基在20-50ps的时间尺度上都表现出非常快的运动，可以用平均值为0.82.+-的一阶参数来表征。0.05。对于包含锥体内自由扩散的模型，这些残基特定的序参数转化为平均锥体半角20.7。+-。3.3度..32个残基也在0.5-4 ns的时间尺度上表现出运动，略低于蛋白质的整体旋转相关时间(8.3 ns)。必须引用这些额外的运动来解释实验和最简单的理论公式之间的差异，在最简单的理论公式中，内部运动仅由两个参数来描述，即广义序参数和有效关联时间[Lipari，G.，and Szabo，A.(1982a)。J.Am化学。SoC。104、4546-4559]。特别是，虽然简单的公式可以解释15N的T1和T2数据，但它不能解释15N-1H的NOE数据，并产生太小或负的NOE的计算值，而观测到的NOE是正的。由于引入了两个比旋转相关时间快并且在时间尺度上相差至少1-2个数量级的内部运动[Glore，G.M.，Szabo，A.，Bax，A.，Kay，L.E.，Driscoll，P.C.和GronenBorn，A.M.(1990)J.Am化学。SoC。112,4989-4991]，这32个残基的所有驰豫数据都可以用两个序参数和两个内部运动中较慢的一个有效关联时间来拟合。这两个运动的一个简单模型是，非常快的运动涉及核心内的轴对称扩散，而较慢的运动包括NH矢量的两个不同方向之间的跳跃。对于这样的模型，跳跃角度(不包括C-末端残基)在15度之间。到69度。平均值为28.6。+-。14.0度..另外42个残基在30-ns-10ms的时间尺度上表现出一些短程运动，这导致了具有不同15N化学位移的不同构象亚态之间的化学交换导致15N谱线加宽。一般来说，0.5-4纳秒和30-纳秒-10毫秒时间尺度上的运动都位于表面可到达的环路和转弯中，连接β-链，以及在链的开始和结束处。此外，一些15N线宽被交换加宽的残基直接参与了与结合的内部水分子的主干氢键，或者靠近这些残基。最后，用1H-15N相关谱、1H-15N异核多量子相干-核Overhauser增强谱和1H-1H核Overhauser增强谱表征了位于分子一个邻接面的至少19个残基的主要物种和次要物种之间慢构象平衡的动力学和平衡性质。
The backbone dynamics of uniformly 15N-labeled interleukin-1.beta. are investigated by using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. 15N T1, T2, and NOE data at a spectrometer frequency of 600 MHz are obtained for 90% of the backbone amide groups. The data provide evidence for motions on three time scales. All the residues exhibit very fast motions on a time scale of .ltorsim.20-50 ps that can be characterized by a single-order parameter with an average value of 0.82 .+-. 0.05. For a model comprising free diffusion within a cone, these residue-specific order parameters translate to an average cone semiangle of 20.7 .+-. 3.3.degree.. Thirty-two residues also display motions on a time scale of 0.5-4 ns, slightly less than the overall rotational correlation time of the protein (8.3 ns). These additional motions must be invoked to account for the discrepancy between experiment and the simplest theoretical formulation in which the internal motions are described by only two parameters, a generalized order parameter and an effective correlation time [Lipari, G., and Szabo, A. (1982a). J. Am. Chem. Soc. 104, 4546-4559]. In particular, while the simple formulation can account for the 15N T1 and T2 data, it fails to account for the 15N-1H NOE data and yields calculated values for the NOEs that are either too small or negative, whereas the observed NOEs are positive. With the introduction of two internal motions that are faster than the rotational correlation time and differ in time scales by at least 1-2 order of magnitude [Glore, G. M., Szabo, A., Bax., A., Kay, L. E., Driscoll, P. C., and Gronenborn, A. M. (1990) J. Am. Chem. Soc. 112, 4989-4991], all the relaxation data for these 32 residues can be fitted by two order parameters and an effective correlation time for the slower of the two internal motions. A simple model for these two motions is one in which the very fast motion involves axially symmetric diffusion within a core, while the slower motion comprises jumps between two different orientations of the NH vector. For such a model the jump angle (excluding the C-terminal residue) ranges from 15.degree. to 69.degree. with a mean value of 28.6 .+-. 14.0.degree.. Another 42 residues are characterized by some short of motion on the 30-ns-10-ms time scale, which results in 15N line broadening due to chemical exchange between different conformational substates with distinct 15N chemical shifts. In general, the motions on both the 0.5-4-ns and 30-ns-10-ms time scales are located in surface-accessible loops and turns connecting the .beta.-strands, as well as at the beginning and end of strands. In addition, some of the residues whose 15N line widths are exchange broadened are directly involved in backbone hydrogen bonding with bound internal water molecules or are in close proximity to residues that are. Finally, the kinetic and equilibrium properties of a slow conformational equilibrium between a major and a minor species, involving at least 19 residues and located on one contiguous face of the molecule, are characterized by using 1H-15N correlation spectroscopy, 1H-15N heteronuclear multiple quantum coherence-nuclear Overhauser enhancement spectroscopy, and 1H-1H nuclear Overhauser enhancement spectroscopy.