Fluorescence and 13C NMR determination of side-chain and backbone dynamics of synthetic melittin and melittin analogues in isotropic solvents.
Fluorescence and 13C NMR determination of side-chain and backbone dynamics of synthetic melittin and melittin analogues in isotropic solvents.
复制标题
荧光和 13C NMR 测定各向同性溶剂中合成蜂毒肽和蜂毒肽类似物的侧链和主链动力学。
DOI：
10.1021/bi00447a053
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Prendergast,FG
中科院分区：
文献类型：
--
作者：
Weaver,AJ;Kemple,MD;Prendergast,FG
Szabo，A.(1982)J.化学。SoC。104,4546-4570]，稳态荧光各向异性和寿命数据的可用性增加了ThT2和NOE数据，以提供关于这些多肽中荧光团动力学的定量信息。提出了一种结合使用荧光和核磁共振数据来获得描述多肽和蛋白质中13C标记荧光团局部运动的参数的技术和模型无关的值的方法。蜂毒素和蜂毒素类似物的动力学被发现与从前一篇论文中提供的CD、荧光和核磁共振光谱信息推断的结构特征一致(Weaver等人，1989)。特别是，随机卷曲的多肽单体的迁移率非常相似，而侧链以及聚集态或寡聚体中的多肽主链运动在不同的类似物中明显不同。对于蜂毒素本身，实验确定的单体和四聚体的总旋转相关时间与基于溶剂可及蛋白质表面积的预测值非常一致。选择性13C标记的蜂毒素Trp-19和Gly-12残基的局部动力学也与多肽结构一致。在无规卷曲的蜂毒素单体中，一个特定的运动模型表明色氨酸侧链在0-y键附近以大约±71的角度移动，关联时间为159±24 ps。在蜂毒素四聚体中，吲哚部分在空间上更加受限，翻转角为±37，但运动速度增加，关联时间为56±8ps。Trp-19侧链的限制迁移率与从蜂毒素四聚体的X射线结构推断的运动限制是一致的。这些结果表明，蛋白质侧链的运动，甚至像吲哚一样大的部分，都可以在皮秒时间尺度上发生，并且这些运动与分子动力学模拟得出的结果合理地相似。生物分子动力学的分子动力学模拟的出现[参见Karplus和McCammon(1981)和Levy(1986)]已经产生了对大分子中皮秒运动的实验检测和定量的需要。这一认识，考虑到这种运动对蛋白质结构和功能的可能意义，为发展能够证明多肽和蛋白质中局部运动的存在以及量化局部运动的幅度和时间尺度的实验方法提供了充分的理由。最近的出版物证明了荧光(Petrich等人，1987；MacKerell等人，1987)和核磁共振(Weiner等人，1987；莱斯等人，1987；Rule等人，1987；Brown等人，1988)光谱分析在研究蛋白质内部运动方面的有效性。应用这些方法所固有的一个限制是，当只使用一种技术时，对实验数据的准确性存在不确定性。然而，原则上，核磁共振和荧光可以结合使用来研究大分子中荧光基团的动力学。
Szabo, A.(1982) J. Am. Chem. Soc. 104, 4546-4570], the availability of steady-state fluorescence anisotropy and lifetime data augment ThT2, and NOE data to provide quantitative information about fluorophore dynamics in these peptides. A method is presented for using combined fluorescence and NMR data to obtain technique-and model-independent values for parameters describing local motion of 13C-labeled fluorophores in peptides and proteins. The dynamics of melittin and melittin analogues are found to be consistent with structural characteristics inferred from CD, fluorescence, and NMR spectral information presented in the preceding paper (Weaver et al., 1989). Inparticular, the mobility of the random coil peptide monomers is shown to be quite similar, while side-chain as well as peptide backbone motion in the aggregated or oligomeric species differs markedly among the analogues. For melittin itself, experimentally determined overall rotational correlation times for the monomer and tetramer agree verywell with values predicted on the basis of solvent-accessible protein surface area. The local dynamics of selectively 13C-labeled Trp-19 and Gly-12 residues of melittin are also found to be consistent with peptide structure. In random coil melittin monomer, a specific model for the motion indicates that the Trp side chain moves through an approximate angle of±71 about the 0-y bond with a correlation time of 159±24 ps. In melittin tetramer, the indole moiety is spatially more confined with a flip angle of±37, yet demonstrates an increased rate of motion with a correlation time of 56±8 ps. The constrained mobility of the Trp-19 side chain is consistent with motional constraints inferred from the X-ray structure of melittin tetramer. These results show that protein side-chain motion, even of moieties as large as indole, can occur on the picosecond time scale and that these motions are reasonably similar to those inferredfrom molecular dynamics simulations. e advent of molecular dynamics simulations of biomolecule dynamics [for reviews, see Karplus and McCammon (1981) and Levy (1986)] has generated a need for experimental de-tection and quantitationof picosecond motions in macro-molecules. This realization, taken with the likely significance of such motions to protein structureand function, provides ample justification for the development of experimental methods able to demonstrate the existence of, and to quantify the amplitude and time scale of, local motions in peptides and proteins. Recent publications demonstrate the usefulness of fluorescence (Petrich et al., 1987; MacKerell et al., 1987) and NMR (Weiner et al., 1987; Rice et al., 1987; Rule et al., 1987; Brown et al., 1988) spectroscopies for the study of internal motions in proteins. One limitation inherent in the application of these methods is the uncertainty regarding the accuracy of the experimental data when only one technique is employed. In principle, however, NMR and fluorescence can be used conjointly to study the dynamics of a fluorescent group in a macromolecule.