Conformation of prion protein repeat peptides probed by FRET measurements and molecular dynamics simulations
Conformation of prion protein repeat peptides probed by FRET measurements and molecular dynamics simulations
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DOI：
10.1016/s0006-3495(04)74303-9
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发表时间：
2004-04-01
影响因子：
3.4
通讯作者：
Gready, JE
中科院分区：
文献类型：
--
作者：
Gustiananda, M;Liggins, JR;Gready, JE
我们报告的稳态荧光共振能量转移（FRET）实验和分子动力学（MD）模拟相结合的使用，调查的朊病毒蛋白（PrP）重复系统的构象分布。FRET首次用于探测7个模型肽中供体Trp残基和受体丹磺酰基之间的距离，作为温度和pH的函数，所述7个模型肽含有1至3个来自有袋负鼠的PrP的第二个十重复序列（PHPGGSNWGQ）（n）G和1个和2个人PrP共有八重复序列（PHGGGWGQ）（n）G。在多重复肽中，通过用Phe替换其他Trp来制备单Trp突变体。由于先前的工作已经表明PrP重复不采用单一的优选稳定构象，FRET值是反映供体-受体距离异质性的平均值。的构象分布的T-依赖性，并推导出平均dansyl-Trp距离，直接从MD模拟的有袋动物dansyl-PHPGGSNWGQG肽。结果表明，FRET和MDT依赖的距离之间的良好协议，并证明显着的灵敏度和重现性的FRET方法在这第一次使用的一组无序肽。基于这些结果，我们提出了一个包含阳离子-π或π-π His-Trp相互作用的模型来解释T-（5- 85 degreesC）和pH-（6.0，7.2）对距离的依赖性，其中HW i，i + 4或WH i，i + 4分离序列比HW i，i + 6或WH i，i + 6分离序列更稳定。该模型具有采用松散折叠构象的肽，其中丹磺酰基-Trp距离远小于完全延伸构象的估计值，例如，对于1-3个十重复，分别类似于16对33，类似于21对69，和类似于22对106埃，并且对于1-2个八重复，分别类似于14对25和类似于19对54埃。这项研究表明，FRET与MD相结合，以前只报道过一次的组合的有用性。通过模拟对柔性肽的构象分布进行初始“映射”可以帮助设计和解释使用稳态强度方法的实验，并指示如何使用时间分辨或各向异性方法。
We report the combined use of steady-state fluorescence resonance energy transfer (FRET) experiments and molecular dynamics (MD) simulations to investigate conformational distributions of the prion protein (PrP) repeat system. FRET was used for the first time to probe the distance, as a function of temperature and pH, between a donor Trp residue and an acceptor dansyl group attached to the N-terminus in seven model peptides containing one to three repeats of the second decarepeat of PrP from marsupial possum (PHPGGSNWGQ)(n)G, and one and two human PrP consensus octarepeats (PHGGGWGQ)(n)G. In multirepeat peptides, single-Trp mutants were made by replacing other Trp(s) with Phe. As previous work has shown PrP repeats do not adopt a single preferred stable conformation, the FRET values are averages reflecting heterogeneity in the donor-acceptor distances. The T-dependence of the conformational distributions, and derived average dansyl-Trp distances, were obtained directly from MD simulation of the marsupial dansyl-PHPGGSNWGQG peptide. The results show excellent agreement between the FRET and MD T-dependent distances, and demonstrate the remarkable sensitivity and reproducibility of the FRET method in this first-time use for a set of disordered peptides. Based on the results, we propose a model involving cation-pi or pi-pi His-Trp interactions to explain the T- (5-85degreesC) and pH- (6.0, 7.2) dependencies on distance, with HW i, i + 4 or WH i, i + 4 separations in sequence being more stable than HW i, i + 6 or WH i, i + 6 separations. The model has peptides adopting loosely folded conformations, with dansyl-Trp distances very much less than estimates for fully extended conformations, for example, similar to16 vs. 33, similar to21 vs. 69, and similar to22 vs. 106 Angstrom for 1-3 decarepeats, and similar to14 vs. 25 and similar to19 vs. 54 Angstrom for 1-2 octarepeats, respectively. The study demonstrates the usefulness of combining FRET with MD, a combination reported only once previously. Initial "mapping" of the conformational distribution of flexible peptides by simulation can assist in designing and interpreting experiments using steady-state intensity methods, and indicating how time-resolved or anisotropy methods might be used.