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 (PHPGGSN>WGQ)nG, and one and two human PrP consensus octarepeats (PHGGG>WGQ)nG. 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-PHPGGSN>WGQG 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-π or π-π His-Trp interactions to explain the T- (5–85°C) 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, ∼16 vs. 33, ∼21 vs. 69, and ∼22 vs. 106 Å for 1–3 decarepeats, and ∼14 vs. 25 and ∼19 vs. 54 Å 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.
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机译:我们报告结合使用稳态荧光共振能量转移(FRET)实验和分子动力学(MD)模拟来研究the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残基和连接至N末端的受体丹磺酰基之间的距离,该距离是温度和pH的函数,该模型肽包含1-3个重复序列的第二个脱十足动物。有袋负鼠(PHPGGSN > W strong> GQ)nG的PrP,以及一和两个人类PrP共有八角豆(PHGGG > W strong> GQ)nG的PrP。在多重复肽中,通过用Phe替代其他Trp来制备单Trp突变体。如先前的工作所示,PrP重复序列没有采用单个优选的稳定构象,因此FRET值是反映供体-受体距离异质性的平均值。直接从有袋动物dansyl-PHPGGSN > W strong> GQG肽段的MD模拟中获得构象分布的T依赖性以及推导的平均dansyl-Trp距离。结果表明,FRET和MD T依赖性距离之间具有极好的一致性,并证明了FRET方法在首次用于一系列无序肽组中的显着灵敏度和可重复性。根据结果,我们提出了一个包含阳离子-π或π-πHis-Trp相互作用的模型,以解释T-(5-85°C)和pH-(6.0,7.2)对距离的依赖性,HW i,i + 4或WH i,i + 4序列比HW i,i + 6或WH i,i + 6序列更稳定。该模型的肽采用松散折叠的构象,其dansyl-Trp距离远小于完全扩展构象的估计值,例如,对于1-3,约16对33,约21对69和22对106Å。脱皮豌豆,1-2个八角豆的分别为〜14 vs. 25和〜19 vs. 54Å。这项研究证明了将FRET与MD结合的有用性,该结合以前仅报道过一次。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,并指出如何使用时间分辨或各向异性方法。
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