Betz S F, Raleigh D P, DeGrado W F, Lovejoy B, Anderson D, Ogihara N, Eisenberg D
Chemical and Physical Sciences Department, DuPont Merck Pharmaceutical Company, Wilmington DE 19880-0328, USA.
Fold Des. 1996;1(1):57-64. doi: 10.1016/S1359-0278(96)00012-0.
The design of amino acid sequences that adopt a desired three-dimensional fold has been of keen interest over the past decade. However, the design of proteins that adopt unique conformations is still a considerable problem. Until very recently, all of the designed proteins that have been extensively characterized possess the hallmarks of the molten globular state. Molten globular intermediates have been observed in both equilibrium and kinetic protein folding/stability studies, and understanding the forces that determine compact non-native states is critical for a comprehensive understanding of proteins. This paper describes the solution and early solid state characterization of peptides that form molten globular ensembles.
RESULTS & CONCLUSIONS: Crystals diffracting to 3.5 A resolution have been grown of a 16-residue peptide (alpha 1A) designed to form a tetramer of alpha-helices. In addition, a closely related peptide, alpha 1, has previously been shown to yield crystals that diffract to 1.2 A resolution. The solution properties of these two peptides were examined to determine whether their well defined crystalline conformations were retained in solution. On the basis of an examination of their NMR spectra, sedimentation equilibria, thermal unfolding, and ANS binding, it is concluded that the peptides form alpha-helical aggregates with properties similar to those of the molten globule state. Thus, for these peptides, the process of crystallization bears many similarities to models of protein folding. Upon dissolution, the peptides rapidly assume compact molten globular states similar to the molten globule like intermediates that are formed at short times after refolding is initiated. Following a rate-determining nucleation step, the peptides crystallize into a single or a small number of conformations in a process that mimics the formation of native structure in proteins.
在过去十年中，设计能呈现所需三维折叠结构的氨基酸序列备受关注。然而，设计具有独特构象的蛋白质仍然是一个相当大的问题。直到最近，所有经过广泛表征的设计蛋白质都具有熔球态的特征。在蛋白质折叠/稳定性的平衡和动力学研究中均观察到了熔球态中间体，了解决定紧密非天然状态的作用力对于全面理解蛋白质至关重要。本文描述了形成熔球态聚集体的肽段的溶液状态及早期固态表征。
已培养出衍射分辨率达3.5埃的16残基肽（α1A）晶体，该肽段设计用于形成α-螺旋四聚体。此外，此前已证明一种密切相关的肽α1能产生衍射分辨率达1.2埃的晶体。对这两种肽段的溶液性质进行了研究，以确定它们在溶液中是否保留了明确的晶体构象。基于对其核磁共振谱、沉降平衡、热解折叠和ANS结合的研究，得出结论：这些肽段形成了具有与熔球态相似性质的α-螺旋聚集体。因此，对于这些肽段，结晶过程与蛋白质折叠模型有许多相似之处。溶解后，这些肽段迅速形成紧密的熔球态，类似于在重折叠开始后短时间内形成的类似熔球态的中间体。在一个决定速率的成核步骤之后，这些肽段结晶形成单一或少数几种构象，这一过程类似于蛋白质中天然结构的形成。
