Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA; Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Methods. 2024 May;225:1-12. doi: 10.1016/j.ymeth.2024.02.007. Epub 2024 Feb 29.
Elucidating the folding energy landscape of membrane proteins is essential to the understanding of the proteins' stabilizing forces, folding mechanisms, biogenesis, and quality control. This is not a trivial task because the reversible control of folding is inherently difficult in a lipid bilayer environment. Recently, novel methods have been developed, each of which has a unique strength in investigating specific aspects of membrane protein folding. Among such methods, steric trapping is a versatile strategy allowing a reversible control of membrane protein folding with minimal perturbation of native protein-water and protein-lipid interactions. In a nutshell, steric trapping exploits the coupling of spontaneous denaturation of a doubly biotinylated protein to the simultaneous binding of bulky monovalent streptavidin molecules. This strategy has been evolved to investigate key elements of membrane protein folding such as thermodynamic stability, spontaneous denaturation rates, conformational features of the denatured states, and cooperativity of stabilizing interactions. In this review, we describe the critical methodological advancement, limitation, and outlook of the steric trapping strategy.
阐明膜蛋白的折叠能量景观对于理解蛋白质的稳定力、折叠机制、生物发生和质量控制至关重要。这不是一项简单的任务，因为在脂质双层环境中，折叠的可逆控制本质上是困难的。最近，已经开发出了新的方法，每种方法在研究膜蛋白折叠的特定方面都具有独特的优势。在这些方法中，空间位阻捕获是一种通用的策略，允许对膜蛋白折叠进行可逆控制，同时最小化对天然蛋白-水和蛋白-脂相互作用的干扰。简而言之，空间位阻捕获利用了双生物素化蛋白自发变性与大单价链霉亲和素分子同时结合的偶联。该策略已被用于研究膜蛋白折叠的关键要素，如热力学稳定性、自发变性速率、变性状态的构象特征以及稳定相互作用的协同性。在这篇综述中，我们描述了空间位阻捕获策略的关键方法学进展、局限性和展望。
