Cobbert Jacqueline D, DeMott Christopher, Majumder Subhabrata, Smith Eric A, Reverdatto Sergey, Burz David S, McDonough Kathleen A, Shekhtman Alexander
Department of Chemistry, University at Albany, Albany, NY.
Wadsworth Center, NY State Department of Health, Albany, NY.
Sci Rep. 2015 Mar 24;5:9402. doi: 10.1038/srep09402.
Intrinsically disordered proteins (IDPs) or unstructured segments within proteins play an important role in cellular physiology and pathology. Low cellular concentration, multiple binding partners, frequent post-translational modifications and the presence of multiple conformations make it difficult to characterize IDP interactions in intact cells. We used peptide aptamers selected by using the yeast-two-hybrid scheme and in-cell NMR to identify high affinity binders to transiently structured IDP and unstructured segments at atomic resolution. Since both the selection and characterization of peptide aptamers take place inside the cell, only physiologically relevant conformations of IDPs are targeted. The method is validated by using peptide aptamers selected against the prokaryotic ubiquitin-like protein, Pup, of the mycobacterium proteasome. The selected aptamers bind to distinct sites on Pup and have vastly different effects on rescuing mycobacterial proteasome substrate and on the survival of the Bacille-Calmette-Guèrin, BCG, strain of M. bovis. This technology can be applied to study the elusive action of IDPs under near physiological conditions.
内在无序蛋白质（IDP）或蛋白质内的无结构片段在细胞生理和病理过程中发挥着重要作用。细胞内浓度低、结合伴侣多样、频繁的翻译后修饰以及多种构象的存在使得在完整细胞中表征IDP相互作用变得困难。我们利用通过酵母双杂交系统筛选的肽适配体和细胞内核磁共振技术，以原子分辨率鉴定与瞬时结构化IDP和无结构片段具有高亲和力的结合物。由于肽适配体的筛选和表征均在细胞内进行，因此仅针对IDP的生理相关构象。通过使用针对分枝杆菌蛋白酶体的原核泛素样蛋白Pup筛选的肽适配体对该方法进行了验证。所选的适配体与Pup上的不同位点结合，对拯救分枝杆菌蛋白酶体底物以及对牛分枝杆菌卡介苗（BCG）菌株的存活具有截然不同的影响。该技术可应用于研究在接近生理条件下IDP难以捉摸的作用。
