PDF(Pubmed)
Abstract：
Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools.
摘要：
掌握跨人类细胞膜的选择性分子运输对医疗保健生物技术提出了巨大的挑战，同时为药物输送提供了突破的前景。基因治疗,和诊断成像。霍乱毒素B亚基（CTB）具有成为这些应用有用的货物转运蛋白的潜力。CTB是一种强大的蛋白质，适合于多种应用的重新设计；然而，蛋白质重新设计主要集中在蛋白质的N和C末端的修饰上。充分利用合理的重新设计需要详细了解表面残基对蛋白质稳定性和结合活性的贡献。这里,我们对CTB的58个表面残基进行了基于Rosetta的计算饱和扫描，包括GM1结合位点，分析配体结合和无配体的结构，以破译对蛋白质稳定性和GM1亲和力的突变影响。差示扫描荧光法和等温滴定量热法的补充实验结果为这些位置之间的40个丙氨酸突变体提供了解链温度和GM1结合亲和力。结果表明，CTB可以适应不同的突变，同时保持其稳定性和配体结合亲和力。这些突变可能允许寡糖结合特异性的修饰，以改变其细胞靶向，改变B亚基的细胞内路由，或通过改变蛋白质稳定性影响其保质期和体内半衰期。我们预计这里呈现的突变空间图将作为未来CTB重新设计的基石，为创新生物技术工具的发展铺平道路。
