Abstract:

Most natural proteins tend to be marginally stable, which allows them to gain flexibility for biological functions. However, marginal stability is often associated with protein misfolding and aggregation under stress conditions, presenting a challenge for protein research and applications such as proteins as biocatalysts and therapeutic agents. In addition, protein instability has been increasingly recognized as one of the major factors causing human diseases. For example, the formation of toxic protein aggregates is the hallmark of many neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Therefore, optimizing protein folding and maintaining protein homeostasis in cells are long-standing goals for the scientific community. Confronting these challenges, various methods have been developed to stabilize proteins. In this review, we classify and summarize various techniques for engineering protein stability, with a focus on strategies for optimizing protein sequences or cellular folding environments. We first outline the principles of protein folding, and describe factors that affect protein stability. Then, we describe two main approaches for protein stability engineering, namely, computational design and directed evolution. Computational design can be further classified into structure-based, phylogeny-based, folding energy calculation-based and artificial intelligence-assisted methods. We present the principles of several methods under each category, and also introduce easily accessible web-based tools. For directed evolution approaches, we focus on library-based, high-throughput screening or selection techniques, including cellular or cell-free display and stability biosensors, which link protein stability to easily detectable phenotypes. We not only introduce the applications of these techniques in protein sequence optimization, but also highlight their roles in identifying novel folding factors, including molecular chaperones, chemical chaperones, and inhibitors of protein aggregation. Moreover, we demonstrate the applications of protein stability engineering in biomedicine and pharmacotherapeutics, including identifying small molecules to stabilize disease-related, aggregation-prone proteins, obtaining conformation-fixed and stable antigens for vaccine development, and targeting protein stability as a means to control protein homeostasis. Finally, we look forward to the trends and prospects of protein stabilization technologies, and believe that protein stability engineering will lead to a better understanding of protein folding processes to facilitate the development of precision medicine. {L-End}

Key words: protein folding, protein stability, protein stability engineering, rational design, computational design, directed evolution

摘要：

天然蛋白质具有临界稳定性的特征，这种较低的稳定性使蛋白质结构具有足够的灵活性，从而支持其发挥生物学功能。然而，临界稳定性使得蛋白质遭受胁迫压力后极易发生错误折叠并失去功能，导致天然蛋白质往往无法满足科学研究与工业应用的需求。此外，体内蛋白质在错误折叠后产生的聚集沉淀被认为是多种疾病发生发展的原因，包括阿尔兹海默病、帕金森综合征等。因此，优化蛋白质的稳定性是科学研究与工程应用领域亟待解决的关键问题。本文从蛋白质的折叠与稳定性机制出发，聚焦于序列优化与折叠环境优化两种改善蛋白质稳定性的手段，综述了基于理性设计、计算机辅助设计改善蛋白质稳定性的研究方法，介绍了用于高通量筛选蛋白质稳定化突变体或折叠相关因子的定向进化技术。通过多项蛋白质序列改良、折叠环境优化的案例介绍，展示了蛋白质稳定化技术在蛋白质工程与生物医药领域的广阔应用，包括酶的稳定化设计、疫苗蛋白质的构象控制、分子伴侣与蛋白质聚集抑制剂的筛选、蛋白质稳态药物的开发等。最后，展望了蛋白质稳定化技术未来的研究方向与前景，定制化的蛋白质稳定性检测技术将会迎来蓬勃发展。

关键词: 蛋白质折叠, 蛋白质稳定性, 蛋白质稳定化工程, 理性设计, 计算机辅助设计, 定向进化