Applicability of Instability Index for In vitro Protein Stability Prediction

作者：Dilani G. Gamage, Ajith Gunaratne, Gopal R. Periyannan, Timothy G. Russell · 发表于：Protein and Peptide Letters · 年份：2019 · DOI：10.2174/0929866526666190228144219 · 被引用次数：242 · 研究领域：Protein purification and stability、Protein Structure and Dynamics、Advanced Proteomics Techniques and Applications

BACKGROUND: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. OBJECTIVE: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. METHODS: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. RESULTS: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in v...