Stabilization of Multimeric Proteins via Intersubunit Cyclization

Abstract

Proteins with high catalytic efficiency and selectivity under mild conditions have long been appreciated by industrial and medicinal fields. These proteins, which are commonly multimeric, often possess low stability, impeding wider application. Currently, strategies to improve the stability of multimeric proteins concentrate on enhancing the interaction at internal interface of the subunits. In this report, we confirmed that the largely underestimated subunit terminal ends are as significant as the internal interface for protein stability. By connecting both the terminal ends and internal interface of subunits, the tetrameric alcohol dehydrogenase (ADH) protein can been cyclized into a rigid form with significantly improved thermostability and resilience. The improvement in the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment () and melting temperature ( ) of the modified protein was 18°C and 23.3°C, respectively, which is superior to the results achieved by normal protein engineering. Our study provided a novel strategy to effectively improve the stability of multimeric proteins, which is suitable not only for the short-chain dehydrogenase/reductase (SDR) family but also other classes of proteins with close terminal ends. Industrially interesting proteins are generally multimeric proteins; however, their applications are often restricted due to low stability caused by the natural tendency of subunit disassociation. Current approaches targeting this problem mainly focus on enhancing the internal interfaces of the subunits to avoid their disassociation. In this study, we identified and confirmed the external interface to be significant for improving the stability of multimeric proteins. By connecting the terminal ends and internal interface with disulfide bonds, we found that the multimeric protein ADH cyclized into a robust monomeric-like form, resulting in superior thermostability compared to traditional protein engineering. This intersubunit cyclization approach is efficient and easy to perform, providing a novel method for engineering many important classes of multimeric proteins.

Quantitative proteomic analysis reveals that serine/threonine kinase is involved in Streptococcus suis virulence and adaption to stress conditions

“…In S. equi subsp . zooepidemicus ATCC35246, 5'-Nucleotidase (5NuC) was found to directly degrade the NET DNA backbone to deoxyadenosine, negatively influencing macrophage phagocytic activity, while the mutant strain Δ5nuc exhibited lower virulence and a weaker ability to spread from blood to organs than the WT strain (Ma et al 2017 ).Similar findings were reported in S. pyogenes for 5'-nucleotidase A (S5nA)(Zheng et al 2015 ). Therefore, our results are in agreement those of previous studies showing SsSTK mutant strains displayed reduced ability to adhere to epithelial cells, increased immune evasion and increased sensitivity to phagocytosis (Zhu et al 2014 ; Zhang et al 2017 ).…”

“…Additionally, the putative IgA-specific zinc metalloproteinase (ZmpC or IgA1) (Zhang et al 2011 ; Dumesnil et al 2018 ), translation initiation factor 2 (HP0272 or SadP) (Ferrando et al 2017 ), chaperone protein DnaJ (Zhang et al 2015 ), and sensor histidine kinase TCS VarS (Zheng et al 2018a ; Zhong et al 2018 ) were significantly up-regulated in the mutant strain. Another 43 VFs that have been reported in other pathogens were identified as DEPs in the ∆stk , including putative 5'-nucleotidase (5NuC) (Zheng et al 2015 ; Ma et al 2017 ), trypsin-like serine protease (HtrA) (Backert et al 2018 ) and metalloendopeptidas (PepO) (Agarwal et al 2013 , 2014 ). These VFs were mainly assigned into eight classes: (i) amino acid transport and metabolism (12 proteins); (ii) posttranslational modification, protein turnover, chaperones (10 proteins); (iii) nucleotide transport and metabolism (9 proteins); (iv) transcription (8 proteins); (v) inorganic ion transport and metabolism (6 proteins); (vi) cell wall/membrane/envelope biogenesis (6 proteins); (vii) general function prediction (3 proteins); and (viii) other proteins of unknown function (7 proteins) (Tables 1 and 2 ).…”

Characterization of Sorbitol Dehydrogenase SmoS fromSinorhizobium meliloti1021

“…2). Tetrameric configurations are reported most often and likely represent the majority of SDR protein structures in solution [43].…”

Engineering Leifsonia Alcohol Dehydrogenase for Thermostability and Catalytic Efficiency by Enhancing Subunit Interactions

“…[15] Like many other multimeric proteins, [16] ADHs have a natural tendency to dissociate their subunits, leading to rapid inactivation. [17] Thus, prevention of their disassociation is the priority for stabilizing these interesting enzymes. In our previous study, we identified a highly capable ADH (LnADH) from Leifsonia.…”

“…In our previous study, we identified a highly capable ADH (LnADH) from Leifsonia. [17] LnADH is able to use NADH as a cofactor rather than the more expensive NADPH. Moreover, it is able to use the solvent isopropanol to recycle the generation of NADH, thus significantly reducing the cost and improving the solubility of substrates.…”