Investigation of the kinetically stable sub-proteomes of Vibrio cholerae and Vibrio harveyi

Abstract

Certain proteins possess high energy barriers that prevent them from unfolding and are therefore categorized as kinetically stable. Kinetically stable proteins (KSPs) remain trapped in a folded form that imparts resistance to detergents, denaturants and proteolysis. High-throughput methods have been designed with detergent resistance in mind, differentiating between KSPs and non-kinetically stable proteins based on resistance to the detergent sodium dodecyl sulfate (SDS). With application of the diagonal two-dimensional SDS-PAGE method (D2D), only 50 proteins from E. coli have been identified and characterized so far, making it difficult to elucidate the structural basis, conservation and biological roles of kinetic stability. By studying other biological systems, the database of KSPs can be expanded for more complex studies. The genus Vibrio is closely related to E. coli¸ but occupies a slightly different biological niche. V. cholerae and V. harveyi were selected as representative species of Vibrio for the proteomics analysis of its KSPs using D2D SDS-PAGE and mass spectrometry. V. cholerae is a well-documented human pathogen with a preference for brackish environments. V. harveyi is a representative of the marine species of Vibrio and is noted as a significant pathogen in the aquaculture industry. The goal of this study was to gain new knowledge and insight about the roll biological roles of kinetic stability in halophiles and pathogens, the conservation of kinetic stability between species, and the structural basis of this biophysical property of proteins.

The structural properties of the KSPs in Vibrio were similar to those of E. coli, suggesting a bias of KSPs towards topological complexity and oligomeric structures. Functional analysis revealed several outer membrane proteins (OMPs), oxidoreductases, metabolic enzymes and essential proteins involved in translation and transcription, suggesting biological roles for kinetic stability in cellular metabolism, essential processes and protection from environmental stress. Additionally, three KSPs – OmpU, OmpT and long-chain-fatty-acid-CoA ligase – were identified and traced back to the virulence pathways of V. cholerae, thereby also implicating kinetic stability in Vibrio bacteria virulence. This work strongly supports a link between topological complexity and oligomeric structure with kinetic stability, and advances the long-term goal of predicting kinetic stability from a protein’s three-dimensional structure. In addition, certain biological functions or pathways in bacteria seem to require kinetic stability, in particular those linked to stress response and virulence. Finally, the high overlap of KSPs in V. cholerae and E. coli suggest the abundance and type of KSPs present in bacteria may correlate with their ability to adapt and survive in harsher environments.

Application of D2D SDS-PAGE revealed more SDS-resistant (i.e. kinetically stable) proteins in V. cholerae than in V. harveyi. To identify these proteins, the spots below the gel diagonal were excised and tryptic in-gel digestion generated peptide fragments that were analyzed by mass spectrometry. Subsequent Mascot analysis led to the identification of 16 KSPs in V. cholerae and seven in V. harveyi. Interestingly, 10/16 (63%) and 3/7 (43%) of the KSPs in V. cholerae and V. harveyi overlap with those in E. coli, respectively. In contrast, only one KSP is shared between the two Vibrio bacteria. The functional and structural properties of all the KSPs identified were compared between the Vibrios, and also to the 50 KSPs previously identified in E. coli. Structural analysis of the closest homologous structures of the KSPs of Vibrio revealed that a large majority, 70%, of the KSPs from Vibrio possessed mixed α-helical and β-sheet secondary structures and all were part of multimeric quaternary structures.