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Outer membrane protein evolution: Past, present, and future
Dhar, Rik
Dhar, Rik
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Abstract
Outer membrane proteins are found in the peripheral membranes of Gram-negative bacteria, mitochondria, and chloroplast. They are required for essential functions such as small molecule transport, structural support, surface binding, and enzymatic catalysis, as well as forming the basis for pathogenesis. Understanding how these proteins have evolved in nature and how they compare to globular proteins of similar structure will help in engineering these proteins for new applications for research and industry.The scope of this dissertation covers the evolution of outer membrane proteins, beginning with an examination of their evolutionary past and diversification (Chapters 2 and 3). Next, a comparative analysis of all current structurally resolved outer membrane proteins with soluble proteins of similar structure (Chapter 4), is covered. Lastly, our attempts to create engineered outer membrane proteins with new functions and methods developed for the directed evolution of future outer membrane proteins are reported (Chapter 5). The evolutionary landscape of outer membrane proteins created on the basis of several previous studies is presented here (Chapter 2). In protein evolution, diversification is driven by gene duplication. This is evident from the presence of repeating topologies in various proteins including the outer membrane beta-barrels, where the beta-hairpin is the repeating unit. While gene duplication is known to be the main driver of outer membrane protein diversification, a computational study from our group revealed a surprising result where topology changes can also lead to strand accretion in outer membrane beta-barrels. One such topology change found between 16- and 18-stranded beta-barrels is the loop to beta-hairpin transition. The loop-to-hairpin transition is experimentally validated through the study of an evolutionary protein pair selected from the above computational study (Chapter 3). This pair consists of the 16-stranded beta-barrel is PorB from Neisseria gonorrhoeae and the 18-stranded beta-barrel is MOMP from Campylobacter jejuni. A chimeric combination of the two is created by replacing loop L3 of PorB with the sequentially matched protein region of MOMP. The resulting mutant protein is then evaluated for stability and characterized using biochemical and biophysical assays. This chimeric protein is found to be stable and shows evidence for strand accretion, thereby providing experimental evidence for an alternate mode of evolutionary diversification in outer membrane proteins. To understand what features are dependent on their environment rather than the fold, outer membrane beta-barrel proteins are compared to structurally similar soluble beta-barrel proteins using computational tools (Chapter 4). The key differences between them are identified by first creating non-redundant datasets of experimentally resolved protein structures, followed by a comparison of their relative size, shape, amino acid composition, hydrophobicity, and periodicity. We found that membrane beta-barrels are generally larger than soluble beta-barrels and membrane beta-barrels are inside-out soluble beta-barrels in terms of hydrophobicity. These features provide insight into how membrane barrels maintain their fold and function in the membrane environment. The third section focuses on the rational designing of a fluorescent outer membrane protein, based on the structural similarity between GFP and transmembrane domain of type-V autotransporters found in the outer membrane (Chapter 5). Our attempt to impart gain of fluorescence function in these designed proteins is presented here. This includes optimization of methods for directed evolution in outer membrane proteins and addressing several challenges related to this process. One such challenge is the lack of an intrinsic fluorescence selection system. A novel solution to this challenge is proposed, supported by some preliminary data. Using custom-built light-emitting devices and through heterologous expression of a light-dependent proton pump, the host organism's growth is made dependent on fluorescence. These methods described in this section could be used in future studies of the directed evolution of outer membrane proteins using fluorescence. Further, a fluorescent outer membrane protein would also enable the study of the membrane environment using microscopy and other fluorescence-based techniques.
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Date
2022-12-31
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University of Kansas
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Keywords
Biochemistry, Biophysics, Beta barrels, Outer Membrane, Protein design, Protein evolution