Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Poster Abstracts

38-POS Board 10 USING ANCESTRAL SEQUENCE RECONSTRUCTION TO UNDERSTAND MEMBRANE TRANSPORTER STRUCTURE, FUNCTION, AND MECHANISM Krishna Reddy 1 ; Olga Boudker 1 ; 1 Weill Cornell Medical College, New York, NY, USA Through evolution, membrane transporters adapt their functional properties to environmental changes and physiological needs. Phylogenetic analyses can reconstruct the evolutionary history of a protein family, inferring ancestral protein sequences before and after the emergence of novel functionality. Biochemical characterization of these sequences can reveal subtle changes in protein properties required for functional diversification that are not evident in traditional sequence comparisons of extant proteins. Ancestral sequence reconstruction (ASR) is a powerful approach to understanding evolution, but its applications to membrane proteins remain limited. We used ASR to uncover the mechanism of functional diversification in glutamate transporters, which have subfamilies coupling substrate import to different ion gradients. Specifically, we recapitulated an evolutionary transition from Na+- to H+-coupling using phylogenetics to reveal the architectural differences between transporters with divergent ion-coupling mechanisms. We then purified an ancestral Na+-coupled transporter to homogeneity, enabling functional characterization in reconstituted proteoliposomes and structural analysis by cryo-EM. The ancestral transporter displays unique structural and functional properties not observed in extant transporters thus far, providing a potential mechanistic basis for functional diversification. Our results provide a framework in which evolution-guided analyses uncover novel structure- function relationships in membrane proteins.

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