Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Wednesday Speaker Abstracts

ELUCIDATING THE SUBSTRATE SPECIFICITIES OF THE TWO MAJOR FUNCTIONAL SUBTYPES IN THE SMALL MULTIDRUG RESISTANCE (SMR) FAMILY Olive E. Burata 1,2 ; Randy B Stockbridge 1,2,3 ; 1 University of Michigan, Program in Chemical Biology, Ann Arbor, MI, USA 2 University of Michigan, Molecular, Cellular, and Developmental Biology, Ann Arbor, MI, USA 3 University of Michigan, Biophyics, Ann Arbor, MI, USA The small multidrug resistance (SMR) family contains the smallest membrane transport proteins found in both bacteria and archaea. The family is functionally dominated by two major subtypes: the quaternary ammonium compound subtype (Qac) which transports a chemically diverse range of biocides, and the guanidinium exporter subtype (Gdx) which selectively transports the essential bacterial metabolite, guanidinium. Here, we 1) determined an x-ray crystal structure of a representative Qac protein, EmrE and 2) using high-throughput mutagenesis, directed evolution, electrophysiology, and cell resistance assays, identified key molecular determinants of the specificity differences between the Gdx and Qac subtypes. DESIGNED PROTON CHANNELS REVEAL MECHANISMS FOR PROTON CHANNEL SELECTIVITY AND CONDUCTIVITY Huong T. Kratochvil ; William F DeGrado 1 ; 1 University of California-San Francisco, Pharmaceutical Chemistry, San Francisco, CA, USA Selective and fine-tuned proton conductance is critical for many biocatalytic and bioenergetic processes. Proton channel proteins precisely control proton transport through proton conduction pathways comprised of hydrogen-bonding networks of nanoconfined water, polar sidechains and backbone carbonyls that are interspersed in well-packed hydrophobic segments that would appear to be barriers to conduction. We hypothesize that these apolar constrictions can transiently expand to accommodate hydrogen-bonding chains of water molecules, and it is the fleeting nature of these water wires that ultimately facilitate highly selective conduction of protons over other ions. To critically test our hypothesis, we turn to protein design. Starting from a minimalist membrane-spanning pentameric bundle with a dry pore, we systematically introduce polar residues, such as Gln, to key pore-lining positions to modulate the lengths of these apolar tracts. From X-ray crystallographic structures of these designs, these polar residues appear to facilitate the formation of water wires within the pore. Furthermore, liposomal proton flux assays reveal that these designs are indeed conductive for protons with selectivity of > 10 6 over K + and Na + . Molecular dynamics simulations reveal water permeation with the introduction of the Gln mutations, further corroborating our hypothesis. Through this combination of design, experiment, and simulation, we are able to dissect the physical principles that underlie the formation of these hydrogen bonding networks and their roles in proton selectivity and conduction.

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