Biophysical Society Thematic Meeting | Stockholm 2022

Physical and Quantitative Approaches to Overcome Antibiotic Resistance

Poster Abstracts

28-POS Board 28 SUBSTRATE TRANSLOCATION ACROSS BACTERIAL SUGAR TRANSPORTER: FROM SINGLE-MOLECULE DETECTION TO BIOLOGICAL RELEVANCE Devika Vikraman 1,2 ; Smrithi R Krishnan 1,2 ; Kozhinjampara R Mahendran 1 ; 1 Rajiv Gandhi Centre for Biotechnology, Transdisciplinary Research Program, Thiruvananthapuram, India 2 Manipal Academy of Higher Education, Manipal, India The passage of biomolecules across the bacterial cell is facilitated by porins that form hydrophilic channels in the outer membrane. Substrate-specific porins are evolved naturally to uptake specialized essential molecules and act as a regulated gateway. We focus on a sugar specific porin called CymA present in the outer membrane of bacteria Klebsiella oxytoca. The crystallographic structure revealed a peculiar geometry comprising a monomeric beta-barrel with the presence of an N terminus segment inside the barrel lumen, constricting the pore diameter. We quantified the translocation kinetics of cyclic sugars of different charge, size, and symmetry across native and truncated CymA, a counterpart, devoid of the N terminus segment, using single-channel recordings. The chemically divergent cyclic hexasaccharides bind to the native and truncated CymA with different interaction affinities with translocation more rapidly through truncated pore. In contrast, larger cyclic octasaccharides completely bind to native and truncated CymA, interestingly, with distinct binding kinetics in agreement with liposome assays. This highlights the importance of symmetry match with pore axis as hexasaccharides fits in to the pore to translocate efficiently whereas heptasaccharides and octasaccharides enters the pore and completely occludes the lumen. This can shed light to understand the basic mechanism behind efficient molecular uptake across bacterial membrane transporters. For example, the occlusion of pore lumen by octasaccharides can be developed as a blocker against the multi-drug resistant pathogens. Finally, we introduced native CymA as a large nanopore sensor for simultaneously sensing a variety of biomolecules such as polypeptides, highlighting the structural and functional versatility of membrane porins. Ref: Vikraman, D.; Satheesan, R.; Kumar, K. S.; Mahendran, K. R. Nanopore Passport Control for Substrate-Specific Translocation. ACS Nano 2020, 14, 2285 2295.

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