Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

27-POS Board 27 EXTENDING CENTRAL DOGMA ONE STEP FURTHER: INSERTION OF MEMBRANE PROTEINS Alexey Ladokhin 1 ; Victor Vasquez Montes 1 ; Mykola Rodnin 1 ; Alexander Kyrychenko 1,2 ; Mauricio Vargas Uribe 1,3 ; 1 KUMC, Biochemistry and Molecular Biology, Kansas City, KS, USA 2 Karazin Kharkiv National University, Institute of Chemistry , Kharkiv, Ukraine 3 Universidad Austral de Chile, Biochemistry and Microbiology, Valdivia, Chile The conversion of a protein structure from a water-soluble to membrane-inserted form is one of the least understood cellular processes. Examples include the cellular action of various bacterial toxins and colicins, tail-anchor proteins and multiple proteins of the Bcl-2 family, bearing pro- apoptotic and anti-apoptotic functions. In our lab we study anti-apoptotic regulator Bcl-xL and diphtheria toxin translocation (T) domain. The latter undergoes conformational change in response to endosomal acidification, inserts into the lipid bilayer and translocates its own N- terminus and the attached catalytic domain of the toxin across the membrane. Our goal is to describe at the molecular level the mechanisms of protonation-dependent conformational switching of the T domain, which serves as a model for membrane insertion/translocation transitions of structurally related proteins (e.g., Bcl-xL, Bax and other apoptotic regulators). Here we present our progress toward this objective, including structural, kinetic and thermodynamic characterization of the insertion pathway of the T domain using both experimental (e.g., Fluorescence Correlation Spectroscopy, FRET, Depth-Dependent Fluorescence Lifetime Quenching, NMR, X-ray crystallography, HD exchange Mass Spectrometry) and computational approaches (e.g., All-Atom Molecular Dynamics Simulation). Supported by NIH GM126778.

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