With the increasing number of drug resistant bacterial strains, it is essential to develop new antibiotics to efficiently fight against such pathogenic strains. Many of these pathogenic bacteria are Gram-negative and contain two membranes (inner and outer), which encapsulate the cellular content and provide protection against harsh environmental conditions and toxic chemicals. Even though the outer membrane (OM) facilitates the uptake of small molecules, the intake of lager molecules, such as essential nutrients including rare metal ions and antibiotics (&gt;600 Da), are mediated by the OM transporters. Bacteriocins are antibiotics produced by some bacteria under stress conditions, targeting similar bacterial species to eliminate the competition for nutrients, including rare minerals essential for survival. Colicin is one class of bacteriocins produced by Escherichia coli (E. coli) against related E. coli strains. This class of bacteriocins is the most widely studied using molecular biological, biochemical and biophysical techniques, and several structural models have been generated using crystallography for colicin bound to their OM receptors and transporters. However, our current knowledge regarding the mechanisms and associated conformational changes that take place during the binding of colicin to the OM receptor and its uptake into the cell is limited. In our present work, we have combined site directed spin labeling with electron paramagnetic resonance (EPR) spectroscopy to investigate conformational changes associated with receptor binding step using colicin E3 receptor binding domain (colicin E3R) with its OM receptor, BtuB. Briefly, we have mutated selected positions of colicin E3R into cysteine and expressed, purified and spin labeled these cysteine mutants. Wild type (WT) BtuB, either in live E. coli cells or in reconstituted bilayer systems, were used to investigate colicin E3R mutants’ binding with BtuB and conformational changes of colicin E3R mutants related to OM receptor binding events. In the future, we are aiming to investigate conformational changes of receptor binding step using spin labeled BtuB at specific extracellular loops, along with spin labeled colicins, to detect distance distributions associated with receptor binding and how receptor bound colicin dynamic changes as to screen for its OM transporter.

Exploring conformational changes associated with antimicrobial agent, colicin E3 during receptor binding on targeted bacteria

256th National Meeting (2018)

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