Poster Session 6, 4:15 PM - 5:00 PM: Campus Center Auditorium [A61]

Presenter: Sammy Redha Mounsif

Faculty Sponsor: Lynmarie K. Thompson

School: UMass Amherst

Research Area: Biochemistry and Molecular Biology

ABSTRACT

Many bacteria exhibit chemotaxis – movement according to chemical gradients. Chemotactic bacteria utilize a well-conserved protein complex to detect molecules in their environment and use this information to control their swimming patterns. This signaling complex contains three components: membrane-spanning receptors bind two cytoplasmic proteins, the histidine kinase CheA and coupling protein CheW. Although there are good structural models for the CheA kinase core, its activity depends on transient interactions with two additional domains, P1 and P2, connected to the core by flexible linkers. Autophosphorylation occurs when the catalytic P4 domain phosphorylates a histidine on the highly mobile P1 domain. Recent computational and NMR studies have begun to characterize the transient P1/P4’ interactions critical for activity. Here, we investigate the use of single molecule (sm)FRET as well as ensemble FRET studies of CheA in solution to observe the conformation, length, and frequency of P1/P4’ interactions involved in kinase regulation and catalysis. Specific labeling of Cys residues on CheA mutants has been achieved. Ensemble FRET and smFRET in a control sample with a known distance across the CheA dimer interface will be measured. Similar experiments will then be performed at the P1/P4’ interface, in the absence and presence of ATP. This will illuminate the frequency and length of transient interactions which activate or inhibit the kinase, while distinguishing between these interactions and determining their relative frequency.