Abstract
Förster resonance energy transfer (FRET) provides a powerful tool for monitoring intermolecular interactions and a sensitive technique for studying Å-level protein conformational changes. One system that has particularly benefited from the sensitivity and diversity of FRET measurements is the maturation of the Shigella type III secretion apparatus (T3SA) needle tip complex. The Shigella T3SA delivers effector proteins into intestinal cells to promote bacterial invasion and spread. The T3SA is comprised of a basal body that spans the bacterial envelope and a needle with an exposed tip complex that matures in response to environmental stimuli. FRET measurements demonstrated bile salt binding by the nascent needle tip protein IpaD and also mapped resulting structural changes which led to the recruitment of the translocator IpaB. At the needle tip IpaB acts as a sensor for host cell contact but prior to secretion, it is stored as a heterodimeric complex with the chaperone IpgC. FRET analyses showed that chaperone binding to IpaB’s N-terminal domain causes a conformational change in the latter. These FRET analyses, with other biophysical methods, have been central to understanding T3SA maturation and will be highlighted, focusing on the details of the FRET measurements and the relevance to this particular system.
Highlights
Förster resonance energy transfer (FRET) has provided a powerful tool that is readily applied to monitoring interactions between molecules as would occur upon binding of a ligand to a protein or the association of a protein to another protein
FRET has provided a powerful tool for studying biological systems including bacterial pathogenesis at a molecular level
This review focuses on the use of FRET in studying the stepwise maturation of the Shigella T3SA tip complex
Summary
Förster (non-radiative) resonance energy transfer (FRET) has provided a powerful tool that is readily applied to monitoring interactions between molecules as would occur upon binding of a ligand to a protein or the association of a protein to another protein. High-resolution microscopy techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM), for example, are capable of achieving resolution on the nanometer scale and have been extensively used for the study of membrane organization [14,15], macromolecular protein complex formation and architecture [16,17], and pathogen/host relationships [18] to name just a few While these techniques offer orders of magnitude improvement in spatial resolution over traditional optical fluorescence detection and have proven invaluable in many studies, sample preparation is extensive and the samples must be imaged under vacuum, thereby limiting its use in dynamic solution-based applications. We will focus primarily on this work, beginning with a review of T3SSs followed by a discussion of FRET and its relevant applications
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