Functional force mapping of membrane-associated protein complexes.
Published: 2004
Language: eng
Number of pages: 301
Description: Molecular recognition is governed by subtle structural and chemical cues between complementary binding partners. Understanding and ultimately controlling these interactions underpins efforts in fields from structural biology, biochemistry, to combinatorial chemistry and protein engineering. While traditional approaches to studying protein-protein interactions and molecular recognition phenomena have provided in-depth molecular scale details on equilibrium binding affinities and the local atomic structure of the binding interfaces, the actual force(s) involved in binding remain largely unknown. We have coupled the ability of scanning probe microscopy (SPM) to provide true three-dimensional images with its high force sensitivity into a new imaging modality termed 'topography targeted force mapping microscopy' (T2FMM) and have applied this technique to perform functional force mapping of membrane associated protein complexes. This approach allows us to physically map the local structure of the binding interface of a membrane protein or receptor and by using a ligand tethered to the scanning tip, measure the forces between the ligand and its complementary binding site as a function of spatial location. But do the observed binding forces necessarily reflect the forces associated with a fully functional membrane protein that is activated upon the binding of a ligand? To address this question, we have combined T2FMM with total internal reflectance fluorescence (TIRF) spectroscopy and microscopy to allow in situ correlation between (1) topography (AFM imaging), (2) interaction forces (AFM force measurements), and (3) functional activity (fluorescence) for individual membrane protein complexes. Our studies have demonstrated the ability of this technique to provide novel insights into the structure of both integral membrane proteins and membrane-associated proteins in a native membrane environment. It also allows the localization of membrane receptors within specific regions of a supported planar lipid bilayer (SPB) system, as well as the co-localization of receptor molecules and membrane-associated proteins upon binding to the membrane surface. Force mapping studies performed using T2FMM and the well-characterized model membrane-associated protein system of cholera toxin have now provided framework necessary to perform 'functional' force mapping of ligand-membrane protein complexes, such as the insulin-insulin receptor (IR) complex, by T2FMM, while simultaneously monitoring the activation of the IR upon insulin binding though correlated fluorescence measurements.
Notes: Adviser: C. M. Vip.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2498.