Grants and Contracts per year
Grants and Contracts Details
This research project focuses on a novel approach to monitor the assembly and trafficking of nicotinic receptors at the single molecule level. Nicotinic receptors cannot be purified outside of the cellular environment making them inaccessible to common single molecule techniques that requite the purification and reconstitution of proteins. This has limited studies on the structure and function of nicotinic receptors involved in nicotine addiction. We will intitally utilize receptors isolated in cell derived vesicles that can then be incorporated with substrate arrays capable of supporting integral membrane protein. Isolation of these vesicles in conjunction with nanostructures will provide a new method for the study of relationship between the structure and dynamics of function of previously inaccessible membrane proteins. Understanding the molecular level properties of receptor function requires the ability to resolve dynamics of individual biomolecules. Single-molecule studies in live cells are extremely limited due to poor spatial resolution and a lack of imaging sensitivity. The use of nanostructures allows us to create an array of nanoscale observation volumes containing isolated integral membrane proteins. This imparts three key advantages: (1) by limiting the excitation volume only molecules in the observation volume will be excited which virtually eliminates background fluorescence; (2) the nanostructure will provide a 10 to 100-fold increase in fluorescence detection due to plasmonic interactions as well as control over the directionality of emitted light, and (3) the nano-observation volume isolates membrane receptors for long periods of time allowing dynamics to be extracted. We will then extend these studies within the context of nicotine addiction by isolating vesicles from different regions of the brain of nAChR GFP knock-mice. We will be able to correlate the stoichiometry of receptors to specific brain regions. These studies will for the first time resolve structure/function relationships of integral membrane proteins at the single receptor level.
|Effective start/end date||7/1/15 → 6/30/17|
- National Institute on Drug Abuse: $371,893.00
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- 1 Finished
7/1/15 → 6/30/17
Project: Research project