Projects and Grants per year
Grants and Contracts Details
Description
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.
Status | Finished |
---|---|
Effective start/end date | 7/1/15 → 6/30/17 |
Funding
- National Institute on Drug Abuse: $371,893.00
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Projects
- 1 Finished
-
Single molecule determination of nAChR structural assembly for therapeutic targeting
Pauly, J. & Richards, C.
National Institute on Drug Abuse
7/1/15 → 6/30/17
Project: Research project