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                            University of Wisconsin Milwaukee
UWM Digital Commons
	December 2013
Protein Association in Living Cells Using Fret Spectrometry: Application to G-Protein Coupled Receptors
	Suparna Patowary
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University of Wisconsin Milwaukee
UWM Digital Commons

Theses and Dissertations

December 2013

Protein Association in Living Cells Using Fret
Spectrometry: Application to G-Protein Coupled
Receptors
Suparna Patowary
University of Wisconsin-Milwaukee

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(2013). Theses and Dissertations. 639.
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studies. Choosing the FRET pair which has well separated spectrum is a solution to the

spectral cross-talk, however, this decreases the spectral overlap integral (J(πœ†)) (3) of the

fluorophores reducing the ability to detect FRET signals. Many of the intensity-based

measurements methods rely on emission and detection filters to measure the fluorescence

intensity of one fluorophore in presence and absence of the other. These filter-based

methods need at least two different detection wavelengths: one, πœ†π‘’π‘š,1, at which donor to

acceptor emission ratio is maximum (i.e. acceptor emission in negligible) and at other,

πœ†π‘’π‘š,2 where it is minimum (donor emission is negligible). Accordingly, we can

approximate equation (3.14) as:



πΌπ‘š(πœ†π‘’π‘₯, πœ†π‘’π‘š,1) β‰… 𝐼
𝐷𝐴(πœ†π‘’π‘₯, πœ†π‘’π‘š,1) = 𝐼

𝐷(πœ†π‘’π‘₯, πœ†π‘’π‘š,1) βˆ’ 𝐼
𝐷(πœ†π‘’π‘š,1, 𝐹𝑅𝐸𝑇) (3.15)

and

πΌπ‘š(πœ†π‘’π‘₯, πœ†π‘’π‘š,2) β‰… 𝐼
𝐴𝐷(πœ†π‘’π‘₯, πœ†π‘’π‘š,2) = 𝐼

𝐴(πœ†π‘’π‘₯, πœ†π‘’π‘š,2) + 𝐼
𝐴(πœ†π‘’π‘š,2, 𝐹𝑅𝐸𝑇) (3.16)



Equations (3.15) and (3.16) are used to calculate the apparent FRET efficiencies in terms

of donor quenching and acceptor sensitized emission (3, 9, 10) as:



πΈπ‘Žπ‘π‘
π·π‘ž

≑
𝐼𝐷(πœ†π‘’π‘š,1, 𝐹𝑅𝐸𝑇)

𝐼𝐷(πœ†π‘’π‘₯, πœ†π‘’π‘š,1)
= 1 βˆ’

𝐼𝐷𝐴(πœ†π‘’π‘₯, πœ†π‘’π‘š,1)

𝐼𝐷(πœ†π‘’π‘₯, πœ†π‘’π‘š,1)
(3.17)



πΈπ‘Žπ‘π‘
𝐴𝑠𝑒 ≑

𝐼𝐴(πœ†π‘’π‘š,2, 𝐹𝑅𝐸𝑇)

𝐼𝐴(πœ†π‘’π‘₯, πœ†π‘’π‘š,1)
=

𝐼𝐴𝐷(πœ†π‘’π‘₯, πœ†π‘’π‘š,2)

𝐼𝐴(πœ†π‘’π‘₯, πœ†π‘’π‘š,2)
βˆ’ 1 (3.18)

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The disadvantage of the above method is spectral bleed-through (see detail

discussion in section 2.1) (9). Additionally, in order to determine the intensities from

donors in the absence of acceptors (𝐼𝐷) and the acceptors in absence of donors (𝐼𝐴), one

needs to perform acceptor photobleaching and separate measurement of samples

containing acceptors only (9, 17), respectively. In the method based on acceptor

photobleaching, the donor is also bleached to a certain extent, and this can affect the

apparent FRET efficiency determination. Additionally, the same sample cannot be used

more than once in experiments relying on acceptor photobleaching, which prevents the

applicability of this method to dynamic studies that require monitoring the evolution of

the protein complex in time.



3.1.4 Determination of FRET efficiency from spectrally resolved fluorescence

intensity measurements

In this section, we will overview the theory of the method of spectrally resolved FRET

imaging (12), and we will also introduce an original contribution, which is related to

extraction of spectral components from a composite spectrum consisting of three

different species of fluorescent molecules.



The proteins of interest are tagged by the fluorescent molecules that act as donors

and acceptors and are transfected to the cells. Cells co-expressing both donor and

acceptor tagged proteins are excited at the excitation wavelength of donor and emission

spectrum is collected at every pixel of the scanning area. In order to extract the donor and

acceptor signals from the composite spectrum at every pixel, first we measure the

Page 194

179







ο‚· 2011-2013: Quaternary structure determination of G-Protein coupled receptors
(GPCRs) using spectrally resolved FΓΆrster resonance energy transfer method and

Monte-Carlo simulations; under Prof. ValericΓ£ Raicu, Physics Department,

UWM, USA



ο‚· 2005-2006: Solar Neutrino Problem; under Dr. Poulose Poulose, Associate
Professor, IITG, India





Teaching Experience


ο‚· Fall 2008-Summer 2009, Spring 2010: Physics 108 (Laboratory for Physics in
Everyday Life), Physics Department, UWM



ο‚· Fall 2009: Physics 121 (General Physics Laboratory), Physics Department, UWM




Publications


Journals


1. S Patowary, L F Pisterzi, J D Holz, J A Oliver, J W Wells and V Raicu.
Experimental verification of FΓΆrster Resonance Energy Transfer (FRET) theory

using optical micro-spectroscopy (OptiMiS) and fluorescence reference standards,

(under preparation)



2. S Patowary, E Alvarez-Curto, T R Xu, J D Holz, J A Oliver, G Milligan and
V Raicu. The muscarinic M3 acetylcholine receptor exists as two differently sized

complexes at the plasma membrane, Biochem. J. 452 (2013) 303 312


3. D R Singh, M M Mohammad, S Patowary, M R Stoneman, J A Oliver, L
Movileanu and V Raicu. Determination of the quaternary structure of a bacterial

ATP-binding cassette (ABC) transporter in living cells, Integr. Biol. 5 (2013) 312-
323



4. M R Stoneman, S Patowary, D R Singh, L Komarova, L G Westrick, J A Oliver,
and V Raicu. Quantifying the efficiency of various FRET constructs using

BioTechniques 52 (2012) 191 195


5. M R Stoneman, S Patowary, M T Roesch, D R Singh, V Strogolov, J A Oliver, V
Raicu. Determination of the Stoichiometry, structure, and distribution in living

cells of protein complexes from analysis of single-molecular complexes,

Multiphoton Microscopy in the Biomedical Sciences X, Proceedings of SPIE,
7569 (2010), 756913-1 756913-8

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180







Contributed Conference Presentations


1. S Patowary, E Alvarez-Curto, T R Xu, J D Holz, J A Oliver, G Milligan and
V Raicu. Probing the stoichiometry and geometry of M3 acetylcholine receptors
at the plasma membrane, poster presentation at Biophysical Society Annual
Meeting, 2013, Philadelphia, PA, USA


2. S Patowary, E Alvarez-Curto, J A Oliver, G Milligan and V Raicu. Quaternary
structure determination of the M3 muscarinic acetylcholine receptors based on

spectral-FRET, poster presentation at Biophysical Society Annual Meeting, 2012,
San diego, CA, USA


3. S Patowary, L F Pisterzi, M R Stoneman, V Strogolov, J A Oliver, J W Wells
and v Raicu. Accurate FRET measurements and testing of the theory for

multimeric complexes using reference fluorescence standards, poster presentation

at Biophysical Society Annual Meeting, 2011, Baltimore, MD, USA


4. S Patowary, L F Pisterzi, M R Stoneman, V Strogolov, J A Oliver, J W Wells
and V Raicu. Experimental testing of the FRET theory for multimeric complexes

using reference fluorescence standards, poster presentation at FRET at 65-One
Day Symposium, University of Virginia, Charlottesville, VA, 2011


5. M R Stoneman, S Patowary, M Roesch, M Dey and V Raicu. In vivo monitoring
of agonist-induced relative movements between G-protein coupled receptor

segments in oligomeric complexes using spectrally resolved FRET. poster

presentation at Biophysical Society Annual Meeting, 2011, Baltimore, MD, USA

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