The proposed studies describe an innovative approach for detecting protein association to spatially localized surface-immobilized small molecule (ligand) and protein microarrays via chiral imaging with second harmonic generation (SHG). The use of microarray-based protein assays has the potential to dramatically increase the analysis of global protein populations within living organisms. The practical implementation of these technologies will have a dramatic impact in proteomics, pharmacological screening of drug candidates, and the investigation of protein-protein interactions. Most conventional microarray-based strategies for detecting protein-ligand, and protein-protein association or for the screening of protein populations are based on fluorescence methods to visualize the interaction of interest. Such methodologies require the fluorescent labeling of a large population of proteins with fluorophores. The inability to accurately perform this operation in a controlled manner for the complete protein output of a cell or organism is a major drawback to currently proposed techniques for high throughput protein screening. This proposal will address the hypothesis that the intrinsic chirality of proteins can be used to """"""""image"""""""" their association on patterned microarrays. This hypothesis will initially be tested by exploring the ability to monitor protein association to ligands presented on the outer leaflet of planar supported lipid bilayers, utilizing the proteins' native chirality as a spectroscopic probe. Then, SHG chiral imaging for protein-ligand based microarray assay will be examined using photolithographic and micro-contact printing methods to produce ordered arrays of ligands for protein adsorption studies. Finally, the ability of chiral SHG imaging to measure protein-protein interactions on patterned protein microarrays will be assessed. In order to fully characterize the spatial arrangement of interfacial protein species in these studies, SHG microscopy will be used in combination with fluorescence microscopy to provide a precise evaluation of the proposed chiral SHG imaging methodology. The proposed studies represent a novel experimental approach that should provide answers to a growing number of questions concerning detection for high throughput proteome screening. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068120-02
Application #
6743691
Study Section
Special Emphasis Panel (ZRG1-BECM (01))
Program Officer
Edmonds, Charles G
Project Start
2003-05-01
Project End
2008-04-30
Budget Start
2004-05-01
Budget End
2005-04-30
Support Year
2
Fiscal Year
2004
Total Cost
$197,340
Indirect Cost
Name
University of Utah
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Tran, Renee J; Sly, Krystal L; Conboy, John C (2017) Applications of Surface Second Harmonic Generation in Biological Sensing. Annu Rev Anal Chem (Palo Alto Calif) 10:387-414
Stokes, Grace Y; Conboy, John C (2014) Measuring selective estrogen receptor modulator (SERM)-membrane interactions with second harmonic generation. J Am Chem Soc 136:1409-17
Sly, Krystal L; Conboy, John C (2014) Determination of multivalent protein-ligand binding kinetics by second-harmonic correlation spectroscopy. Anal Chem 86:11045-54
Sly, Krystal L; Mok, Sze-Wing; Conboy, John C (2013) Second harmonic correlation spectroscopy: a method for determining surface binding kinetics and thermodynamics. Anal Chem 85:8429-35
Smith, Kathryn A; Conboy, John C (2012) A simplified sum-frequency vibrational imaging setup used for imaging lipid bilayer arrays. Anal Chem 84:8122-6
Sly, Krystal L; Nguyen, Trang T; Conboy, John C (2012) Lens-less surface second harmonic imaging. Opt Express 20:21953-67
Nguyen, Trang T; Sly, Krystal L; Conboy, John C (2012) Comparison of the energetics of avidin, streptavidin, neutrAvidin, and anti-biotin antibody binding to biotinylated lipid bilayer examined by second-harmonic generation. Anal Chem 84:201-8
Smith, Kathryn A; Conboy, John C (2011) Using micropatterned lipid bilayer arrays to measure the effect of membrane composition on merocyanine 540 binding. Biochim Biophys Acta 1808:1611-7
Nguyen, Trang T; Conboy, John C (2011) High-throughput screening of drug-lipid membrane interactions via counter-propagating second harmonic generation imaging. Anal Chem 83:5979-88
Nguyen, Trang T; Rembert, Kelvin; Conboy, John C (2009) Label-free detection of drug-membrane association using ultraviolet-visible sum-frequency generation. J Am Chem Soc 131:1401-3

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