Biophotonics: Biophotonics Based on Liquid Crystals: New Principles for Profiling Regulatory Signaling Proteins and Their Post-Translational Modifications

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

Whereas decoding the entire genomes of procaryotic and eucaryotic organisms was a major biological challenge of the nineties, this new decade seeks to capture an understanding of the protein interactions of a cell. This understanding has the potential to yield enormous societal benefit via predictions of total cellular responses to new pharmaceutical compounds, infectious agents, environmental toxins, or disruptions in particular regulatory pathways. The research described in this proposal aims to address this grand challenge by establishing a fundamentally new and highly sensitive optical measurement technique that permits rapid, simultaneous detection and quantification of multiple protein molecules involved in regulating intracellular signaling pathways. The research described in this proposal fuses principles of optics in liquid crystalline media with the design of nanostructured surfaces to create a fundamentally new optical tool that has the potential to be broadly applicable to studies aimed at unraveling the complex behavior of cellular systems at the protein level. The first goal is to establish the use of biophotonic methods based on liquid crystals for analysis of key regulatory signaling pathways in cells. Such a technology could impact drug discovery, pharmacodynamic monitoring, regulation of gene expression, protein-protein interactions, and our understanding of carcinogenesis. The second broad goal is to demonstrate that liquid crystals can form the basis of a general and powerful methodology that permits detection of post-translational modifications to proteins. Because the identification of specific chemical modifications of a protein can reveal its cellular location, biological lifespan and activation status, classification of proteins by their post-translational modification would be enormously useful in studies elucidating the complex behavior of cells. We propose to explore the use of liquid crystals to monitor cellular expression and activation of regulatory signaling proteins such as Ras, and to detect the interaction of Ras with target proteins in the multiple signaling pathways that mediate its biological activity. Although our initial focus will be on detecting the binding of Ras to anti-Ras IgG and, when activated, its binding to the down stream effector molecular RAF1, this system is prototypical and will, therefore, be generalizable to a broad class of regulatory signaling proteins. We propose to demonstrate the generality of the methodology by using liquid crystals to screen simultaneously for post-translational modifications of multiple proteins involved in a common regulatory pathway. First, we will design surfaces that permit coupling of biomolecular recognition events between total Ras and anti-Ras IgG, and between activated Ras and RAF1, to the orientations of liquid crystals. Second, we will determine the sensitivity and capability to quantitate photonic methods based on liquid crystals using as examples detection of total Ras and activated Ras. Third, we will integrate photonic principles based on liquid crystals with microfluidics so as to minimize sample size and enable detection of minute amounts of captured proteins (~104 molecules). We will provide simultaneous measurement of total Ras and activated Ras, and integrate the delivery of the sample and the liquid crystal. Fourth, we will demonstrate the generality of the above described methodology based on liquid crystals by rapidly screening for the post-translational modifications of multiple proteins involved in a common regulatory pathway (MAPK, JNK, ERK 1/2). Although the principles we propose to develop are broadly applicable to fundamental studies in cell and molecular biology, they also have the potential for a tremendous impact on biomedical investigators. One such example is in the detection of the activation states of intracellular regulatory proteins. It is the activation state of these proteins that determines which intracellular signaling cascades are initiated, and these in turn determine the fundamental behaviors of cells. As such, the rapid determination of the activation states of these regulatory proteins will represent a boon to the study of diverse medical conditions that result from inappropriate signaling events such as those linked to neoplastic transformation and immune mediated disorders.