Kinetic Analysis Of The G Protein Cascade Using Surface Plasmon Resonance

In addition to the main focus on Gb5-RGS7, we work on applications of surface plasmon resonance (SPR) for G protein studies. SPR is a relatively novel method which is one of the few techniques that monitors protein-protein interactions in real time. The measurements of the apparent affinity (Kd) and the on- and off- rates can be used for modeling signal transduction. Since virtually all kinds of proteins can be studied by SPR, the binding parameters for interactions in an entire G protein pathway could be obtained by one technique and thus discrepancies between constants obtained by different methods could be avoided. We chose the phototransduction cascade because it is well characterized at the physiologic level and the proteins are readily available. In the course of these studies, we found that in addition to kinetic measurements, SPR is very useful for the studies of regulation of protein-protein interactions by low molecular weight compounds and for structure-function analysis. For example, SPR has allowed us to gain the following new insights into molecular details of GRK (G protein-coupled Receptor Kinase) function:

1. ATP and ADP regulate the interaction of GRKs with Ca2+-binding proteins,
2. GRK binding increases the affinity of Ca2+-binding proteins to Ca2+,
3. All GRKs have two independent high-affinity sites for Ca2+-calmodulin.

We will continue to use SPR to advance our research on RGS proteins. We are particularly interested in SPR at lipid surfaces, a technique that has emerged in the past two years. It allows binding to be measured under conditions resembling the native environment, providing "real" rather than apparent kinetic constants.

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