; R o o t E n t r y F y J @ C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l w J w J 4 @ ? @ A B C D E F G H F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; 9507357 Brautigan The overall goal of this research is to understand the molecular basis for the high affinity binding and the modulation of substrate specificity of type 1 protein phosphatase (PP1) by its glycogen binding regulatory subunit called RG. Anchor proteins bind cAMP dependent protein kinase with nanomolar affinity by a relatively short sequence with a helical structure. This project will test the hypothesis that in a similar way a helical segment in RG is the recognition site for PP1. Extremely well conserved among all eukaryotes, from yeasts to humans, PP1 has been shown to play an essential role in mitosis, as well as in regulating intermediary metabolism and maintaining integrity of the cytoskeleton. It is thought that the catalytic subunit of PP1 can carry out these diverse biological missions by forming distinctive heterodimers with regulatory subunits that, like RG, target the PP1 to intracellular locales, alter its substrate specificity, and become phosphorylated as a means of modulating these functions. The objectives of this research plan are to: 1. generate, express, and purify a bacterial fusion protein containing the functional N terminal 40 kDa domain of RG linked to glutathione S transferase (GST). Show the correct folding of the recombinant protein by circular dichroic spectra and by limited digestion with proteases; 2. establish assays for the binding of PP1 catalytic subunit to this GST RG -fusion protein, and to the RG domain cleaved from GST after purification, using solid phase adsorption; 3. produce truncated versions of the RG domain as fusion proteins and analyze them for their ability to bind PP1. Make single amino acid substitutions in putative helices in the RG domain by site directed mutagenesis to precisely map the interaction site with PP1. These experiments will test the specific hypothesis that one of four putative helices in RG is the primary binding site for PP1. The results will provide new information by identifying the protein structures that bind PP1, and the portion of RG that can modify PP1 specificity, representing a fundamental advance in our understanding of the operation of this essential enzyme. The details of these protein protein interactions could prove valuable in designing reagents to immobilize PP1 or other enzymes used in manufacturing processes. Solid phase, active PP1 could be used as a way to detect toxins in water or other samples, because several classes of fresh water and marine toxins are potent inhibitors. %%% The goal of this research is to understand the molecular basis for the function of a regulatory subunit of protein phosphatase type 1 (PP1), all essential enzyme highly conserved among all eukaryotes. Different regulatory subunits target PP1 to specific intracellular sites such as glycogen, myosin, or chromosomes. This research will provide new fundamental information about the molecular structure of a protein protein interface that immobilizes an active form of an important regulatory enzyme. *** @ @ ; Oh +' 0 $ H l S u m m a r y I n f o r m a t i o n ( > D h R:WWUSERTEMPLATENORMAL.DOT marcia steinberg Sharon Alston @ k : @ @ i J @ q Microsoft Word 6.0 3 ; e = e j j j j j j j L & 9
