In the USA approximately 25 million patients/year undergo surgery using general anesthetics having very low therapeutic indices and whose molecular mechanisms remain unknown, hampering the design of improved agents. The broad long-term objective of the proposed research is to define the molecular determinants of those general anesthetic binding sites that effect the function of proteins. The specific protein to be studied is protein kinase C (PKC). Volatile Anesthetics and long chain alcohols are known to affect the activity of activated PKC.
We aim to test the hypothesis that anesthetics act on the regulatory domain (C1) at the two diacylglycerol (DAG) binding sites (C1A & B) by locating general anesthetic binding sites on PKC by photolabeling them with recently developed photoactivatable anesthetic alcohols. Photolabeled residues will be identified by mass spectrometry. Encouraging preliminary results show these agents to interact with the isolated second cysteine-rich (C1B) regulatory subdomain of PKC6 at a tyrosine adjacent to the C1B phorbol binding site and that the C1A fragment exhibits different binding characteristics. The crystal structure of the C1B fragment has been published, and we will determine its structure with various general anesthetics bound. Designed mutations within this binding pocket will be made in order to determine the principles governing anesthetic-protein interactions at the molecular level. Similar studies will be undertaken with C1A. In the intact C1 domain, we will test the hypothesis that interactions between the C1A and C1B subdomains contribute to the anesthetic binding sites. The importance of allosteric interactions between the anesthetic and DAG sites at C1A & C1B in the C1 domain will be assessed by crystallizing it complexed with and without a phorbol ester bound and in the presence and absence of anesthetics. Structural changes will be interpreted by measuring anesthetic and phorboI-C1 interactions in parallel.
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|Shanmugasundararaj, Sivananthaperumal; Lehle, Simon; Yamodo, Herve I et al. (2012) The location and nature of general anesthetic binding sites on the active conformation of firefly luciferase; a time resolved photolabeling study. PLoS One 7:e29854|
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|Das, Joydip; Zhou, Xiaojuan; Miller, Keith W (2006) Identification of an alcohol binding site in the first cysteine-rich domain of protein kinase Cdelta. Protein Sci 15:2107-19|
|Das, Joydip; Addona, George H; Sandberg, Warren S et al. (2004) Identification of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase Cdelta. J Biol Chem 279:37964-72|