Cdc42Hs, a member of the Ras superfamily signal transduction proteins, binds guanine nucleotides and acts as a molecular timing switch in multiple signal transduction pathways. This proposal is centered around studying the structure and dynamics of Cdc42Hs forms associated with its oncogenic potential and will provide atomic level information into mechanisms that regulate this activity. The Cdc42Hs constructs that will be studied are, A) Cdc42Hs(nucleotide-free) and, B) Cdc42Hs(F28L_?L8). Defining the structure and dynamics of the nucleotide-depleted form of Cdc42Hs will shed light on the conformation(s) of Cdc42Hs that facilitate the binding of protein effectors, such as Dbl, that stimulate oncogenic activity. The study of the structure and dynamics of Cdc42Hs(F28L_?L8) should reveal information on interactions in Cdc42Hs that block oncogenic activity caused by the single mutant Cdc42Hs(F28L). The immediate goals of this research are to use NMR and fluorescence as structural techniques to gain a better understanding of the processes by which changes in Cdc42Hs and its interaction with regulators affect various important signal transduction pathways that lead to cancer. The long-term goals of this proposed research are to gain a better understanding of how protein structure and dynamics correlate with aberrant cell function, which will lay the foundation for future research into the development of a rational anti-cancer drug design program. Cdc42Hs is a member of the Ras superfamily of proteins, which have been implicated in cancers in the colon, breasts, and lungs. These studies will provide information on particular aspects of specific cell-signaling processes that may be subsequently used in the development of new anti-cancer treatments that are specific and may be less toxic than traditional chemotherapy approaches. I have a background in the use of fluorescence spectroscopy in the study of biological molecules and have used NMR spectroscopy to study structure of small peptides in solution. The mentored phase of this proposal will provide me with the opportunity to gain experience in several uses of these techniques that have not yet been mastered that will be crucial to my transition to independent investigation. The Department of Molecular Medicine at Cornell University has great experience in the area of applying physical methods to biological processes, and in particular, signal transduction. It is an excellent setting for this project, and also to foster the development of my career. The research career development plan of this proposal is two-phased. Phase I (Mentored) will allow for the study of the nucleotide-free construct of Cdc42Hs. The experience with the structural tools, data analysis procedures, and scientific collaboration in this phase will foster the necessary preparation for a successful transition into Phase II (Independent), during which time I will study the structure and dynamics of Cdc42Hs(F28L_?L8), leading to further research in the structural biology of biomolecules related to cancer.
