Structural characterization of two-component signal transduction proteins and calbindin D28k

by 1977- Kojetin, Douglas John

KOJETIN, DOUGLAS J. Structural Characterization of Two-Component Signal Transduction Proteins and Calbindin D28k . (Under the direction of Professor John Cavanagh.) The studies described involve the structural analysis of proteins involved in signal transduction pathways. In the first study, the metal binding properties of the initiation of sporulation response regulator Spo0F was studied using a variety of biophysical and biochemical techniques. The experiments show that most of the divalent metals studied, including and Ca2+, Mg2+ and Mn2+, which display primarily 1:1 binding, allow for favorable conditions for phosphotransfer between Spo0F and its cognate kinase KinA. In contrast, Spo0F binds up to three Cu2+ ions and the presence of this metal does not allow for the phosphotransfer reaction to occur. In the second study, a comparative modeling study of the OmpR sub-family of response regulators from B. subtilis and E. coli was performed and used to suggest the possibility of sub-classes within this related domain family based on regions of the response regulator regulatory domain that is known to interact with cognate four-helix bundle HisKA/Hpt domains. In the third study, the structural refinement of the fourhelix bundle LuxU phosphotransferase from V. harveyi is described using a combination of dipolar couplings and water-based explicit refinement. In the fourth and last study, the development of a solution structure of Ca2+-loaded calbindin D28k, an EF-hand calcium binding protein, and the interaction between peptides derived from ran-binding protein M and myo-inositol monophosphatase are described. Structural Characterization of Two-Component Signal Transduction Proteins and Calbindin D28k by Douglas John Kojetin a dissertation submitted to the graduate faculty of north carolina state university in partial fulfillment of the requirements for the degree of doctor of philosophy biochemistry raleigh 2005 approved by: Dr. John Cavanagh chair of advisory committee Dr. Dennis T. Brown Dr. William L. Miller Dr. Charles H. Opperman Dr. Ronald A. Venters