Crystallization Studies of Calmodulin Binding Targets

Abstract

In a recent scientific study, 70 new calmodulin (CaM) binding targets have been identified in human brain.(1) For this research project, peptides corresponding to the CaM binding portion of a number of these targets, including, LAT1, RPS2, STIM1, CaMkv, and Rab11b, have been synthesized and purified. In a couple of cases, including Rab 11b, the full length protein, has also been expressed and purified. The ultimate aim is to obtain an understanding of how CaM can regulate so many targets with such selectivity. Toward this goal we are collecting target binding data as well as structures of a range of peptides and proteins in complex with CaM using x-ray crystallography. The peptides described in this work have been synthesized using Fmoc solid peptide synthesis and the Rab11b protein was expressed in E. Coli and purified using a series of column chromatography steps. Crystallization conditions have been optimized for a specific subset of these complexes. Diffraction data were obtained for CaM:LAT1 at the Max Lab synchrotron facility in Lund, Sweden. The data obtained was then refined at Colgate University, NY, employing the software, Refmac5, to furnish a crystal structure with a resolution of 2.1 A and an R-factor of 25%. We will also present preliminary binding data of the LAT1 sequence bound to CaM, determined to be in the uM range, and obtained by isothermal titration calorimetry (ITC).1. “Integrated protein array screening and high throughput validation of 70 novel neural calmodulin binding proteins” O’Connell, DJO.; Bauer, MC, O’Brien J, Johnson, WM,∗ Divizio CA,∗ O’Kane S, Berggard T, Merino A, Akerfeldt KS, Linse S, Cahill DJ Molecular & Cellular Proteomics 9, 1118-1132 (2010).