title: Protein species-specific characterization of conformational change induced by multisite phosphorylation.
abstract: Phosphorylation is a central mechanism for regulating the structure and function of proteins in the cell, but accurate characterization of a specific protein phospho-species is challenging due to the difficulty of separating it from other species, as well as the limitations of the traditional structural methods. By using selective top-down ETD, we were able to identify six specific phospho-species of calmodulin (CaM). Phosphorylation of CaM at four sites by CK2 was found to follow a sequential order, with Ser81 as the first, Thr79 as the second, and Ser101 or Thr117 as the third. By combining top-down ETD with hydrogen/deuterium exchange, the impact of phosphorylation on CaM's structure was elucidated in a species-specific manner. A negligible structural effect was observed for mono-phosphorylation at Ser81, or di-phosphorylation at Ser81-Thr79, or tri-phosphorylation at Ser81-Thr79-Ser101 or Ser81-Thr79-Thr117. However, it was found that a significant phosphorylation-induced conformational change in CaM was caused by simultaneous phosphorylation at Ser101 and Thr117. The dramatically increased deuterium incorporation for residues between 102 and 119 strongly suggests that the structure of this region has been greatly changed.
authors.affiliation: University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada.
authors.forename: Jingxi
authors.initials: J
authors.lastname: Pan
authors.affiliation: MRM Proteomics Inc., 4464 Markham Street, Suite #2108, Victoria, BC V8Z 7X8, Canada.
authors.forename: Suping
authors.initials: S
authors.lastname: Zhang
authors.affiliation: University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada; Department of Biochemistry & Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada. Electronic address: christoph@proteincentre.com.
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
mesh_terms: D002147:Calmodulin / Q000737:chemistry*; D006801:Humans; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D002147:Calmodulin; D010750:Phosphoproteins; D011994:Recombinant Proteins
keywords: Calmodulin; Electron transfer dissociation; Hydrogen/deuterium exchange; Phosphorylation; Post-translational modification; Top-down