The browser you are using is not supported by this website. All versions of Internet Explorer are no longer supported, either by us or Microsoft (read more here: https://www.microsoft.com/en-us/microsoft-365/windows/end-of-ie-support).

Please use a modern browser to fully experience our website, such as the newest versions of Edge, Chrome, Firefox or Safari etc.

Portrait of Sara Snogerup Linse

Sara Linse

Professor

Portrait of Sara Snogerup Linse

Integrated protein array screening and high throughput validation of 70 novel neural calmodulin binding proteins

Author

  • David O'Connell
  • Mikael Bauer
  • John O'Brien
  • Winifred M Johnson
  • Catherine A Divizio
  • Sara L O'Kane
  • Tord Berggård
  • Alejandro Merino
  • Karin Akerfeldt
  • Sara Linse
  • Dolores J Cahill

Summary, in English

Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labelled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (K(D) = 1 muM) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments, of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (koff = 10(3) s(-1)) and high affinity (K(D) = 1 muM), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We have developed a microarray of the identified target proteins with which we can characterise the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage gated channel Kv6.1, (residues 474-493), CaM kinase-like vesicle-associated protein (302-316), EF-hand domain family member A2 (202-216) and phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (400-415).

Department/s

  • Biophysical Chemistry
  • MultiPark: Multidisciplinary research focused on Parkinson´s disease

Publishing year

2010

Language

English

Pages

1118-1132

Publication/Series

Molecular & Cellular Proteomics

Volume

9

Issue

6

Document type

Journal article

Publisher

American Society for Biochemistry and Molecular Biology

Topic

  • Physical Chemistry

Status

Published

ISBN/ISSN/Other

  • ISSN: 1535-9484