Evaluating Calmodulin-Protein Interactions by Rapid Photoactivated Cross-Linking in Live Cells Metabolically Labeled with Photo-Methionine.

This work addresses the question of how the Ca sensor protein calmodulin shapes cellular responses to Ca signals. Proteins interacting with affinity tagged calmodulin were captured by rapid ( ≈ 7 s) photoactivated cross-linking under basal conditions, after brief removal of extracellular Ca and during a cytosolic [Ca] transient in cells metabolically labeled with a photoreactive methionine analog. Tagged adducts were stringently enriched, and captured proteins were identified and quantified by LC-MS/MS. A set of 489 proteins including 27 known calmodulin interactors was derived. A threshold for fractional capture was applied to define a high specificity group of 170 proteins, including 22 known interactors, and a low specificity group of 319 proteins. Capture of ∼60% of the high specificity group was affected by manipulations of Ca, compared with ∼20% of the low specificity group. This suggests that the former is likely to contain novel interactors of physiological significance. The capture of 29 proteins, nearly all high specificity, was decreased by the removal of extracellular Ca, although this does not affect cytosolic [Ca]. Capture of half of these was unaffected by the cytosolic [Ca] transient, consistent with high local [Ca]. These proteins are hypothesized to reside in or near microdomains of high [Ca] supported by the Ca influx.