ENGINEERING CALPASTATIN TO DEVELOP A SENSOR TO DETECT ACTIVE CALPAIN

Calpains, Ca2+-activated cysteine proteases are essential for early embryonic development and function in signal transduction, cell adhesion, and apoptosis. Calpains also contribute to cataractogenesis, myocardial infarctions, and neurodegenerative diseases such as Alzheimer's. The various methods currently available to demonstrate these roles do not directly identify spatial or temporal activation of calpain in cells. Therefore, a tool to detect active calpain in situ will be useful. Calpastatin is the ubiquitous, endogenous inhibitor that specifically binds the active conformation of the conventional calpains. Calpastatin consists of four homologous domains each containing three subdomains A, B, and C. The crystal structure of a calpastatin C peptide mimic bound to the homodimer of calpain domain VI in the presence of Ca2+ provides a model for the structure of calpastatin subdomains A and C bound to calpain domains IV and VI (56). This structure predicts the calpain domain IV carboxy-terminus is approximately 31 A from the bound calpastatin domain I amino-terminus and the calpastatin domain I subdomain A amino-terminus is approximately 50 A from the calpastatin domain I subdomain C carboxy-terminus when bound to calpain. Both are distances that should allow fluorescence resonance energ