Detecting the active conformation of calpain with calpastatin-based reagents.
The specific, calcium-dependent, high affinity interaction between calpain and its endogenous inhibitor calpastatin was exploited to selectively detect the calcium-bound, catalytically competent, conformation of calpain in vitro. Modification of calpastatin domain-1 (Val(114)-Ser(270)) or its N-terminal fragment (Val(114)-Pro(202)), at selected unique cysteine residues with maleimide-AlexaFluor546 did not compromise calpastatin function (inhibition of calpain) or its binding with calpain. Ca(2+)-dependent binding between catalytically dead calpain-2 (Cys(105)Ala) fused with eGFP and these fluorigenic calpastatin peptides generates fluorescent resonance energy transfer (FRET). The FRET signal documents proximity of calpain-2, C-terminally linked fluorophore to specific sites within calpastatin when the proteins form a complex. These results provide important insights into the calcium-dependent interaction between calpain and calpastatin and for holo-calpain-2 in solution experimentally validate some key features of their predicted interactions. These data also provide proof of concept that the calpastatin-based reagents may be useful to selectively detect the active conformation of calpain.
Croall
Dorothy E
DE
5735 Hitchner Hall, Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, Maine 04469-5375, USA. dorothy.croall@umit.maine.edu
Vanhooser
Lisa M
LM
Cashon
Robert E
RE
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; C033668:calpastatin; D002154:Calpain; D002118:Calcium
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