A fluorescence spectroscopy study of the calpactin I complex and its subunits p11 and p36: calcium-dependent conformation changes.
A fluorescence study of the calpactin I complex, a heterotetramer composed of two molecules of p36 and two molecules of p11, and its subunits, was performed to clarify their conformation. The analysis of the fluorescence characteristics of the single Trp of p36, in the absence of Ca(2+), shows that: (i) in the complex, Trp is buried within the protein matrix and subjected to static quenching from nearby groups; (ii) for p36 the results are similar, but Trp seems even more shielded than in the complex. Adding Ca(2+) to the calpactin I complex, or to p36, shifts the Trp emission maximum wavelengths, and increases the quantum yields which reflect a conformational change, burying the Trp in a more hydrophobic environment. In the presence and even in the absence of Ca(2+), the binding of phosphatidylserine liposomes induces a conformational change, detected by fluorescence measurements. The Ca(2+) dissociation constants, as determined by fluorescence titrations, are similar for the complex and p36 (KD approximately 0.5 x 10(-3) M). The affinity is enhanced a 1000-times in the presence of negatively charged phospholipids. In p11, both Try residues are located in a hydrophobic environment and the protein fluorescence does not change upon Ca(2+) addition.
Pigault
C
C
U.A. CNRS 491, Université Louis Pasteur, Cedex, France.
Follénius-Wund
A
A
Lux
B
B
Gérard
D
D
D000818:Animals; D017302:Annexins; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002417:Cattle; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008970:Molecular Weight; D010743:Phospholipids / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan; D014443:Tyrosine
D017302:Annexins; D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010743:Phospholipids; D014443:Tyrosine; D014364:Tryptophan; D002118:Calcium