Cation binding and conformation of tryptic fragments of Nereis sarcoplasmic calcium-binding protein: calcium-induced homo- and heterodimerization.
Nereis sarcoplasmic calcium-binding protein (NSCP) is a compact 20-kDa protein that competitively binds three Ca2+ or Mg2+ ions and displays strong positive cooperativity. Its three-dimensional structure is known. It thus constitutes a good model for the study of intramolecular information transduction. Here we probed its domain structure and interaction between domains using fragments obtained by controlled proteolysis. The metal-free form, but not the Ca2+ or Mg2+ form, is sensitive to trypsin proteolysis and is preferentially cleaved at two peptide bonds in the middle of the protein. The N-terminal fragment 1-80 (T1-80) and the C-terminal fragment 90-174 (T90-174) were purified to electrophoretic homogeneity. T1-80, which consists of a paired EF-hand domain, binds one Ca2+ with Ka = 3.1 x 10(5) M-1; entropy increase is the main driving force of complex formation. Circular dichroism indicates that T1-80 is rich in secondary structure, irrespective of the Ca2+ saturation. Ca2+ binding provokes a difference spectrum which is similar to that observed in the intact protein. These data suggest that this N-terminal domain constitutes the stable structural nucleus in NSCP to which the first Ca2+ binds. T90-174 binds two Ca2+ ions with Ka = 3.2 x 10(4) M-1; the enthalpy change contributes predominantly to the binding process. Metal-free T90-174 is mostly in random coil but converts to an alpha-helical-rich conformation upon Ca2+ binding. Ca2+ binding to T1-80 provokes a red-shift and intensity decrease of the Trp fluorescence but a blue-shift and intensity increase in T90-174.(ABSTRACT TRUNCATED AT 250 WORDS)
Durussel
I
I
Department of Biochemistry, University of Geneva, Switzerland.
Luan-Rilliet
Y
Y
Petrova
T
T
Takagi
T
T
Cox
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011077:Polychaeta; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin; D014961:X-Ray Diffraction
D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D014357:Trypsin; D008274:Magnesium; D002118:Calcium