Coupling of ligand binding and dimerization of helix-loop-helix peptides: spectroscopic and sedimentation analyses of calbindin D9k EF-hands.
Isolated Ca2+-binding EF-hand peptides have a tendency to dimerize. This study is an attempt to account for the coupled equilibria of Ca2+-binding and peptide association for two EF-hands with strikingly different loop sequence and net charge. We have studied each of the two separate EF-hand fragments from calbindin D9k. A series of Ca2+-titrations at different peptide concentrations were monitored by CD and fluorescence spectroscopy. All data were fitted simultaneously to both a complete model of all possible equilibrium intermediates and a reduced model not including dimerization in the absence of Ca2+. Analytical ultracentrifugation shows that the peptides may occur as monomers or dimers depending on the solution conditions. Our results show strikingly different behavior for the two EF-hands. The fragment containing the N-terminal EF-hand shows a strong tendency to dimerize in the Ca2+-bound state. The average Ca2+-affinity is 3.5 orders of magnitude lower than for the intact protein. We observe a large apparent cooperativity of Ca2+ binding for the overall process from Ca2+-free monomer to fully loaded dimer, showing that a Ca2+-free EF-hand folds upon dimerization to a Ca2+-bound EF-hand, thereby presenting a preformed binding site to the second Ca2+-ion. The C-terminal EF-hand shows a much smaller tendency to dimerize, which may be related to its larger net negative charge. In spite of the differences in dimerization behavior, the Ca2+ affinities of both EF-hand fragments are similar and in the range lgK = 4.6-5.3.
Julenius
Karin
K
Department of Biophysical Chemistry, Lund University, Lund, Sweden.
Robblee
James
J
Thulin
Eva
E
Finn
Bryan E
BE
Fairman
Robert
R
Linse
Sara
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D064026:Calbindins; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D003488:Cyanogen Bromide / Q000737:chemistry; D019281:Dimerization; D020832:EF Hand Motifs; D008024:Ligands; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D064026:Calbindins; D008024:Ligands; D010446:Peptide Fragments; D064030:S100 Calcium Binding Protein G; D003488:Cyanogen Bromide; D002118:Calcium