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Coupling of ligand binding and dimerization of helix-loop-helix peptides: Spectroscopic and sedimentation analyses of calbindin D-9k EF-hands

Julenius, Karin LU ; Robblee, James; Thulin, Eva LU ; Finn, Bryan E; Fairman, Robert and Linse, Sara LU (2002) In Proteins 47(3). p.323-333
Abstract
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 D-9k. 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... (More)
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 D-9k. 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 IgK = 4.6-5.3. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Molecular, Models, Biological, Ligands, EF Hand Motifs, Dimerization, Cyanogen Bromide : chemistry, Comparative Study, Circular Dichroism, Cattle, Vitamin D-Dependent : metabolism, Calcium-Binding Protein, Vitamin D-Dependent : chemistry, Calcium : metabolism, Binding Sites, Amino Acid Sequence, Animal, Molecular Sequence Data, Peptide Fragments : chemistry, Protein Binding, Sequence Alignment, Spectrometry, Fluorescence
in
Proteins
volume
47
issue
3
pages
323 - 333
publisher
John Wiley & Sons
external identifiers
  • scopus:0037093642
  • wos:000175265700008
  • pmid:11948786
ISSN
0887-3585
DOI
10.1002/prot.10080
language
English
LU publication?
yes
id
5becffc5-fdf0-463b-96b4-a1320e595234 (old id 107547)
date added to LUP
2007-06-29 11:50:47
date last changed
2017-01-01 06:58:03
@article{5becffc5-fdf0-463b-96b4-a1320e595234,
  abstract     = {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 D-9k. 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 IgK = 4.6-5.3.},
  author       = {Julenius, Karin and Robblee, James and Thulin, Eva and Finn, Bryan E and Fairman, Robert and Linse, Sara},
  issn         = {0887-3585},
  keyword      = {Molecular,Models,Biological,Ligands,EF Hand Motifs,Dimerization,Cyanogen Bromide : chemistry,Comparative Study,Circular Dichroism,Cattle,Vitamin D-Dependent : metabolism,Calcium-Binding Protein,Vitamin D-Dependent : chemistry,Calcium : metabolism,Binding Sites,Amino Acid Sequence,Animal,Molecular Sequence Data,Peptide Fragments : chemistry,Protein Binding,Sequence Alignment,Spectrometry,Fluorescence},
  language     = {eng},
  number       = {3},
  pages        = {323--333},
  publisher    = {John Wiley & Sons},
  series       = {Proteins},
  title        = {Coupling of ligand binding and dimerization of helix-loop-helix peptides: Spectroscopic and sedimentation analyses of calbindin D-9k EF-hands},
  url          = {http://dx.doi.org/10.1002/prot.10080},
  volume       = {47},
  year         = {2002},
}