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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
; ; ; ; and
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 Inc.
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
2016-04-01 16:10:54
date last changed
2022-01-28 17:55:25
@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}},
  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}},
  language     = {{eng}},
  number       = {{3}},
  pages        = {{323--333}},
  publisher    = {{John Wiley & Sons Inc.}},
  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}},
  doi          = {{10.1002/prot.10080}},
  volume       = {{47}},
  year         = {{2002}},
}