Coupling of ligand binding and dimerization of helix-loop-helix peptides: Spectroscopic and sedimentation analyses of calbindin D-9k EF-hands
(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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https://lup.lub.lu.se/record/107547
- author
- Julenius, Karin LU ; Robblee, James ; Thulin, Eva LU ; Finn, Bryan E ; Fairman, Robert and Linse, Sara LU
- organization
- publishing date
- 2002
- 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}}, }