Skip to main content

Lund University Publications

LUND UNIVERSITY LIBRARIES

Förster resonance energy transfer studies of calmodulin produced by native protein ligation reveal inter-domain electrostatic repulsion.

Hellstrand, Erik LU ; Kukora, Stephanie ; Shuman, Cynthia LU ; Steenbergen, Sara ; Thulin, Eva LU ; Kohli, Anita ; Krouse, Beth ; Linse, Sara LU and Akerfeldt, Karin S (2013) In The FEBS Journal 280(11). p.2675-2687
Abstract
This study explores the influence of long-range intra-protein electrostatic interactions on the conformation of calmodulin in solution. Ensemble Förster resonance energy transfer (FRET) is measured for calmodulin with a fluorophore pair incorporated specifically with a donor at residue 17 and an acceptor at position 117. This construct was generated by a combination of solid phase peptide synthesis, cloning, expression and native chemical ligation. This labelling method has not previously been used with calmodulin and represents a convenient method for ensuring the explicit positioning of the fluorophores. The ensemble FRET experiments reveal significant electrostatic repulsion between the globular domains in the calcium-free protein. At... (More)
This study explores the influence of long-range intra-protein electrostatic interactions on the conformation of calmodulin in solution. Ensemble Förster resonance energy transfer (FRET) is measured for calmodulin with a fluorophore pair incorporated specifically with a donor at residue 17 and an acceptor at position 117. This construct was generated by a combination of solid phase peptide synthesis, cloning, expression and native chemical ligation. This labelling method has not previously been used with calmodulin and represents a convenient method for ensuring the explicit positioning of the fluorophores. The ensemble FRET experiments reveal significant electrostatic repulsion between the globular domains in the calcium-free protein. At low salt, calmodulin has a relatively extended conformation and the distance between the domains is further increased by denaturation, by heat or by non-ionic denaturants. The repulsion between domains is screened by salt and is also diminished by calcium binding, which changes the protein net charge from -23 to -15. Compared with the calcium-free form at low salt, the FRET efficiency for the calcium-bound form has, on average, increased 10-fold. The conformation of the calcium form is insensitive to salt screening. These results imply that when the two globular domains of calmodulin interact with target, there is no significant free energy penalty due to electrostatic interactions. (Less)
Please use this url to cite or link to this publication:
author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
The FEBS Journal
volume
280
issue
11
pages
2675 - 2687
publisher
Wiley-Blackwell
external identifiers
  • wos:000319413400012
  • pmid:23552119
  • scopus:84878257862
  • pmid:23552119
ISSN
1742-464X
DOI
10.1111/febs.12269
language
English
LU publication?
yes
id
717870a8-2396-4a9c-b74b-d5fd01a1d64a (old id 3734241)
date added to LUP
2016-04-01 10:37:37
date last changed
2022-04-04 19:48:46
@article{717870a8-2396-4a9c-b74b-d5fd01a1d64a,
  abstract     = {{This study explores the influence of long-range intra-protein electrostatic interactions on the conformation of calmodulin in solution. Ensemble Förster resonance energy transfer (FRET) is measured for calmodulin with a fluorophore pair incorporated specifically with a donor at residue 17 and an acceptor at position 117. This construct was generated by a combination of solid phase peptide synthesis, cloning, expression and native chemical ligation. This labelling method has not previously been used with calmodulin and represents a convenient method for ensuring the explicit positioning of the fluorophores. The ensemble FRET experiments reveal significant electrostatic repulsion between the globular domains in the calcium-free protein. At low salt, calmodulin has a relatively extended conformation and the distance between the domains is further increased by denaturation, by heat or by non-ionic denaturants. The repulsion between domains is screened by salt and is also diminished by calcium binding, which changes the protein net charge from -23 to -15. Compared with the calcium-free form at low salt, the FRET efficiency for the calcium-bound form has, on average, increased 10-fold. The conformation of the calcium form is insensitive to salt screening. These results imply that when the two globular domains of calmodulin interact with target, there is no significant free energy penalty due to electrostatic interactions.}},
  author       = {{Hellstrand, Erik and Kukora, Stephanie and Shuman, Cynthia and Steenbergen, Sara and Thulin, Eva and Kohli, Anita and Krouse, Beth and Linse, Sara and Akerfeldt, Karin S}},
  issn         = {{1742-464X}},
  language     = {{eng}},
  number       = {{11}},
  pages        = {{2675--2687}},
  publisher    = {{Wiley-Blackwell}},
  series       = {{The FEBS Journal}},
  title        = {{Förster resonance energy transfer studies of calmodulin produced by native protein ligation reveal inter-domain electrostatic repulsion.}},
  url          = {{http://dx.doi.org/10.1111/febs.12269}},
  doi          = {{10.1111/febs.12269}},
  volume       = {{280}},
  year         = {{2013}},
}