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Conformational Ensembles of Calmodulin Revealed by Nonperturbing Site-Specific Vibrational Probe Groups

Kelly, Kristen L.; Dalton, Shannon R.; Wai, Rebecca B.; Ramchandani, Kanika; Xu, Rosalind J.; Linse, Sara LU and Londergan, Casey H. (2018) In Journal of Physical Chemistry A 122(11). p.2947-2955
Abstract

Seven native residues on the regulatory protein calmodulin, including three key methionine residues, were replaced (one by one) by the vibrational probe amino acid cyanylated cysteine, which has a unique CN stretching vibration that reports on its local environment. Almost no perturbation was caused by this probe at any of the seven sites, as reported by CD spectra of calcium-bound and apo calmodulin and binding thermodynamics for the formation of a complex between calmodulin and a canonical target peptide from skeletal muscle myosin light chain kinase measured by isothermal titration. The surprising lack of perturbation suggests that this probe group could be applied directly in many protein-protein binding interfaces. The infrared... (More)

Seven native residues on the regulatory protein calmodulin, including three key methionine residues, were replaced (one by one) by the vibrational probe amino acid cyanylated cysteine, which has a unique CN stretching vibration that reports on its local environment. Almost no perturbation was caused by this probe at any of the seven sites, as reported by CD spectra of calcium-bound and apo calmodulin and binding thermodynamics for the formation of a complex between calmodulin and a canonical target peptide from skeletal muscle myosin light chain kinase measured by isothermal titration. The surprising lack of perturbation suggests that this probe group could be applied directly in many protein-protein binding interfaces. The infrared absorption bands for the probe groups reported many dramatic changes in the probes' local environments as CaM went from apo- to calcium-saturated to target peptide-bound conditions, including large frequency shifts and a variety of line shapes from narrow (interpreted as a rigid and invariant local environment) to symmetric to broad and asymmetric (likely from multiple coexisting and dynamically exchanging structures). The fast intrinsic time scale of infrared spectroscopy means that the line shapes report directly on site-specific details of calmodulin's variable structural distribution. Though quantitative interpretation of the probe line shapes depends on a direct connection between simulated ensembles and experimental data that does not yet exist, formation of such a connection to data such as that reported here would provide a new way to evaluate conformational ensembles from data that directly contains the structural distribution. The calmodulin probe sites developed here will also be useful in evaluating the binding mode of calmodulin with many uncharacterized regulatory targets.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Physical Chemistry A
volume
122
issue
11
pages
9 pages
publisher
The American Chemical Society (ACS)
external identifiers
  • scopus:85044403286
ISSN
1089-5639
DOI
10.1021/acs.jpca.8b00475
language
English
LU publication?
yes
id
45489c3d-446f-49ec-8df5-8d4b3026d1ab
date added to LUP
2018-05-22 09:31:27
date last changed
2019-10-15 06:38:02
@article{45489c3d-446f-49ec-8df5-8d4b3026d1ab,
  abstract     = {<p>Seven native residues on the regulatory protein calmodulin, including three key methionine residues, were replaced (one by one) by the vibrational probe amino acid cyanylated cysteine, which has a unique CN stretching vibration that reports on its local environment. Almost no perturbation was caused by this probe at any of the seven sites, as reported by CD spectra of calcium-bound and apo calmodulin and binding thermodynamics for the formation of a complex between calmodulin and a canonical target peptide from skeletal muscle myosin light chain kinase measured by isothermal titration. The surprising lack of perturbation suggests that this probe group could be applied directly in many protein-protein binding interfaces. The infrared absorption bands for the probe groups reported many dramatic changes in the probes' local environments as CaM went from apo- to calcium-saturated to target peptide-bound conditions, including large frequency shifts and a variety of line shapes from narrow (interpreted as a rigid and invariant local environment) to symmetric to broad and asymmetric (likely from multiple coexisting and dynamically exchanging structures). The fast intrinsic time scale of infrared spectroscopy means that the line shapes report directly on site-specific details of calmodulin's variable structural distribution. Though quantitative interpretation of the probe line shapes depends on a direct connection between simulated ensembles and experimental data that does not yet exist, formation of such a connection to data such as that reported here would provide a new way to evaluate conformational ensembles from data that directly contains the structural distribution. The calmodulin probe sites developed here will also be useful in evaluating the binding mode of calmodulin with many uncharacterized regulatory targets.</p>},
  author       = {Kelly, Kristen L. and Dalton, Shannon R. and Wai, Rebecca B. and Ramchandani, Kanika and Xu, Rosalind J. and Linse, Sara and Londergan, Casey H.},
  issn         = {1089-5639},
  language     = {eng},
  month        = {03},
  number       = {11},
  pages        = {2947--2955},
  publisher    = {The American Chemical Society (ACS)},
  series       = {Journal of Physical Chemistry A},
  title        = {Conformational Ensembles of Calmodulin Revealed by Nonperturbing Site-Specific Vibrational Probe Groups},
  url          = {http://dx.doi.org/10.1021/acs.jpca.8b00475},
  volume       = {122},
  year         = {2018},
}