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European Biophysics Journal

, Volume 40, Issue 4, pp 565–576 | Cite as

The presence of membranes or micelles induces structural changes of the myristoylated guanylate-cyclase activating protein-2

  • Stephan Theisgen
  • Lars Thomas
  • Thomas Schröder
  • Christian Lange
  • Michael Kovermann
  • Jochen Balbach
  • Daniel Huster
Original Paper

Abstract

Guanylate cyclase-activating proteins (GCAPs) are neuronal Ca2+ sensors that play a central role in shaping the photoreceptor light response and in light adaptation through the Ca2+-dependent regulation of the transmembrane retinal guanylate cyclase. GCAPs are N-terminally myristoylated, and the role of the myristoyl moiety is not yet fully understood. While protein lipid chains typically represent membrane anchors, the crystal structure of GCAP-1 showed that the myristoyl chain of the protein is completely buried within a hydrophobic pocket of the protein, which stabilizes the protein structure. Therefore, we address the question of the localization of the myristoyl group of GCAP-2 in the absence and in the presence of lipid membranes as well as DPC detergents (as a membrane substitute amenable to solution state NMR). We investigate membrane binding of both myristoylated and nonmyristoylated GCAP-2 and study the structure and dynamics of the myristoyl moiety of GCAP-2 in the presence of POPC membranes. Further, we address structural alterations within the myristoylated N-terminus of GCAP-2 in the presence of membrane mimetics. Our results suggest that upon membrane binding the myristoyl group is released from the protein interior and inserts into the lipid bilayer.

Keywords

Lipid modification GCAP-2 Membrane-protein interaction Order parameter 

Abbreviations

GC

Guanylate cyclase

GCAP-2

Guanylate cyclase-activating protein-2

NCS

Neuronal calcium sensor

HSQC

Heteronuclear single quantum coherence

POPC

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

DPC

Dodecylphosphocholine

IPTG

Isopropyl β-d-1-thiogalactopyranoside

OD

Optical density

GMP

Guanosine-5′-monophosphate

cGMP

Cyclic GMP

GTP

Guanosine-5′-triphosphate

EDTA

Ethylenediaminetetraacetic acid

HPLC

High performance liquid chromatography

FID

Free induction decay

TFA

Trifluoroacetic acid

INEPT

Insensitive nuclei enhanced by polarization transfer

DTT

Dithiothreitol

MOPS

3-(N-morpholino)propanesulfonic acid

MES

2-(N-morpholino)ethanesulfonic acid

Tris

Tris(hydroxymethyl)aminomethane

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

PDB

Protein databank

NMR

Nuclear magnetic resonance

MWCO

Molecular weight cut-off

TCEP

Tris(2-carboxyethyl)phosphine

WATERGATE

Water suppression by gradient-tailored excitation

GARP

Globally optimized alternating phase rectangular pulse

WALTZ

Wideband alternating-phase low-power technique for zero-residual-splitting

Cmc

Critical micelle concentration

Notes

Acknowledgments

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, HU 720/10-1). M.K. and J.B. were supported by grants from DFG (SFB 610) and J.B. by the Exzellenznetzwerk Biowissenschaften of Sachsen-Anhalt. J.B. thanks the European Development Fund of the European Union.

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Copyright information

© European Biophysical Societies' Association 2011

Authors and Affiliations

  • Stephan Theisgen
    • 1
  • Lars Thomas
    • 1
  • Thomas Schröder
    • 2
  • Christian Lange
    • 2
  • Michael Kovermann
    • 3
  • Jochen Balbach
    • 3
  • Daniel Huster
    • 1
  1. 1.Institute of Medical Physics and BiophysicsUniversity of LeipzigLeipzigGermany
  2. 2.Institute of Biochemistry/BiotechnologyMartin-Luther-University Halle-WittenbergHalle (Saale)Germany
  3. 3.Biophysics Research GroupMartin-Luther-University Halle-WittenbergHalle (Saale)Germany

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