Mapping Protein Interfaces by Chemical Cross-Linking and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Application to a Calmodulin/Adenylyl Cyclase 8 Peptide Complex

Abstract

Chemical cross-linking—an established technique in protein chemistry—has re-emerged, in combination with mass spectrometric analysis of the reaction products, as a valuable tool to identify interacting amino acid sequences in protein complexes. In the present study, we are mapping the interface of the calcium-dependent complex between calmodulin (CaM) and a peptide derived from the C-terminal region of adenylyl cyclase 8 (AC 8). Cross-linking reactions are performed using the two amine-reactive, isotope-labeled (d₀ and d₄) cross-linkers BS³ (bis[sulfosuccinimidyl]suberate) and BS²G (bis[sulfosuccinimidyl]glutarate) as well as the “zero-length” cross-linker (EDC, ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride). After separation of the cross-linking reaction mixtures by one-dimensional gel electrophoresis (sodium dodecyl sulphate polyacrylamide gel) and in-gel digestion of the cross-linked complexes, the resulting peptide mixtures are analyzed by nano-high-performance liquid chromatography/nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The identified intermolecular cross-linking products will give further insight into calmodulin/adenylyl cyclase 8 interaction.

Keywords

nano-HPLC ion cyclotron resonance mass spectrometry protein interaction chemical cross-linking isotope-labeled cross-linkers calmodulin adenylyl cyclase

Get full access to this article

View all access options for this article.

References

Engen

J.R.

Smith

D.L.

, in Methods in Molecular Biology, Vo l. 146: Mass Spectrometry of Proteins and Peptides, Ed by Chapman

J.R.

, Humana Press Inc., Totowa, New Jersey, USA, p. 95 (2000).

Loo

J.A.

, “Studying noncovalent protein complexes by electrospray ionization mass spectrometry”, Mass Spectrom. Rev. 16, 1 (1997).

Sinz

, “Chemical Cross-Linking and Mass Spectrometry for Mapping Three-Dimensional Structures of Proteins and Protein Complexes”, J. Mass Spectrom. 38, 1225 (2003).

Back

J.W.

de Jong

Muijsers

A.O.

de Koster

C.G.

, “Chemical cross-linking and mass spectrometry for protein structural modeling”, J. Mol. Biol. 331, 303 (2003).

Fenn

J.B.

Mann

Meng

C.K.

Wong

S.F.

Whitehouse

C.M.

, “Electrospray ionization for mass spectrometry of large biomolecules”, Science 46, 64 (1989).

Mann

Talbo

, “Developments in matrix-assisted laser desorption ionization peptide mass spectrometry”, Curr. Opin. Biotech. 7, 11 (1996).

Comisarov

M.B.

Marshall

A.G.

, “Fourier transform ion cyclotron resonance spectroscopy”, Chem. Phys. Lett. 25, 282 (1974) .

Marshall

A.G.

, “Milestones in Fourier transform ion cyclotron resonance spectrometry technique development”, Int. J. Mass Spectrom. 200, 331 (2000).

Dihazi

G.H.

Sinz

, “Mapping low-resolution three-dimensional protein structures using chemical cross-linking and Fourier transform ion-cyclotron resonance mass spectrometry”, Rapid Commun. Mass Spectrom. 17, 2005 (2003).

10.

Kruppa

G.H.

Schoeniger

Young

M.M.

, “A top down approach to protein structural studies using chemical cross-linking and Fourier transform mass spectrometry”, Rapid Commun. Mass Spectrom. 17, 155 (2003).

11.

Crivici

Ikura

, “Molecular and structural basis of target recognition by calmodulin”, Annu. Rev. Biophys. Biomol. Struct. 24, 85 (1995).

12.

Carafoli

, “Calcium signaling: A tale for all seasons”, Proc. Natl. Acad. Sci. USA 99, 1115 (2002).

13.

Babu

Y.S.

Sack

J.S.

Greenhough

T.J.

Bugg

C.E.

Means

A.R.

Cook

W.J.

, “3-Dimensional structure of calmodulin”, Nature 315, 37 (1985).

14.

Babu

Y.S.

Bugg

C.E.

Cook

W.J.

, “Structure of calmodulin refined at 2.2 Å resolution”, J. Mol. Biol. 204, 191 (1988).

15.

Persechini

Kretsinger

R.H.

, “The central helix of calmodulin functions as a flexible tether”, J. Biol. Chem. 263, 12175 (1988).

16.

Barbato

Ikura

Kay

M.L.E.

Pastor

R.W.

Bax

, “Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two dimensional NMR spectroscopy: The central helix is flexible”, Biochemistry 31, 5269 (1992).

17.

Comte

Maulet

Cox

A.J.

, “Ca²⁺-dependent high-affinity complex formation between calmodulin and melittin”, Biochem. J. 209, 269 (1983).

18.

Cooper

D.M.F.

, “Calmodulin-binding sites on adenylyl cyclase type VIII”, J. Biol. Chem. 274, 8012 (1999).

19.

Bähler

Rhoads

, “Calmodulin signaling via the IQ motif”, FEBS Lett. 513, 107 (2002).

20.

Espreafico

E.M.

Cheney

R.E.

Matteoli

Nascimento

A.A.C.

Decamilli

P.V.

Larson

R.E.

Mooseker

M.S.

, “Primary Structure and Cellular Localization of Chicken Brain Myosin-V (P190), an Unconventional Myosin with Calmodulin Light Chains”, J. Cell Biol. 119, 1541 (1992).

21.

Laemmli

U.K.

, “Cleavage of Structural Proteins During Assembly of Head of Bacteriophage-T4”, Nature 227, 680 (1970).

22.

Jensen

O.N.

Shevchenko

Mann

, in: Protein Structure, A Practical Approach, Ed by Creighton

T.E.

, Oxford University Press, Oxford, UK, p. 48 (1997).

23.

BioAPEX User's Manual, Vol. 1.1. Bruker Daltonics, Billerica, MA, USA (1996).

24.

Peri

Steen

Pandey

, “GPMAW—A software tool for analyzing proteins and peptides”, Trends Biochem. Sci. 26, 687 (2001).

25.

Schulz

D.M.

Ihling

Clore

G.M.

Sinz

, “Mapping the Topology and Determination of a Low Resolution Three-Dimensional Structure of the Calmodulin-Melittin Complex by Chemical Cross-Linking and High-Resolution FTICR Mass Spectrometry: Direct Demonstration of Multiple Binding Modes”, Biochemistry 43, 4703 (2004).

26.

Kalkhof

Ihling

Mechtler

Sinz

, “Chemical Cross-Linking and High-Performance Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Protein Interaction Analysis: Application to a Calmodulin/Target Peptide Complex”, Anal. Chem. 77, 495 (2005).

27.

Hermanson

G.T.

, Bioconjugate Techniques. Academic Press Inc., San Diego, USA (1996).

28.

Novak

Young

M.M.

Schoeniger

J.S.

Kruppa

G.H.

, “A top-down approach to protein structure studies using chemical cross-linking and Fourier transform mass spectrometry”, Eur. J. Mass Spectrom. 9, 623 (2003).

29.

Zubarev

R.A.

Kelleher

N.L.

McLafferty

F.W.

, “Electron capture dissociation of multiply charged protein cations. A nonergodic process”, J. Am. Chem. Soc. 120, 3265 (1998).