Probing the E/K Peptide Coiled-Coil Assembly by Double Electron–Electron Resonance and Circular Dichroism

Biochemistry, vol. 60, pp. 19–30

Abstract

Double electron-electron resonance (DEER, also known as PELDOR) and circular dichroism (CD) spectroscopies were explored for the purpose of studying the specificity of the conformation of peptides induced by their assembly into a self-recognizing system. The E and K peptides are known to form a coiled-coil heterodimer. Two paramagnetic TOAC α-amino acid residues were incorporated into each of the peptides (denoted as K** and E**), and a three-dimensional structural investigation in the presence or absence of their unlabeled counterparts E and K was performed. The TOAC spin-labels, replacing two Ala residues in each compound, are covalently and quasi-rigidly connected to the peptide backbone. They are known not to disturb the native structure, so that any conformational change can easily be monitored and assigned. DEER spectroscopy enables the measurement of the intramolecular electron spin-spin distance distribution between the two TOAC labels, within a length range of 1.5-8 nm. This method allows the individual conformational changes for the K**, K**/E, E**, and E**/K molecules to be investigated in glassy frozen solutions. Our data reveal that the conformations of the E** and K** peptides are strongly influenced by the presence of their counterparts. The results are discussed with those from CD spectroscopy and with reference to the already reported nuclear magnetic resonance data. We conclude that the combined DEER/TOAC approach allows us to obtain accurate and reliable information about the conformation of the peptides before and after their assembly into coiled-coil heterodimers. Applications of this induced fit method to other two-component, but more complex, systems, like a receptor and antagonists, a receptor and a hormone, and an enzyme and a ligand, are discussed.

Authors 9

1. Elena A. Golysheva

   Novosibirsk State University · Institute of Chemical Kinetics and Combustion

   Affiliation as printed

   Novosibirsk State University, Novosibirsk 630090, Russian Federation

   V. V. Voevodsky Institute of Chemical Kinetics and Combustion, Novosibirsk 630090, Russian Federation

2. Aimee L. Boyle Aachen Leiden Institute of Chemistry, Gorlaeus Laboratories

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands

3. Barbara Biondi

   University of Padua

   Affiliation as printed

   Department of Chemical Sciences, University of Padova, 35131 Padova, Italy

   Institute of Biomolecular Chemistry, Padova Unit, CNR, 35131 Padova, Italy

4. Paolo Ruzza

   University of Padua

   Affiliation as printed

   Department of Chemical Sciences, University of Padova, 35131 Padova, Italy

   Institute of Biomolecular Chemistry, Padova Unit, CNR, 35131 Padova, Italy

5. Alexander Kros Aachen Leiden Institute of Chemistry, Gorlaeus Laboratories

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands

6. Jan Raap corresponding Aachen Leiden Institute of Chemistry, Gorlaeus Laboratories

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands

7. Claudio Toniolo

   University of Padua

   Affiliation as printed

   Department of Chemical Sciences, University of Padova, 35131 Padova, Italy

   Department of Chemistry, University of Padova, 35131 Padova, Italy

   Institute of Biomolecular Chemistry, Padova Unit, CNR, 35131 Padova, Italy

8. Fernando Formaggio

   University of Padua

   Affiliation as printed

   Department of Chemical Sciences, University of Padova, 35131 Padova, Italy

   Institute of Biomolecular Chemistry, Padova Unit, CNR, 35131 Padova, Italy

9. Sergei A. Dzuba

   Novosibirsk State University · Institute of Chemical Kinetics and Combustion

   Affiliation as printed

   Novosibirsk State University, Novosibirsk 630090, Russian Federation

   V. V. Voevodsky Institute of Chemical Kinetics and Combustion, Novosibirsk 630090, Russian Federation