Hindered rotation of a cofactor methyl group as a probe for protein-cofactor interaction.
Exploring protein-cofactor interactions on a molecular level is one of the major challenges in modern biophysics. Based on structural data alone it is rarely possible to identify how subtle interactions between a protein and its cofactor modulate the protein's reactivity. In the case of enzymatic processes in which paramagnetic molecules play a certain role, EPR and related methods such as ENDOR are suitable techniques to unravel such important details. In this contribution, we describe how cryogenic-temperature ENDOR spectroscopy can be applied to various LOV domains, the blue-light sensing domains of phototropin photoreceptors, to gain information on the direct vicinity of the flavin mononucleotide (FMN) cofactor by analyzing the temperature dependence of methyl-group rotation attached to C(8) of the FMN's isoalloxazine ring. More specifically, mutational studies of three amino acids surrounding the methyl group led to the identification of Asn425 as an important amino acid that critically influences the dark-state recovery of Avena sativa LOV2 domains. Consequently, it is possible to probe protein-cofactor interactions on a sub-angstrom level by following the temperature dependencies of hyperfine couplings.
Brosi
Richard
R
Fachbereich Physik, Institut für Experimentalphysik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany.
Illarionov
Boris
B
Mathes
Tilo
T
Fischer
Markus
M
Joshi
Monika
M
Bacher
Adelbert
A
Hegemann
Peter
P
Bittl
Robert
R
Weber
Stefan
S
Schleicher
Erik
E
D018554:Avena; D016825:Chlamydomonas reinhardtii; D003067:Coenzymes / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005486:Flavin Mononucleotide / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D056949:Phototropins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012399:Rotation
D003067:Coenzymes; D056949:Phototropins; D005486:Flavin Mononucleotide