Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1.
Phototropins (phot1 and phot2) are blue light receptor kinases that control a range of photoresponses that serve to optimize the photosynthetic efficiency of plants. Light sensing by the phototropins is mediated by a repeated motif at the N-terminal region of the protein known as the LOV domain. Bacterially expressed LOV domains bind flavin mononucleotide noncovalently and are photochemically active in solution. Irradiation of the LOV domain results in the formation of a flavin-cysteinyl adduct (LOV390) which thermally relaxes back to the ground state in the dark, effectively completing a photocycle that serves as a molecular switch to control receptor kinase activity. We have employed a random mutagenesis approach to identify further amino acid residues involved in LOV-domain photochemistry. Escherichia coli colonies expressing a mutagenized population of LOV2 derived from Avena sativa (oat) phot1 were screened for variants that showed altered photochemical reactivity in response to blue light excitation. One variant showed slower rates of LOV390 formation but exhibited adduct decay times 1 order of magnitude faster than wild type. A single Ile --> Val substitution was responsible for the effects observed, which removes a single methyl group found in van der Waals contact with the cysteine sulfur involved in adduct formation. A kinetic acceleration trend was observed for adduct decay by decreasing the size of the isoleucine side chain. Our findings therefore indicate that the steric nature of this amino acid side chain contributes to stabilization of the C-S cysteinyl adduct.
Christie
John M
JM
Plant Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, University Avenue, Glasgow, Scotland, UK. J.Christie@bio.gla.ac.uk
Corchnoy
Stephanie B
SB
Swartz
Trevor E
TE
Hokenson
Mark
M
Han
In-Seob
IS
Briggs
Winslow R
WR
Bogomolni
Roberto A
RA
D001120:Arginine / Q000235:genetics; D002942:Circular Dichroism; D056931:Cryptochromes; D003545:Cysteine / Q000235:genetics; D019020:Directed Molecular Evolution; D004553:Electric Conductivity; D004926:Escherichia coli / Q000235:genetics; D005420:Flavoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007532:Isoleucine / Q000235:genetics; D008027:Light; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000502:physiology*; D010777:Photochemistry; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D014633:Valine / Q000235:genetics
D056931:Cryptochromes; D005420:Flavoproteins; D010940:Plant Proteins; D007532:Isoleucine; D001120:Arginine; D014633:Valine; D008239:Lysine; D003545:Cysteine; D010100:Oxygen