Conformational heterogeneity and propagation of structural changes in the LOV2/Jalpha domain from Avena sativa phototropin 1 as recorded by temperature-dependent FTIR spectroscopy.
Phototropins control phototropism, chloroplast movement, stomatal opening, and leaf expansion in plants. Phototropin 1 (phot1) is composed of a kinase domain linked to two blue light-sensing domains, LOV2 and LOV1, which bind flavin mononucleotide. Disruption of the interaction between the LOV2 domain and a helical segment named Jalpha, joining LOV to the kinase domain, induces the subsequent kinase activity of phototropin 1 and further-downstream signal transduction. Here we study the effects of temperature and hydration on the light-triggered signal propagation in the phot1 LOV2 domain of Avena sativa (AsLOV2/Jalpha), using Fourier transform infrared spectroscopy to unravel part of the molecular mechanism of phototropin 1. We report that AsLOV2/Jalpha shows an intense signal in the amide I and II regions, arising mainly from beta-sheet changes and the unbinding of the Jalpha helix from the Per-ARNT-Sim core and its subsequent partial unfolding. Importantly, these structural changes only occur under conditions of full hydration and at temperatures above 280 K. We characterized a newly isolated low-hydration intermediate that shows a downshift of high-frequency amide I signals and that possibly corresponds to loop tightening, without large beta-sheet or Jalpha structural changes. In addition, we report a heterogeneity in AsLOV2/Jalpha involving two different C(4)=O conformer populations, coexisting in the dark state and characterized by C(4)=O carbonyl frequencies at 1712 cm(-1) and 1694 cm(-1) that are attributable to a single H-bond and two H-bonds at this site, respectively. Such conformers display slightly shifted absorption spectra and cause a splitting of the 475-nm band in the ultraviolet/visible spectra of LOV domains at low temperature.
Alexandre
Maxime T A
MT
Department of Biophysics, Faculty of Sciences, Vrije Universiteit, 1081HV Amsterdam, The Netherlands.
van Grondelle
Rienk
R
Hellingwerf
Klaas J
KJ
Kennis
John T M
JT
D018554:Avena; D056931:Cryptochromes; D005420:Flavoproteins / Q000737:chemistry*; D006358:Hot Temperature; D006860:Hydrogen Bonding; D008027:Light; D008956:Models, Chemical; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D014867:Water / Q000737:chemistry
D056931:Cryptochromes; D005420:Flavoproteins; D010940:Plant Proteins; D014867:Water