Limits...
Light-induced structural changes of the LOV2 domains in various phototropins revealed by FTIR spectroscopy

View Article: PubMed Central - PubMed

ABSTRACT

Phototropin (Phot), a blue-light photoreceptor in plants, consists of two FMN-binding domains (named LOV1 and LOV2) and a serine/threonine (Ser/Thr) kinase domain. We have investigated light-induced structural changes of LOV domains, which lead to the activation of the kinase domain, by means of light-induced difference FTIR spectroscopy. FTIR spectroscopy revealed that the reactive cysteine is protonated in both unphotolyzed and triplet-excited states, which is difficult to detect by other methods such as X-ray crystallography. In this review, we describe the light-induced structural changes of hydrogen-bonding environment of FMN chromophore and protein backbone in Adiantum neo1-LOV2 in the C=O stretching region by use of 13C-labeled samples. We also describe the comprehensive FTIR analysis of LOV2 domains among Arabidopsis phot1, phot2, and Adiantum neo1 with and without Jα helix domain.

No MeSH data available.


(a) Difference FTIR spectra of native neo1-LOV2 (dotted lines) and neo-LOV2 reconstituted with the FMN and apoprotein (solid lines) in the 1800–950 cm−1 region at 295K. (b and c) Difference FTIR spectra of neo1-LOV2 reconstituted with unlabeled FMN (solid lines), [4,10a-13C2]FMN (dotted line in b) and [2-13C]FMN (dotted line in c). (d and e) Double-difference spectra of those in Figure 3b and c, where the labeled spectra (dotted lines) are subtracted from the unlabeled spectra (solid lines). This figure is modified from Ref 51.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5036776&req=5

f3-7_89: (a) Difference FTIR spectra of native neo1-LOV2 (dotted lines) and neo-LOV2 reconstituted with the FMN and apoprotein (solid lines) in the 1800–950 cm−1 region at 295K. (b and c) Difference FTIR spectra of neo1-LOV2 reconstituted with unlabeled FMN (solid lines), [4,10a-13C2]FMN (dotted line in b) and [2-13C]FMN (dotted line in c). (d and e) Double-difference spectra of those in Figure 3b and c, where the labeled spectra (dotted lines) are subtracted from the unlabeled spectra (solid lines). This figure is modified from Ref 51.

Mentions: We first examined whether the FMN chromophore is properly reconstituted into the original binding site. Figure 3a compares difference FTIR spectra of the S390 and unphotolyzed states between the native (dotted lines) and reconstituted (solid lines) neo1-LOV2 measured at 295 K. The reconstituted neo1-LOV2 sample was prepared by mixing the unlabeled FMN and apoprotein as described in elsewhere51. Both spectra coincide very well with each other although the spectral deviation was observed. Figure 3a provides criteria of experimental accuracy in the following measurements.


Light-induced structural changes of the LOV2 domains in various phototropins revealed by FTIR spectroscopy
(a) Difference FTIR spectra of native neo1-LOV2 (dotted lines) and neo-LOV2 reconstituted with the FMN and apoprotein (solid lines) in the 1800–950 cm−1 region at 295K. (b and c) Difference FTIR spectra of neo1-LOV2 reconstituted with unlabeled FMN (solid lines), [4,10a-13C2]FMN (dotted line in b) and [2-13C]FMN (dotted line in c). (d and e) Double-difference spectra of those in Figure 3b and c, where the labeled spectra (dotted lines) are subtracted from the unlabeled spectra (solid lines). This figure is modified from Ref 51.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036776&req=5

f3-7_89: (a) Difference FTIR spectra of native neo1-LOV2 (dotted lines) and neo-LOV2 reconstituted with the FMN and apoprotein (solid lines) in the 1800–950 cm−1 region at 295K. (b and c) Difference FTIR spectra of neo1-LOV2 reconstituted with unlabeled FMN (solid lines), [4,10a-13C2]FMN (dotted line in b) and [2-13C]FMN (dotted line in c). (d and e) Double-difference spectra of those in Figure 3b and c, where the labeled spectra (dotted lines) are subtracted from the unlabeled spectra (solid lines). This figure is modified from Ref 51.
Mentions: We first examined whether the FMN chromophore is properly reconstituted into the original binding site. Figure 3a compares difference FTIR spectra of the S390 and unphotolyzed states between the native (dotted lines) and reconstituted (solid lines) neo1-LOV2 measured at 295 K. The reconstituted neo1-LOV2 sample was prepared by mixing the unlabeled FMN and apoprotein as described in elsewhere51. Both spectra coincide very well with each other although the spectral deviation was observed. Figure 3a provides criteria of experimental accuracy in the following measurements.

View Article: PubMed Central - PubMed

ABSTRACT

Phototropin (Phot), a blue-light photoreceptor in plants, consists of two FMN-binding domains (named LOV1 and LOV2) and a serine/threonine (Ser/Thr) kinase domain. We have investigated light-induced structural changes of LOV domains, which lead to the activation of the kinase domain, by means of light-induced difference FTIR spectroscopy. FTIR spectroscopy revealed that the reactive cysteine is protonated in both unphotolyzed and triplet-excited states, which is difficult to detect by other methods such as X-ray crystallography. In this review, we describe the light-induced structural changes of hydrogen-bonding environment of FMN chromophore and protein backbone in Adiantum neo1-LOV2 in the C=O stretching region by use of 13C-labeled samples. We also describe the comprehensive FTIR analysis of LOV2 domains among Arabidopsis phot1, phot2, and Adiantum neo1 with and without Jα helix domain.

No MeSH data available.