Identification of the CO Stretching Vibrations of FMN and Peptide Backbone by ¹³C-Labeling of the LOV2 Domain of Adiantum Phytochrome3

Abstract

Phototropin, a blue-light photoreceptor in plants, has two FMN-binding domains named LOV1 and LOV2. We previously observed temperature-dependent FTIR spectral changes in the C=O stretching region (amide-I vibrational region of the peptide backbone) for the LOV2 domain of Adiantum phytochrome3 (phy3-LOV2), suggesting progressive structural changes in the protein moiety (Iwata, T., Nozaki, D., Tokutomi, S., Kagawa, T., Wada, M., and Kandori, H. (2003) Biochemistry 42, 8183-8191). Because FMN also possesses two C=O groups, in this article, we aimed at assigning C=O stretching vibrations of the FMN and protein by using 13C-labeling. We assigned the C(4)=O and C(2)=O stretching vibrations of FMN by using [4,10a-13C2] and [2-13C] FMNs, respectively, whereas C=O stretching vibrations of amide-I were assigned by using 13C-labeling of protein. We found that both C(4)=O and C(2)=O stretching vibrations shift to higher frequencies upon the formation of S390 at 77-295 K, suggesting that the hydrogen bonds of the C=O groups are weakened by adduct formation. Adduct formation presumably relocates the FMN chromophore apart from its hydrogen-bonding donors. Temperature-dependent amide-I bands are unequivocally assigned by separating the chromophore bands. The hydrogen bond of the peptide backbone in the loop region is weakened upon S390 formation at low temperatures, while being strengthened at room temperature. The hydrogen bond of the peptide backbone in the alpha-helix is weakened regardless of temperature. On the other hand, structural perturbation of the beta-sheet is observed only at room temperature, where the hydrogen bond is strengthened. Light-signal transduction by phy3-LOV2 must be achieved by the progressive protein structural changes initiated by the adduct formation of the FMN.

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“…This frequency has not been observed experimentally for the Z form of glycine. However, a frequency lying in the range 1,641–1,686 cm −1 is observed in different peptides and is assigned to CO stretching 60 The frequency calculated by us corresponding to CO 2 asymmetric stretching vibration for gly. (H 2 O) 3 lies near the lower end of the aforementioned experimentally observed range.…”

“…Vibrational frequencies of N and Z glycine obtained without specific solvent effect and without geometry optimization in aqueous media have been discussed by previous authors 13, 53, 55, 57–60. Here, we would mainly discuss the vibrational frequencies of Z glycine obtained by inclusion of both specific and bulk solvent effects under full geometry optimization in aqueous media.…”

“…The present calculated unscaled vibrational frequencies and their assignments for Z glycine and its complexes with one, two, and three water molecules obtained with full geometry optimization in aqueous media are presented in Table I. The theoretically calculated 13, 57 and experimentally observed 59, 60 vibrational frequencies by other authors are also presented in this table for comparison. The calculated vibrational frequencies from one of the previous studies 57 are scaled by the factor 0.9 while those from another work 13 are unscaled.…”

Fourier-Transform Infrared Study of the Photoactivation Process of Xenopus (6–4) Photolyase

“…We finally obtained the FAD •− minus FAD ox , the FADH • minus FAD ox , and the FADH − minus FAD ox spectra, from which any difference spectrum among the four redox states can be calculated. Detailed structural analysis requires band assignments using isotope-labeling and site-directed mutagenesis, as we performed for bacteriorhodopsin 44,45 and LOV domain, 46,47 while we discuss the molecular mechanism on the activation of (6-4) PHR based on the present FTIR results below.…”

Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain

“…They recorded changes in structure between dark and signalling states, including significant structural perturbation in the β -sheet, associated with an initial reorientation of a highly conserved glutamine residue, with subsequent unfolding of the J α -helix. 19, 23-24 Using step scan FTIR, Heberle and co-workers observed distinct phases in the LOV domain temporal response to formation of A 390 , ranging from microseconds to several milliseconds. 25-26 Other relevant time resolved studies of LOV domains include IR studies on the picosecond timescale and electronic spectroscopy on the femtosecond to nanosecond time domain, which resolved triplet formation and decay.…”