In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells.

Gött-Zink L, Klocke J, Bogeholz L, Kottke T (2020)
The Journal of biological chemistry.

Zeitschriftenaufsatz | E-Veröff. vor dem Druck | Englisch
 
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Abstract / Bemerkung
Proteins are usually studied in well-defined buffer conditions, which differ substantially from those within a host cell. In some cases, the intracellular environment has an impact on the mechanism, which might be missed by in vitro experiments. Infrared difference spectroscopy previously has been applied to study the light-induced response of photoreceptors and photoenzymes in vitro Here, we established the in-cell infrared difference (ICIRD) spectroscopy in the transmission and attenuated total reflection (ATR) configuration to investigate the light-induced response of soluble proteins in living bacterial cells. ICIRD spectroscopy on the light, oxygen, or voltage (LOV) domains of the blue light receptors aureochrome and phototropin revealed a suppression of the response of specific secondary structure elements, indicating that the intracellular environment affects LOV photoreceptor mechanisms in general. Moreover, in-cell fluorescence spectroscopy disclosed that the intracellular environment slows down the recovery of the light-induced flavin adduct. Segment-resolved ICIRD spectroscopy on basic-region leucine zipper (bZIP)-LOV of aureochrome 1a from the diatom Phaeodactylum tricornutum indicated a signal progression from the LOV sensor to the bZIP effector independent of unfolding of the connecting A'alpha-helix, an observation that stood in contrast to in vitro results. This deviation was recapitulated in vitro by emulating the intracellular environment through addition of the crowding agent bovine serum albumin, but not by sucrose polymers. We conclude that ICIRD spectroscopy is a non-invasive, label-free approach for assessing conformational changes in receptors in living cells at ambient conditions. As demonstrated, these near-native responses may deviate from the mechanisms established under in vitro conditions. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
Erscheinungsjahr
2020
Zeitschriftentitel
The Journal of biological chemistry
eISSN
1083-351X
Page URI
https://pub.uni-bielefeld.de/record/2944312

Zitieren

Gött-Zink L, Klocke J, Bogeholz L, Kottke T. In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells. The Journal of biological chemistry. 2020.
Gött-Zink, L., Klocke, J., Bogeholz, L., & Kottke, T. (2020). In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells. The Journal of biological chemistry. doi:10.1074/jbc.RA120.013091
Gött-Zink, L., Klocke, J., Bogeholz, L., and Kottke, T. (2020). In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells. The Journal of biological chemistry.
Gött-Zink, L., et al., 2020. In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells. The Journal of biological chemistry.
L. Gött-Zink, et al., “In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells.”, The Journal of biological chemistry, 2020.
Gött-Zink, L., Klocke, J., Bogeholz, L., Kottke, T.: In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells. The Journal of biological chemistry. (2020).
Gött-Zink, Lukas, Klocke, Jessica, Bogeholz, Lena, and Kottke, Tilman. “In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells.”. The Journal of biological chemistry (2020).

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