Vibrational Assignment of the Ultrafast Infrared Spectrum of the Photoactivatable Flavoprotein AppA

详细信息    查看全文

• 作者：Allison Haigney ; Andras Lukacs ; Richard Brust ; Rui-Kun Zhao ; Michael Towrie ; Gregory M. Greetham ; Ian Clark ; Boris Illarionov ; Adelbert Bacher ; Ryu-Ryun Kim ; Markus Fischer ; Stephen R. Meech ; Peter J. Tonge
• 刊名：The Journal of Physical Chemistry B
• 出版年：2012
• 出版时间：September 6, 2012
• 年：2012
• 卷：116
• 期：35
• 页码：10722-10729
• 全文大小：500K
• 年卷期：v.116,no.35(September 6, 2012)
• ISSN：1520-5207

文摘

The blue light using flavin (BLUF) domain proteins, such as the transcriptional antirepressor AppA, are a novel class of photosensors that bind flavin noncovalently in order to sense and respond to high-intensity blue (450 nm) light. Importantly, the noncovalently bound flavin chromophore is unable to undergo large-scale structural change upon light absorption, and thus there is significant interest in understanding how the BLUF protein matrix senses and responds to flavin photoexcitation. Light absorption is proposed to result in alterations in the hydrogen-bonding network that surrounds the flavin chromophore on an ultrafast time scale, and the structural changes caused by photoexcitation are being probed by vibrational spectroscopy. Here we report ultrafast time-resolved infrared spectra of the AppA BLUF domain (AppA_BLUF) reconstituted with isotopes of FAD, specifically [U-¹³C₁₇]-FAD, [xylene-¹³C₈]-FAD, [U-¹⁵N₄]-FAD, and [4-¹⁸O₁]-FAD both in solution and bound to AppA_BLUF. This allows for unambiguous assignment of ground- and excited-state modes arising directly from the flavin. Studies of model compounds and DFT calculations of the ground-state vibrational spectra reveal the sensitivity of these modes to their environment, indicating they can be used as probes of structural dynamics.