Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675

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• 作者：Patrick E. Konold ; Eunjin Yoon ; Junghwa Lee ; Samantha L. Allen ; Prem P. Chapagain ; Bernard S. Gerstman ; Chola K. Regmi ; Kiryl D. Piatkevich ; Vladislav V. Verkhusha ; Taiha Joo ; Ralph Jimenez
• 刊名：The Journal of Physical Chemistry Letters
• 出版年：2016
• 出版时间：August 4, 2016
• 年：2016
• 卷：7
• 期：15
• 页码：3046-3051
• 全文大小：397K
• 年卷期：0
• ISSN：1948-7185

文摘

Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S₁ lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift.