Water Structural Changes in the L and M Photocycle Intermediates of Bacteriorhodopsin as Revealed by Time-Resolved Step-Scan Fourier Transform Infrared (FTIR) Spectroscopy

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• 作者：Joel E. Morgan ; Ahmet S. Vakkasoglu ; Robert B. Gennis ; Akio Maeda
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：March 13, 2007
• 年：2007
• 卷：46
• 期：10
• 页码：2787 - 2796
• 全文大小：213K
• 年卷期：v.46,no.10(March 13, 2007)
• ISSN：1520-4995

文摘

In previous Fourier transform infrared (FTIR) studies of the photocycle intermediates ofbacteriorhodopsin at cryogenic temperatures, water molecules were observed in the L intermediate, in theregion surrounded by protein residues between the Schiff base and Asp96. In the M intermediate, thewater molecules had moved away toward the Phe219-Thr46 region. To evaluate the relevance of thisscheme at room temperature, time-resolved FTIR difference spectra of bacteriorhodopsin, including thewater O-H stretching vibration frequency regions, were recorded in the micro- and millisecond timeranges. Vibrational changes of weakly hydrogen-bonded water molecules were observed in L, M, and N.In each of these intermediates, the depletion of a water O-H stretching vibration at 3645 cm^-1, originatingfrom the initial unphotolyzed bacteriorhodopsin, was observed as a trough in the difference spectrum.This vibration is due to the dangling O-H group of a water molecule, which interacts with Asp85, andits absence in each of these intermediates indicates that there is perturbation of this O-H group. Theformation of M is accompanied by the appearance of water O-H stretching vibrations at 3670 and 3657cm^-1, the latter of which persists to N. The 3670 cm^-1 band of M is due to water molecules present inthe region surrounded by Thr46, Asp96, and Phe219. The formation of L at 298 K is accompanied by theperturbations of Asp96 and the Schiff base, although in different ways from what is observed at 170 K.Changes in a broad water vibrational feature, centered around 3610 cm^-1, are kinetically correlated withthe L-M transition. These results imply that, even at room temperature, water molecules interact withAsp96 and the Schiff base in L, although with a less rigid structure than at cryogenic temperatures.