Photocycling study techniques

As demonstrated, different time-resolved FTIR techniques allow to study the complete photocycle of bacteriorhodopsin in the entire range from picoseconds to several milliseconds. Infrared difference spectra trace reactions which take place in different parts of the protein molecule. Isotopically labeled proteins or proteins with mutations at specific sites... 

Unlike visual rhodopsins that bleach upon illumination, archaeal rhodopsins exhibit photocycle. This is highly advantageous in ultrafast spectroscopic studies and these techniques have been extensively applied in addition to low-temperature spectroscopy [2,12,13]. In particular, bacteriorhodopsin has been regarded historically as the model system to test new spectroscopic methods. As in visual rhodopsins, the light absorption of archaeal rhodopsins causes formation of red-shifted primary intermediates [68]. The primary K intermediate can be stabilized at 77 K. Time-resolved visible spectroscopy of bacteriorhodopsin reveals the presence of the precursor, called the J intermediate [12,13]. The J intermediate is more red-shifted (7.max -625 nm) than the K intermediate (2rn ix -590 nm). The excited state of bacteriorhodopsin possesses blue-shifted absorption, which decays nonexpo-nentially. The two components of the stimulated emission decay at about 200 and 500 fs [69]. The J intermediate is formed in <500 fs, and converted to the K intermediate within 3 ps [12,69]. 

The enhanced signal-to-noise ratio that is provided by resonance enhancement as well as the reduced complexity of the vibrational spectrum make it possible to perform a wide variety of time-resolved studies to determine the structure of the chromophore in the photocycle intermediates. These approaches are discussed in more detail elsewhere in this volume by Kincaid with emphasis on time-resolved Raman studies of heme proteins. Room-temperature flow methods have been extensively used to obtain time-resolved spectra with time resolution ranging from seconds to microseconds.The basic idea is to flow the sample and then introduce an optical pump beam upstream from the probe to initiate the photochemical cycle. Such experiments have been performed on the millisecond and microsecond time scales. For experiments with time resolution faster than microseconds, it is necessary to convert the setup to a two-pulse, pump-probe technique where the time resolution is established by the delay between the pump and probe laser pulses. The time resolution of this approach can be increased to around 1 psec beyond this point increased time resolution will be achieved only with reduced spectral resolution according to the uncertainty principle. 

The photochemical mechanism of the BP-containing PI was discussed, but it is not still well understood. Scaiano et al. [175] were the earliest to study the photochemical mechanisms of a model compound in solution using conventional and laser time-resolved techniques. In particular, it is important to learn whether the benzophenonediimide behaves as BP or as N-arylphthalimides. They used a model prepared from BTDA and 2-isopropylaniline. This model compound might be subjected to intramolecular photocyclization at the imide carbonyls as reported in the literature [162]. But no... 

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