Enzymatic reactions exhibit remarkable selectivity and efficiency, the likes of which are rarely achieved in bench-top chemical reactions. While it is clear that the biochemical prowess of an enzyme arises from its highly ordered structure, the detailed mechanism by which it functions has proven elusive. This is because proteins are not simply static macromolecules that host an active site, as depicted by their crystal structure;rather, they are dynamic molecules whose choreographed motions can gate the transport of substrate to and from the active site and can modulate over time the activity of that site. To develop a mechanistic understanding of how proteins function, it is essential to study this choreography of life at the atomic level with ultrafast time resolution. This choreography of life is being investigated by time-resolved techniques that are based on the pump-probe method. The pump is usually a laser pulse that triggers, at a well-defined instant of time, the process we wish to investigate. The duration of the laser pulse can be as short as a few tens of femtoseconds, which allows us to access the chemical time scale for molecular motion. After photoexcitation, a time-delayed probe pulse is directed through the pump-illuminated volume to interrogate the system. When the probe pulse is derived from an X-ray source, we can probe the protein structure via Laue diffraction or via Small- and Wide-Angle-X-ray-Scattering (SAXS/WAXS). When the probe pulse is generated in the uv-vis or mid-IR region, we can interrogate the system spectroscopically. The time resolution of the pump-probe method is limited only by the duration of the pump and probe pulses and the timing jitter between them. Each pump-probe measurement produces a time-resolved snapshot of the protein. By stitching together a series of snapshots, we create a movie that, in the case of time-resolved Laue diffraction, provides a near-atomic view of the correlated structure changes triggered by the pump pulse. We can literally watch a protein as it functions! Our efforts to develop time-resolved X-ray methods suitable for investigating protein dynamics and function are summarized in separate annual reports. Since all ultrafast time-resolved protein studies use light as a trigger, these investigations require detailed knowledge of the photophysics of chromophores in proteins, and thereby require ultrafast time resolution (<100 fs). In addition to ultrafast time-resolved studies of proteins, we wish to assess the time-ordered sequence of events that follow laser photoexcitation out to time scales as long as seconds. Finally, we wish to be able to compare dynamics of proteins in solution as well as in crystals. Time-resolved spectroscopy is well suited for all of these studies, and is the focus of this annual report. The pump pulse used to photoexcite the sample can come from multiple laser sources. For example, a home-built Optical Parametric Amplifier (OPA) can be used to generate broadly tunable femtosecond pump pulses whose time of arrival can be varied from femtoseconds to a few ns via an optical delay line. An Optical Parametric Oscillator (OPO) with broad tunability throughout the visible generates 2-3 ns pump pulses that can be delayed electronically out to seconds. A Q-switched, frequency-doubled Nd:YAG laser generates 200-ns pulses at 532 nm that can be delayed electronically out to seconds. A 527-nm CW laser can be electronically gated on and off with an AOM capable of approximately 200 ns switching times. An electronic timing-system was developed to properly synchronize these pump sources to the probe pulses so they can be used independently, or in combination with one another. One of the challenges we are currently working on is accurate determination of the extent of photoactivation, as that quantity is crucial to determine reaction quantum yield as well as absolute basis spectra for putative intermediates along the reaction pathway. Due to the limits of our optical delay line, species generated by ultrashort pulse photoexcitation can be tracked only out to a few ns. To follow the reaction pathway out to longer time scales, we have been forced to use a different pump source, for which the apparent photoproduct yield is invariably different. Thus, the absolute population of intermediates is not easily determined, and the spectra of putative intermediates cannot be determined absolutely. To follow dynamics from femtoseconds to seconds with a common ultrashort pulse requires a second source of amplified pulses: one to generate the pump pulse and the other to generate the probe pulse. Both amplifiers would be seeded by a common oscillator to eliminate timing jitter between their output pulses. With two fully synchronized regenerative amplifiers, we will be able to fully exploit a variety of novel spectroscopic techniques in our LCP femtosecond laser laboratory, including pump-dump-probe methods. Thanks to ARRA funds, we acquired in the summer of 2010 a new regenerative amplifier system to complement our existing home-built regenerative amplifier, which was acquired in 1993. Proper integration of these two complex systems will require a major tear-down and rebuild, and will involve hundreds of optical components. Because this upgrade will impose a lengthy shutdown of our femtosecond laser lab, we have elected to postpone this effort till sometime in fiscal year 2013. In the meantime, we have continued to acquire the motion control components needed to align and operate our expanded laser system, and continue to work on designs for the remaining subsystems needed to properly integrate these two laser systems. Briefly, we aim to use a femtosecond Ti:sapphire oscillator to seed both old and new femtosecond Ti:sapphire regenerative amplifiers. The oscillator generates 10nJ, 100 fs laser pulses at a frequency of 80 MHz. Each regenerative amplifier will boost oscillator pulses up to 2 mJ at a repetition rate of 120 Hz, the maximum frequency at which our CCD detector can be read. The wavelength of these pulses, 780 nm, is near the peak of the gain in Ti:sapphire. The amplified pulses will be used to pump separate OPAs, which convert intense 780 nm pulses to a wide range of wavelengths that can be tuned to the chromophore of interest. Since we plan to pump several OPAs with each regenerative amplifier, we will have various pulses available simultaneously, and can pursue not only pump-probe studies, but also pump-dump-probe studies, a novel method developed in my group in the 1990s while a Professor of Chemistry at Harvard University. The time delay between pulses originating from different regenerative amplifiers will be controlled by both optical and electronic means. The optical delay must span the pulse repetition period for the oscillator, which is 12.5 ns, and corresponds to a distance of 375 cm (when traversed at the speed of light). A double pass optical delay line can achieve this magnitude delay with a travel of 93.75 cm. Hence, we acquired a 1 m linear servo motor translation stage for this task.
We aim to employ large, retroreflector optics in our double pass optical delay line, and will insert this delay line between the oscillator and the regenerative amplifier used to generate the pump pulses. Electronic means will be used to step the pulse delay by 12.5 ns increments, and a combination of optical and electronic means will be used to achieve complete coverage of delays from femtoseconds to seconds. The electronic synchronization capabilities needed for this combined system outstrip the capacity of our current electronic timing system, and will require the development of a new FPGA-based timing system. Thanks to experience gained developing FPGA timing systems for synchrotron beamlines, this task should be reasonably straightforward.

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Kaila, Ville R I; Schotte, Friedrich; Cho, Hyun Sun et al. (2014) Contradictions in X-ray structures of intermediates in the photocycle of photoactive yellow protein. Nat Chem 6:258-9
Cho, Hyun Sun; Schotte, Friedrich; Dashdorj, Naranbaatar et al. (2013) Probing anisotropic structure changes in proteins with picosecond time-resolved small-angle X-ray scattering. J Phys Chem B 117:15825-32
Schotte, Friedrich; Cho, Hyun Sun; Kaila, Ville R I et al. (2012) Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography. Proc Natl Acad Sci U S A 109:19256-61