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. The newly installed laser system includes a femtosecond Ti:sapphire oscillator that seeds a femtosecond Ti:sapphire regenerative amplifier. The oscillator generates 10nJ, 100 fs laser pulses at a frequency of about 80 MHz, and the regenerative amplifier boosts selected 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. Though the oscillator and regenerative amplifier were new, the CW Nd:YLF laser used to pump the oscillator was not. This laser, a Spectra Physics Millennia Xs, had been acquired in 1999, and was refurbished with new laser diodes. Unfortunately, the refurbished pump laser was unstable, and adversely affected the femtosecond oscillator performance. After extensive negotiations with Spectra Physics, they agreed to take responsibility for this problem and replaced the Millennia Xs with a new Millennia V. The stability is now sufficient to proceed with the integration of this laser system into our femtosecond laser lab. The output of this oscillator will be split and used to seed both old and new regenerative amplifiers. The output of these regenerative amplifiers will pump separate OPAs, which convert intense 780 nm pulses to a wide range of wavelengths that can be tuned to the chromophore of interest. To preserve ultrafast time resolution, these pulses must be optically synchronized at the sample location. This synchronization will be achieved with a fast, linear motor translation stage configured as a double-pass optical delay, whose range of travel is sufficient to delay optical pulses by an amount that exceeds one round trip in the oscillator. By amplifying a different pulse from the oscillator pulse train, we can vary the pump-probe arrival times from femtoseconds to seconds with high precision over the entire range of times. The electronic synchronization capabilities needed for this combined system outstrip the capacity of our current electronic timing system, and 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 straightforward, and will be pursued in the coming year.

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Charlier, Cyril; Alderson, T Reid; Courtney, Joseph M et al. (2018) Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell. Proc Natl Acad Sci U S A 115:E4169-E4178
Charlier, Cyril; Courtney, Joseph M; Alderson, T Reid et al. (2018) Monitoring 15N Chemical Shifts During Protein Folding by Pressure-Jump NMR. J Am Chem Soc 140:8096-8099
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