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. We investigate this choreography of life using time-resolved techniques 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 acousto-optic modulator (AOM) capable of approximately 200 ns switching times. These sources can be used independently or in conjunction with one another. One of the challenges we face is accurate determination of the extent of photoactivation, as that quantity is crucial to determine the 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 accurately. 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. 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 (12.5 ns), and will be accomplished with a 1-m long linear servo motor translation stage operated in double-pass mode. Electronic means will be used to step the pulse delay by 12.5 ns increments, with a combination of optical and electronic means capable of achieving complete coverage from femtoseconds to seconds. 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 Field-Programmable-Gate-Array (FPGA) based timing system. To that end, we acquired newer generation Suzaku SZ410 FPGA Boards (XC4VFX12), and with assistance from electronics experts at the Advance Photon Source, have designed a custom printed circuit board (PCB) that will function as an I/O distribution panel for the Suzaku board. The PCB board was custom manufactured in Sept. 2014, and soon after we assembled and tested our third-generation FPGA. With this new timing system, we are able to control independently the relative timing of 24 outputs to a precision down to 10 ps. We have also been developing a new player-piano paradigm for gating the FPGA-generated pulses. This player-piano concept allows us to specify a repeating pattern appropriate for a particular experiment, which defines not only the relative timing of all pulses, but also the repetition frequency for the sequence. The desired repeating pattern is generated by software and represents the sheet music. After loading the music into the FPGA, it plays it deterministically. This approach affords unprecedented flexibility and extensibility in developing and coordinating data acquisition sequences. A failure of the cooling system in Bldg. 5, Rm. B2-10 shut down our femtosecond laser system this past year. The NIH has since acquired a replacement cooling system which became operational in Sep 2015. Once we bypass the internal controller and operate it from our own PID-based temperature controller, we will be able to resume operation of our femtosecond laser laboratory. In conjunction with our 3rd-generation FPGA, we will be able to pursue sophisticated pump-dump-probe studies of proteins in solution and in crystals with high time resolution from femtoseconds to seconds.
| 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 |
