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 enzymes 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 into how enzymes function, it is essential to study this choreography of life with structurally-sensitive methods capable of ultrafast time resolution. We investigate this choreography of life using time-resolved techniques based on the pump-probe method. The pump is often a laser pulse that triggers at a well-defined instant of time the process we wish to investigate. After triggering a structural change, a time-delayed probe pulse is directed through the pump-illuminated volume to interrogate the system. 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; optical pulses generated in our femtosecond laser laboratory can be as short as a few tens of femtoseconds, which allows us to access the chemical time scale for molecular motion. Each pump-probe measurement produces a time-resolved snapshot of the biomolecule. By stitching together a series of snapshots, we create a movie that allows us to literally watch a biomolecule as it functions! In time-resolved NMR studies, a rapid pressure jump triggers a change in a protein's structure, the dynamics of which can be probed with side chain specificity. Our efforts to develop time-resolved methods suitable for investigating biomolecule dynamics and function are summarized here. The Ultrafast Biophysical Chemistry Section of LCP maintains a laser lab in Bldg. 5, Rm. B2-10 with numerous operational laser systems including two femtosecond regenerative amplifier systems, associated home-built Optical Parametric Amplifiers (OPAs), a nanosecond Optical Parametric Oscillator (OPO), and several frequency-doubled Nd:YAG lasers. The OPAs and OPO are capable of producing intense optical pulses over a broad spectral range spanning from the ultraviolet to the mid-infrared. When the pump and probe pulses used to photoexcite and monitor biomolecules are generated with OPAs, the time resolution of the spectroscopic measurement can be less than 100 femtoseconds, and the relative time of arrival can be varied from femtoseconds to a few nanoseconds 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 approximately 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) that is capable of approximately 200 ns switching times. These sources can be used independently or in conjunction with one another. The electronic synchronization capabilities needed to effectively operate this array of laser systems is beyond the capacity of our existing electronic timing system. Hence, we are developing our third-generation Field-Programmable-Gate-Array (FPGA) based timing system, which employs a Suzaku FPGA that is capable of controlling independently the relative timing of up to 24 digital outputs. Briefly, the Suzaku board consists of a Xilinx FPGA chip and its associated micro-Linux processor. To properly sequence the pulses, we developed interrupt handler code capable of responding to interrupts at a rate of approximately 1 kHz without missing a beat. The pulse sequences required for all output channels are coded in measure-long packets, each of which fully specifies the repeating pattern required for all output channels. These packets are generated, indexed, and stored in RAM. The interrupt handler is fed a sequence of indices that specify the order in which the packets are played, and therefore defines the data acquisition sequence in a deterministic fashion. This player-piano paradigm generates pulse sequences appropriate for experiments ranging from time-resolved spectroscopy in our laser lab to time-resolved Laue crystallography or time-resolved SAXS/WAXS studies on the BioCARS beamline at the APS. We have built four identical FPGA boards so we can employ this deterministic approach to data acquisition in time-resolved studies conducted in various locations, including our femtosecond laser lab and the BioCARS X-ray beamline. We are soon ready to integrate our third-generation FPGA into our femtosecond laser laboratory, which should allow us to precisely control the generation and timing of laser pulses originating from several different laser systems, and facilitate time-resolved spectroscopic studies over a broad range of wavelengths and time scales. In a collaboration with Ad Bax, we developed a novel high-pressure apparatus capable of rapidly switching the hydrostatic pressure in a zirconia NMR tube between 1 bar and 2.5 kbar in a few milliseconds. Many proteins spontaneously unfold at high pressure, or can be engineered to do so via mutagenesis. Thus, rapid changes in pressure can trigger protein unfolding or folding. By acquiring 2D and 3D NMR spectra over a series of time delays following the high-pressure transition, the folding/unfolding pathway can be unveiled at an unprecedented level of detail. Over this past year, this instrument, dubbed Icarus-I, has been exploited with a 600 MHz magnet. A second-generation version of the pressure-jump apparatus is being developed with an aim to make it easier to operate, improve its reliability, reduce maintenance requirements, and allow this novel technology to be extended to higher-field magnets, such as the 800 MHz magnet in Bldg. 50. Much progress has been made, and Icarus-II should be operational in FY 2019.
| 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 |
| 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 |
| 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 |
