Ultrafast Biophysical Studies of Biomolecules at the NIH

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications & trials

Abstract

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, and with appropriate software, can tailor their operation to the timing requirements of labs in which they are deployed. We are currently focused on developing Python code to interact with this timing system and all timing critical, computer controlled hardware required to properly sequence all operations required to acquire a data set. We are working on a Methods-based approach whereby a table defines an extensible list of methods, each of which fully specifies the operating parameters required for all components used to acquire data. The FPGA provides hardware trigger pulses to properly synchronize all systems that require precise timing. For systems that can tolerate jitter of a few milliseconds, we are developing a novel approach based on Network Time Protocol (NTP) that precisely synchronizes all computers within our local area network to a millisecond precision, and therefore allows tasks to be started under software control with millisecond precision. The goal is to be able to define a set of high-level instructions that control the data acquisition sequence in a fully deterministic fashion. For example, we may wish to record transient absorption spectra as a function of pump power, polarization, pump-probe time delay, pump-dump time delay, temperature, sample position, and repeat the measurements via nested loops. Though more work remains, we are getting very close to realizing this vision for acquiring data in a deterministic fashion whose signal-to-noise ratio is limited only by photon counting statistics. In a collaboration with Ad Bax, we have 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 pressure transition, the folding/unfolding pathway can be unveiled at an unprecedented level of structural detail. Over this past year, our first-generation instrument, dubbed Icarus-I, has been exploited on Bax's 600 MHz magnet. Two copies of a second-generation version of the pressure-jump apparatus, dubbed Icarus-II, have been developed and were designed to achieve greater ease of operation, improved reliability, and can be deployed on the 800 MHz magnet in Bldg. 50 and the 900 MHz magnet in Bldg. 6. The ability to acquire pressure-jump NMR data on high-field magnets following tens of thousands of pressurization/depressurization cycles without equipment failure has made possible incisive investigations into the mechanism of protein folding and misfolding, the latter of which can lead to devastating amyloid diseases. The ability to structurally characterize intermediates that lead to the formation of these plaques may help provide a structural basis for drug design to suspend the advance of amyloid diseases.