Ultrafast Biophysical Studies of Biomolecules at the NIH

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications, trials & patents

Abstract

In 2024, we introduced a major update of the software used by the Bax group to control our home-built Icarus-I and Icarus-II pressure jump systems. This release was coded by Sean Fitch, a computer science undergraduate who offered his services as a remote Guest Researcher. The GUI developed by Mr. Fitch to operate the home-built pressure-jump systems is very intuitive, the monitoring and logging capabilities of his software package are significantly improved and robust, and his complete software package is accessible on GitHub. This software has been beta tested and is now deployed on all three pressure jump systems in operation at the NIH. This operational standardization facilitates the merging of time-resolved NMR data acquired on all three magnets, as demonstrated in a recent Bax group publication. These instruments play an important role in the research being pursued by members of the Bax group. An important parameter when pursuing time-resolved studies of biomolecules is sample temperature. For example, life prospers at temperatures as low as -1.8˚C for arctic cod to 121˚C for the aptly named Strain 121 Archaea microbe found within a hydrothermal vent in the Northeast Pacific Ocean. Since rates of reaction are strongly temperature dependent, biomolecules have evolved to perform their intended function at the temperature of their environment. To investigate biomolecule structural dynamics over a large temperature range, we developed a novel temperature controller capable of rapidly and precisely controlling the temperature of sample circulating in a capillary from -16 to 120˚C. The temperature controller operates a pair of thermoelectric coolers (TECs) that employ the Peltier effect to pump heat to/from an aluminum nozzle that surrounds the capillary and controls its temperature. One of our modes of data acquisition involves a T-ramp, in which the temperature repeatedly ramps linearly between low and high temperature settings at a slew rate of nearly 1 K⋅s-1. Unfortunately, this temperature cycling damaged the TECs used to control the sample temperature and required frequent replacement, an invasive procedure akin to replacing the engine of a car. We developed a second-generation temperature controller that addressed this problem and further improves its performance. Our new temperature controller employs three coolant reservoirs: low temperature, room temperature, and high temperature, and have demonstrated operation up to 130 °C. The key to TEC longevity is limiting the temperature difference across the TECs during operation to less than 50 ˚C. Indeed, no degradation in performance was observed following a thermal-cycling stress test that consisted of 500 thermal cycles between -10 and 120 °C. This second-generation temperature controller provides another benefit: switching coolant reservoirs when changing the temperature by a large amount speeds the temperature transition and minimizes the dead time suffered when performing T-jump studies. We are putting the finishing touches on a manuscript that describes this instrument in sufficient detail for others to duplicate it capabilities or potentially commercialize it. This manuscript is entitled: ‘Rapid and precise control of capillary temperature from -20 to 130 °C,’ Eli Worth, Valentyn Stadnytskyi, Hyun Sun Cho, Friedrich Schotte, and Philip Anfinrud. We intend to publish this manuscript in Review of Scientific Instruments. As reported last year, we are developing a compact, general-purpose, dual-beam, time-resolved absorption spectrometer capable of probing structural dynamics in photoactive biomolecules over a broad range of temperatures and time scales. Unfortunately, the picosecond pulsed supercontinuum laser, which we employed as a tunable probe source, failed earlier this year and had to be sent back to Austria for warranty repair. We are still awaiting its return. Hence, our planned studies to investigate the quaternary transition of hemoglobin (Hb) after photolysis of its carbon-monoxy form (HbCO) has been put on hold. The aim of this study is to use a short pulse laser to photolyze HbCO at a well-defined point in time, use a lower power but longer duration laser pulse to re-photolyze subunits in which geminate rebinding occurs, and finally use a very long AOM-switched laser pulse to help maintain the photolyzed state during the expected quaternary structure transition. Prior studies have shown that the high-affinity R state is not homogeneous but is represented by multiple R states. Hence, by photolyzing HbCO completely at time zero and maintaining the deoxy state with continued illumination, we aim to characterize the dynamics of quaternary structure transitions from different R states without the complications of geminate recombination. This home-built instrument should prove quite useful to characterize time scales over which biomolecules undergo structural transitions and will help identify sample conditions appropriate for subsequent studies with more advanced techniques, such as time-resolved x-ray scattering and diffraction. The one-dimensional SAXS/WAXS scattering curves acquired with the instrumentation we developed are rich in structural information, but quantitative extraction of that information is challenging. We have taken advantage of the APS shutdown and focused much of our attention on analysis methods capable of extracting the maximum amount of useful information embedded in our data. Fundamentally, biomolecule behavior should be describable according to equilibria between structures whose relative populations can be characterized by enthalpic and entropic contributions to their respective free energies. The rates of transitions between structures are governed by absolute temperature and the free energies of activation barriers between them. Guided by these principles, we are working to develop a general model for predicting scattering curves that account for every detail observed in both temperature-dependent and time-resolved scattering studies and can be parameterized according to fundamental physical properties. We start by putting our experimental data on the same absolute scale. This scaling is possible thanks in part to our development of a partially-transmissive beamstop in our scattering setup, which allows us to determine with high accuracy the integrated number of photons transmitted through our sample in each x-ray scattering image. Scaling is also aided by the magnitude of the strong water ring observed in the WAXS region. Note that traditional SAXS setups lack both of these means of putting data acquired at different times on the same absolute scale. When applying these new, improved methods to data already acquired, we are able to quantify the propensity of proteins to form weakly associated dimers and witness their propensity to acquire or shed ions. We can characterize their thermal expansion coefficient, quantify enthalpic and entropic contributions to dimer formation or protein unfolding, and in some cases, even approximately count the number of ions shed with a biomolecule unfolds. We are testing the analysis tools we are developing on various biomolecules with an aim to converge on a standardized, unified approach for characterizing structure and structural dynamics in a wide variety of biomolecules including protein and RNA. The time-resolved methodology we have developed and continue to refine brings to bear powerful methods for characterizing structure and structural dynamics of biomolecules, which helps provide a structural basis for understanding how biomolecules function from a mechanistic point of view. This fundamental understanding of biomolecule function should help promote a more rational approach to drug development with significant benefits to human health.