DNA is the blueprint for life, but proteins are the macromolecules that make life happen. One of the grand challenges for the 21st century is to develop a molecular-level understanding of protein function. This knowledge will help us better understand how protein dysfunction can lead to disease, and can provide a framework for molecular-based treatment of these diseases.
To that end, we are investigating the relationships between protein structure, dynamics, and function using ultrafast time-resolved laser spectroscopy and x-ray diffraction. These experimental techniques employ a laser "pump" pulse as short as ~50×10-15 seconds (50 femtoseconds) to trigger a photophysical or photochemical reaction and a variably delayed "probe" pulse that measures the spectral or structural evolution of the protein. This pump-probe technique provides the means to acquire "snapshots" of a protein as it executes its targeted function. By monitoring changes that occur over time, from femtoseconds to seconds, we can build a foundation for understanding how proteins execute their targeted function with high efficiency and selectivity.
To probe the structural evolution of an entire protein as it functions, we have developed the technique of picosecond time-resolved x-ray crystallography. This effort began in 1998 as a multinational collaboration with experiments carried out on a time-resolved beam line at the European Synchrotron and Radiation Facility (ESRF) in Grenoble, France. In 2003, we reported in Science the first 150 picosecond time-resolved x-ray structure of a myoglobin mutant. In 2006, we initiated a project to develop the infrastructure required to pursue picosecond time-resolved x-ray diffraction studies of proteins in the USA. This effort, which was pursued on the NIH-supported BioCARS beamline at the Advanced Photon Source (APS) in Argonne, IL, led to the first ~100 ps time-resolved Small- and Wide-Angle X-ray Scattering (SAXS/WAXS) study of a protein in 2010, and the first ~150 ps time-resolved Laue diffraction study of a signaling protein in 2012, both of which were published in the Proceedings of the National Academy of Science, USA. We are concurrently working to extend the time resolution of Laue crystallography down to < 1 ps by taking advantage of the world’s first free electron x-ray laser, the LCLS in Stanford, which is capable of generating intense, monochromatic x-ray pulses of less than 0.1 ps duration.
Our time-resolved Laue crystallography studies allow us to recover near-atomic resolution protein structures on ultrafast time scales. However, the crystal packing forces limit the range of motion accessible to the protein, and can inhibit or even prevent the structural changes needed to fully execute its targeted function. To examine protein structural dynamics in solution, where the full range of protein conformational motion is realized, we have developed the infrastructure needed to acquire picosecond time-resolved Small- and Wide-angle X-ray Scattering (SAXS/WAXS) data. Though the structural information available from these data is at low resolution, it complements our time-resolved crystallographic studies, and provides a more complete picture into the mechanism of protein function.
These time-resolved studies are performed on a large number of molecules at a time, and reveal ensemble-averaged structural changes. To generate a molecular-level understanding into how proteins function, we would prefer to watch a protein function one molecule at a time on ultrafast time scales, a feat that is impossible experimentally, but can be achieved computationally. However, results obtained computationally are only as good as the potentials used to account for the atomic- and molecular-scale interactions. To address their respective limitations, joint analysis of time-resolved structural data obtained from x-ray scattering and ensemble-averaged molecular dynamics simulations performed in the group of Dr. Gerhard Hummer are being pursued to validate the potentials used in the computational analysis and to unveil in mechanistic detail how proteins execute their targeted function. These complementary approaches are providing a glimpse into the function of proteins at an unprecedented level of detail, and take us far in our quest to unveil the biophysical equivalent of pistons, levers, wheels, and gears in Nature’s remarkable nanoscale molecular machines.
DNA provides the blueprint for life, but proteins are the molecules that make life happen. Since many diseases arise from dysfunctional protein activity, a detailed understanding into how proteins function is crucial to develop effective, molecular-based therapies for treating disease.
It has been said that “a picture is worth a thousand words.” Our lab has developed experimental methods that make it possible to take near-atomic resolution snapshots of a protein’s structure with an exposure time as short as 0.1 billionth of a second. We are concurrently working to reduce our exposure time down to less than 1 trillionth of a second. Why so fast? Because the functional protein motions we seek to characterize can occur on ultrafast time scales, they will be smeared out if our exposure time is too long. By stitching together individual snapshots into movies, we can literally watch a protein as it functions, and read chapter one of a protein’s story.
We use ultrafast time-resolved laser spectroscopy and x-ray diffraction to study proteins as they perform their targeted function. These experimental techniques employ the pump-probe method in which a laser pump pulse triggers a structural change in a protein, and a suitably delayed probe pulse records the protein’s absorption spectrum (spectroscopy), or its stucture (x-ray diffraction). By changing the time delay between the pump and probe pulses, we can track time-dependent changes in the protein structure and examine how those changes influence its function. By watching a protein as it functions in real time, we aim to generate a detailed understanding into how it functions, and thereby unveil the biophysical equivalent of pistons, levers, wheels, and gears in Nature’s remarkable nanoscale molecular machines. This knowledge will help us better understand the conditions under which a protein might suffer dysfunction, and provide a molecular basis for treating disease.
Applying our Research
Since many diseases arise from dysfunctional protein activity, a detailed understanding into how proteins function will help medical science develop effective, molecular-based therapies tailored to the individual. When this capability becomes mainstream, the promise of personalized medicine can be fully realized.
Need for Further Study
A combination of experimental and theoretical time-resolved studies of an expanded set of proteins are needed to more fully survey the molecular toolbox exploited by proteins as they execute their targeted function.