Ralph Jimenez' group at JILA uses ultrafast lasers to investigate protein motions in solution. These studies are critical for understanding how enzyme conformations change in response to forces such as light, chemical bonding, voltage changes, or cofactor substitution. Understanding protein motions should yield new information on how proteins recognize and bind to other biomolecules. Protein motion studies will one day shed light on key research topics in molecular biology, including (1) how enzyme inhibitors work, (2) how apoenzymes and cofactors combine to form active enzymes, (3) the conditions that cause proteolytic enzymes to attach a particular protein, (4) the forces that cause subtle chemical changes in particular enzymes, and (5) the effect on enzyme shape of binding to chemicals at sites different from its active site.

Until recently, information about protein structure and function came from the detailed atomic pictures revealed in X-ray crystallographic studies. However, these studies are, in essence, snapshots of a single configuration. Current research is designed to fill in missing information about the vibrations and other dynamic characteristics of proteins to deduce exactly how they work in living systems.

The characterization of protein dynamics is a formidable challenge because biomolecular motions range from ultrafast (in the femtosecond range) to relatively slow (in the range of seconds). The Jimenez group uses both ultrafast laser spectroscopy and laser microscopy to track changes in proteins after exposure to light or other activation. Ultrafast laser spectroscopy is performed with lasers that emit pulses of light 50 femtoseconds or less in duration. It is analogous to using a shutter on a camera, only billions of times faster. The incredible speed is necessary to capture the fastest protein motions. For laser microscopy, the group is developing a novel laser microscope incorporating microfluidics technology. Microfluidics are tiny liquid-handling systems that permit rapid mixing of biological samples, with observation times of seconds or longer. This instrument allows the researchers to observe protein motions in kinetically evolving systems (such as the interactions of an enzyme with its substrate) over 15 orders of magnitude in time.

The Jimenez group is using its laser-based instruments to study the motions of several proteins, including photoactive yellow protein (PYP), FixL, and cytochrome c. PYP is a protein found in bacteria growing in salt water. It changes shape when blue light is present. FixL is an oxygen-sensing protein found in bacteria living in the root nodules of legumes. Its shape depends upon whether or not oxygen is bound inside. Cytochrome c (with its heme cofactor) helps transform food energy into the energy-rich molecule ATP (adenosine triphosphate). It changes shape depending on its energy state. Molecular flexibility in these proteins appears to be important, but quantitative measurements are not yet available. The researchers plan to gather molecular flexibility data to better understand binding, folding, and other conformational transitions in these proteins.

Recently, the Jimenez group initiated studies to quantify the motions of cytochrome c and other porphyrins. To facilitate measurements over a wide range of time scales, they substituted a zinc atom for the iron atom in the cytochrome c. To determine which motions are important biologically, the researchers compared the motions of natural cytochrome c, which has an active, three-dimensional (3D) structure, with the motions of an identical, but denatured protein, which has lost both its 3D structure and biological activity. Their spectroscopic technique clearly differentiated between active, functional cytochrome c and its denatured, nonfunctional cousin, showing quantitatively that the latter is floppier and more disordered than the native protein. It also provided information on the protein's binding with substrates, interaction with solvents, and energy states. In the future, the researchers will seek to gather sufficient information about the motions of cytochrome c to be able to relate them to the changes in entropy that drive the biochemical reactions mediated by this protein.