Biophysics

The Ralph Jimenez group at JILA uses ultrafast laser spectroscopy to investigate protein motions in solution. The lasers produce pulses of light shorter than most of these motions, making it possible to take "snapshots" of protein motion. To better understand protein motion, the group also probes electronic transitions of bound cofactors or ligands. To investigate the structural origins of protein motions, the group chemically modifies the proteins under study or investigates similar proteins found in mutant species.

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 attack a particular protein, (4) the forces that cause subtle chemical changes in particular enzymes, and (5) the "action-at-a-distance" changes at an active site that can occur when a small molecule or protein binds to a remote location in an enzyme.

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, however, because biomolecular motions range from ultrafast (in the femtosecond range) to relatively slow (in the range of seconds). To track changes in proteins after exposure to light or other activation, the Jimenez group uses laser microscopy and ultrafast laser spectroscopy. 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, a topic of great interest to the Jimenez group. The group is also adapting a JILA MONSTR (Multidimensional Optical Nonlinear SpecTRometer) to perform 2D spectroscopy to directly observe proteins motions. The 2D experiments will enable the researchers to resolve motions along different directions within proteins and determine if there are preferred pathways of energy flow in biological molecules.

Microfluidics Devices

The Jimenez group is developing laser microscopy incorporating microfluidics technology for several applications. One effort is focused on developing enhanced fluorescent proteins for single-molecule imaging in cells. The group is generating these proteins in mammalian cells, then screening them for desired optical characteristics with microfluidics-based spectroscopy. The group is using closely related microfluidics technology to develop genetically encoded fluorescence-resonance energy transfer (FRET) sensors to detect zinc (Zn) and other metal ions inside proteins.

Jimenez and his collaborators recently built a new microscope that incorporates a microfluidics network for cell sorting. Microfluidics are tiny liquid-handling circuits that permit precise and rapid mixing of biological samples as well as observation times of seconds or longer. Similar systems permit researchers to sort cells according to such properties as fluorescence, brightness, or photostability. Other microfluidics-based instruments developed in the lab enable the observation of cell deformations due to interactions with laser light.

The Jimenez group is currently investigating red fluorescent proteins (RFPs), which are members of the m-fruit family. This family of proteins (including m-cherry, m-orange, m-plum, or m-banana) fluoresce in the red, orange, purple, or yellow, respectively. The researchers are evaluating a method for rapidly measuring the resistance of RFP mutants to bleaching by an intense laser beam. This technique will be combined with optical trapping to screen and sort a diverse genetic library of cells. The goal is to select RFPs with enhanced photostability.

In another project, the group is investigating zinc (Zn) proteins. Zn is important in such diseases as diarrhea, pneumonia, and sickle cell anemia; but methods to image its concentrations in cells have not yet been developed. The Jimenez group currently studies zinc finger proteins, which stabilize the Zn-protein link into a particular type of "hairpin" structure. The group is tagging these proteins with fluorescent proteins at either end for use as intracellular zinc sensors. These sensors look like beacons whose colors change depending on whether zinc ions are bound. In these studies, the group is developing a microfluidics-based method for selecting members of a zinc sensor genetic library that exhibit higher sensitivity as well as larger and faster fluorescence responses in vivo. The Jimenez group is collaborating with Amy Palmer, in the Chemistry and Biochemistry Department at the University of Colorado on this effort and in the RFP experiments.

Biofuels

Ralph Jimenez and his collaborators are using a microfluidics system to sort algal cells for the production of biofuels. Microalgae have great promise for achieving the goal of producing lipids from sunlight. These lipids can be converted to biodiesel. However, algae require significant development in the laboratory before they will be able to produce meaningful amounts of renewable fuels. The Jimenez group is currently developing a "lab-on-a-chip" microfluidic system to rapidly screen genetic libraries and naturally diverse samples of algae. The researchers are assessing the potential for improving the lipid (fat) content of individual algal strains while optimizing the growth conditions for these strains. The group is using its prototype lab-on-a-chip device to measure photosynthetic activity and correlate this activity with lipid production. This work is resulting in a faster, more precise method for assessing algal strains for biofuels production.

2D Spectroscopy

Ralph Jimenez and Steve Cundiff have created the JILA MONSTR, a precision optical instrument. It works much like a multidimensional nuclear magnetic resonance (NMR) experiment, except that it uses femtosecond optical pulses that are short enough to observe molecular motions. The Jimenez group is applying this new technology to the study of heme proteins, protein-ligand interactions, and protein folding.

Heme proteins are found in all life forms. They perform a variety of biological functions, including electron transfer, oxygen transport, and chemical reactions in which one reactant gains electrons while the other reactant loses electrons. Heme proteins contain a cofactor consisting of a porphyrin with an iron (Fe) or other metallic atom in its center. To better understand exactly how heme proteins perform their important functions in living organisms, the Jimenez group is using the JILA MONSTR to investigate one of two key absorption bands of the heme cofactor. The researchers are attempting to resolve two coupled peaks in the visible absorption spectrum. The peaks are known as Q_x and Q_y. These peaks make up the Q band, which appears between 500 and 600 nm. They are "perpendicular transitions," which means they are sensitive to motions along different directions within the protein.

To study the Q_x and Q_y peaks, the Jimenez group is building a new green femtosecond laser for use with the JILA MONSTR. Next, the researchers will substitute magnesium (Mg) for Fe in the cofactor of a heme protein derived from horseradish. This substitution should make it possible to separate the two peaks and take a closer look at them. In the future, the group would like to study how the asymmetry of the motions in heme proteins is related to their remarkable properties.

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