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Molecular Biophysics

The Ralph Jimenez group specializes in molecular biophysics, a relatively new field that employs concepts and techniques from physics, chemistry, and biology to elucidate the behaviors of biomolecules found in nature. The group’s eclectic research topics include (1) studies of the elastic properties of single cells, (2) the characterization of cellular fluorescent proteins, (3) investigations of protein motions, (4) the use of ultrafast x-rays to make molecular movies of chemical reactions, (5) directed-evolution studies of red fluorescent proteins and algae destined for biofuel production, and (6) mapping the distribution of metal ions in living cells.

Elastic Properties of Cells

Studies of the elastic properties of cells can reveal differences between healthy cells and cells that are infected by parasites, cancerous, or capable of transferring cancer to other parts of the body. Thus, measurements of cellular elasticity may provide a biomarker for detecting disease or assessing its progression. However, meaningful measurements of elasticity must be done at the single cell level to give clinicians the ability to assess levels of infection or malignancy.

For this reason, the Jimenez group has developed a novel high-throughput cytometry technique that can measure fluorescent cell components and cellular characteristics such as the elasticity of single red blood cells. This innovative microfluidic flow cytometer uses a focused laser beam to trap and stretch individual cells as they flow slowly through a microchannel. The device accurately measures the elasticity of about 1.2 red blood cells per second, which is a hundred times faster than previous methods.

Cellular Fluorescent Proteins

Investigation of red fluorescent proteins is a key research focus of the Jimenez group. These proteins are derived from sea anemones and coral. They have a barrel-shaped structure that surrounds and protects a color-producing entity (chromophore) that fluoresces red. The group is interested in acquiring a detailed understanding of the structure and behavior of natural red fluorescent proteins. It is also working on developing a more stable variant of natural red fluorescent protein that may one day be used as a biomarker in thick tissues.

Researchers in the lab employ three innovative technologies in their investigations of red fluorescent proteins: microfluidic flow cytometry, multidimensional electronic spectroscopy, and fluorescence spectroscopy (in studies of pressure-induced changes in protein behavior conducted with the J. Mathias Weber group). Microfluidic flow cytometry is used to quantify light-induced bleaching of mixtures of red fluorescent proteins inside HeLa cells. Cytometry is making it possible for the researchers to work on directing the evolution of “supernatural” red fluorescent proteins that are more stable in the presence of light and are less likely to lose the ability to fluoresce (which can happen with their natural counterparts).

Protein Motions

Investigations of protein motions rely on the Multidimensional Optical Nonlinear SpecTRometer, or JILA MONSTR. These studies focus on animal-derived proteins such as heme mono-oxygenases and red fluorescent proteins. The JILA MONSTR is able to “see” key molecular interactions in the active site of the heme proteins, helping the researchers investigate how protein motions influence enzyme behavior. The MONSTR is also shedding light on changes in the chromophores of red fluorescent proteins. These chromophores are thought to be responsible for switching between higher and lower brightness during fluorescence. Together with related studies of red fluorescent protein mutants, this work may lead to “designer” chromophores that exhibit significantly more fluorescence.

The use of ultrafast x-rays to make molecular movies of chemical reactions is a new research challenge for the group. The group is designing an ultrafast x-ray-based spectrometer to study disordered protein samples such as metal-containing proteins dissolved in solution. Analysis of the ultrafast x-ray spectra will make it possible to precisely measure bond distances in proteins, analyze the excited states of enzyme cofactors, and “image” protein behavior. X-ray fluorescence from targeted atoms inside proteins will allow the researchers to monitor structural and functional changes inside specific proteins and biomolecules. These innovative experiments include researchers from the Kapteyn/Murnane group at JILA and colleagues from NIST Boulder.

Directed Evolution Experiments

Directed-evolution experiments aim (1) to develop red fluorescent proteins that can be more readily studied in the laboratory and (2) develop strains of algae ideally suited for the production of biofuels. Efforts to develop “supernatural” red fluorescent proteins are focused on coming up with proteins that are less likely to convert to nonfluorescent dark states and more likely to resist rapid, irreversible bleaching by light. Both behaviors interfere with cellular imaging of red fluorescent proteins. Starting with natural red fluorescent proteins, the group’s directed evolution experiments use a microfluidic cytometer to sort fluorescent-protein-containing mammalian cells via selective measurements of dark-state conversion and light-induced bleaching. The group plans to engineer new proteins that fluoresce longer and are brighter, resistant to bleaching, or both.

The microfluidic cytometry at the heart of directed evolution experiments is also helping the group meet other biological measurement challenges, including the screening of algae for fat (lipid) content. Lipids are energy-rich biomolecules that could be used to make biodiesel fuels and other petroleum analogs. Lipid-rich algae could become an important future renewable energy resource. The group is initially looking at the lipid content of natural communities of algae found in lakes. Since lipid content affects cellular elasticity, or stretching, the group can screen natural algae one cell at a time in a microfluidic cytometer.

Metal Ion Distribution in Cells

The mapping of metal ion concentrations in cells relies on genetically encoded biosensors. The development of fluorescent protein-based sensors is the target of this research. Such sensors can compensate for signal variations due to cellular components that fluoresce because of the intensity of the excitation laser. The group is working to develop genetically encoded metal sensors with significantly improved optical, physical, and chemical properties. In the future, such metal sensors should be able to detect single metal-ion signals from engineered biosensors inside living cells.

The group is currently designing multiple genetic libraries, each containing as many as 10 million potential sensors and expressing the library members in living cells. Then using microfluidics technology, the researchers induce cellular responses to specific chemicals, measure these responses with laser light, and rapidly screen and sort the most promising cells for further study. Such studies range from investigations of the mechanism of metal-ion sensing through metal-ion transport through cell membranes.