Our goal is to elucidate the active site structures of metalloproteins to gain insight into the biochemical reactions catalyzed by these systems and the basic physics that controls these processes. Of particular interest are iron proteins involved in electron transfer and O₂ metabolism. We probe the state of the iron and its interaction with the environment by Mössbauer and paramagnetic resonance spectroscopy and try to infer reaction mechanisms from the changes observed in the course of the enzymatic cycle. At present we focus on integer spin states and on exchange coupled systems which exhibit new and unusual physical properties.

Heme proteins such as hemoglobin and myoglobin reversibly bind molecular oxygen and transport or store it in the organism. We are studying ligand binding to heme proteins over a wide range in temperature, pressure, viscosity, and time by flash photolysis. A picture emerges in which the biological activity is governed by spatial and temporal fluctuations. We have investigated the conformational substates participating in these fluctuations with, among other methods, x-ray diffraction and studied the binding step at the active centers using infrared spectroscopy and magnetic susceptibility. The results provide insight into the molecular mechanisms of protein function and suggest that the reaction theories used for explaining protein reactions must be generalized.

The Laboratory for Fluorescence Dynamics (LFD), a national biomedical resource, has a dual and equal commitment to foster fluorescence research and to provide service in a user-oriented facility.

Fluorescence Research and Development
The research goal of the LFD is to develop new fluorescence instrumentation, design new theoretical formulations of fluorescence phenomena, and compile appropriate software, with the aim of advancing basic research and biomedical applications. Examples of current projects include: instrumentation (frequency domain fluorometer with lifetime and spectral resolution, laser heterodyning, lifetime fluorescence microscopy), theory (associative and nonassociative anisotropy decay), software (global analysis of multifrequency data sets), optical imaging (near-infrared images of tissue), and applications (two-photon fluorescence correlation spectroscopy). These advances in fluorescence technology are transferred to the user fluorescence laboratory.

Fluorescence Laboratory
The laboratory serves both the campus research community and visiting scientists. To date, core and collaborative research has stressed macromolecular assembly and dynamics, membrane structure/function relationships, and fluorescence microscopy of cells. The LFD houses a spectropolarimeter for circular dichroism measurements. Fluorescence equipment includes high-sensitivity, photon-counting, scanning fluo rometers (with polarization accessory), three laser-based variable multifrequency phase/modulation fluorometers with different excitation wavelength and modulation frequency options, stopped flow and high pressure accessories. Dedicated personal computers assist in data collection and analysis. Ancillary support for biomedical research is housed in a general biochemistry laboratory, which is equipped for biological sample manipulation.

Recent experiments have shown that enzymes are flexible structures. Internal movements in enzymes in a short time scale have been postulated and detected. What the role of the solvent is, whether this peculiar feature of an enzyme is related to its function, and how the internal dynamics change when the substrate binds to the active site are just a few of many interesting questions. A complete time description of an enzyme is the ultimate goal. Many enzymes contain fluorescent probes. Time-resolved emission anisotropy is used to gain information about their local flexibility. A study in different solvents, in dehydrated powders and with substrates and inhibitors, would give a description of the changes induced by these factors in the conformational flexibility.

This project explores the use of frequency-domain methods to obtain near-infrared optical images of thick tissues. The use of near-infrared radiation has been proposed as an attractive alternative to obtain information about the oxygenation state of tissues due to the difference in optical spectra of the oxy- and deoxy- form of hemoglobin. Our frequency-domain approach uses the propagation of high-frequency AM light. In the frequency-domain, propagation of the AM intensity wave in a highly scattering medium is analogous with wave optics. An object immersed in the medium produces deformation of the propagation wavefront of the amplitude modulated wave and results in an easy identification of absorbing and scattering objects such as blood vessels or bone. Computer algorithms display in real-time the wavefront of the AM wave after traversing the tissue.

We have developed a transient infrared spectrometer with microsecond time resolution using the Vanderbilt free-electron laser as a monitoring light source. Measurement of the infrared absorption of carbon monoxide (CO) after laser photodissociation of CO-ligated heme protein enables us to obtain information about the interactions and motions of protein and ligands during recombination.

Our research focuses on the structure, dynamics, and function of biopolymer aggregates, e.g., lipids and water forming membrane bilayers, proteins complexing with DNA and regulating gene expression, and proteins involved in complexes with other proteins. The studies require very-large-scale computer simulations and have become possible through the development of statistical mechanical theory, efficient algorithms, graphics tools, a simulation program, and the group's network of powerful workstations which function as a high-performance parallel computer.