Reusch, Rosetta N.
A.B., Hunter College
A.M., Ph.D., Columbia University
Postdoctoral Fellow, Imperial College, London
Our laboratory is interested in the metabolism and functions of poly-(R)-3-hydroxybutyrate (PHB) and its interactions with other cellular macromolecules. This dynamic and versatile polymer has remarkable solvent properties which enable it to "dissolve" salts of polyanions, such as polynucleotides and inorganic polyphosphates, and transfer them into and through membranes. One of our present studies is of complexes of PHB with inorganic polyphosphate. These complexes form voltage-gated calcium channels in the plasma membranes of E. coli and many other bacteria that display many of the characteristics of protein calcium channels. In this study, we incorporate plasma membrane vesicles or channels extracted from them into planar phospholipid bilayers, and observe the single-channel currents using voltage-clamp techniques. Variables under investigation include lipid composition, temperature, ion composition, channel blockers and agonists and their effects on ion selectivity, current magnitude, and gating kinetics. The channel complexes can be reconstituted from components and examined by the same means. We are also exploring the possibility that these channels are used for transfer of DNA between cells. These studies involve isolation of PHB/DNA complexes from cells undergoing genetic transformation, and in vitro passage of oligonucleotides through PHB channels in bilayers.
Our studies of PHB complexes employ a broad variety of experimental techniques. In addition to electrophysiological methods, we make use of transmission electron microscopy and atomic force microscopy, computer modeling with molecular mechanics and molecular dynamics, and a variety of spectroscopic procedures, including fluorescence, atomic absorption, nuclear magnetic resonance, infra-red, x-ray diffraction, and mass spectroscopy.
In addition to complexes with polyphosphates, PHB also forms covalent bonds to many proteins that have important roles in cell metabolism. There are between 70 and 100 PHB-conjugated proteins in E. coli. Those identified thus far are mainly highly conserved proteins with important roles in transcription, translation, protein folding and protein targeting. We are now engaged in identifying the binding site for PHB on some of these proteins. We plan to mutate the amino acid to which PHB is covalently bound and observe the effect on PHB incorporation and protein function.
Another area of our research is the identification of proteins involved in PHB metabolism, and the genes that encode them. We are engaged in a collaborative effort, using standard biochemistry and molecular biology methods, to sequence the genes and produce clones and mutants for use in studies of the mechanisms of ion channel formation and DNA transfer. Yet another focus of interest is the putative involvement of PHB in human disease processes. We have found that PHB is ubiquitous in plant and animal tissues. Of particular interest is the discovery of PHB in atherogenic lipoproteins in human plasma, which led us to investigate its potential as an atherogenic agent. Preliminary studies indicate a statistically significant association between plasma PHB and "bad" cholesterol, and we are engaged in collaborative studies to further examine this relationship.