Douglas A. Gage Research Interests
The primary area of interest in our laboratory is the biosynthesis and function of plant osmoregulatory compounds, in particular betaines and their sulfonium analogs. To help plants adjust to salt or drought stress, these zwitterionic "compatible osmolytes" can accumulate in the cytoplasm to concentrations of more than 1M without disrupting cellular function. The mechanism by which enzymes and cellular proteins are protected from denaturation in these high concentrations is not well understood.
Our work is currently focused on the osmolyte dimethylsulfoniopropionate (DMSP). In addition to its role in physiological adaptation, DMSP is one of the primary contributors to biogenic sulfur in the atmosphere through its breakdown to dimethylsulfide, a gas whose atmospheric oxidation products are important in cloud formation and potentially in climate regulation. Our biosynthetic studies have determined that this compound is derived from methionine via two distinct pathways in higher plants and marine algae. We are using stable isotope and radioisotope labeling methods to identify intermediates in these pathways. Isolation and characterization of the enzymes involved in DMSP biosynthesis is one near-term objective of this work. A longer term goal is to understand how this pathway is regulated under different environmental conditions, both in the context of plant adaptation to stress and global sulfur cycles.
A second focus of the research in our laboratory is on the development of new analytical methods employing mass spectrometry to characterize protein structure. This work is carried out in conjunction with the core research of the MSU-NIH Mass Spectrometry Facility, a national research resource. We are interested in identifying and localizing posttranslational modifications, such as phosphorylation, glycosylation, acylation, disulfide bonds and proteolytic processing. To approach this problem we have combined enzymatic and/or chemical modification of the target protein with molecular weight determinations by two sensitive mass spectrometric techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). By mapping the predicted masses obtained from protein sequences to the measured masses of the protein or peptide fragments, posttranslational modifications can be characterized. Algorithms are also being developed that allow the mass mapping approach to be used to rapidly identify isolated proteins that are listed in gene or protein sequence databases. In another approach, we use mass spectrometric methods, such as post-source decay (PSD) analysis, to obtain direct sequence information from peptide fragmentation. In order to apply these new methodologies to biological samples, we have been developing techniques to manipulate and analyze picomole to sub-picomole quantities of proteins, including direct MS analysis of proteins on electrophoretic gels or transfer membranes.