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Virginia F. Smith

What am I interested in?

In general, I enjoy using physical and chemical methods to answer biological questions.  Over the years I have studied a range of problems, following my curiosity where it might lead.  Some of the questions I have been pondering lately are

How can a single protein have two different conformations and two different functions?

Image of aconitase converting between its two active forms

Although it was thought for many years that each gene produced a single protein with a single function, this simplistic view has been overturned by the discovery of a number of proteins that perform different functions depending on such factors as cell type, changes in oligomeric state, or the cellular concentration of a certain ligand.  One such protein that has two distinct jobs is the mammalian iron-responsive element protein 1 (IRP-1), which plays an important role in the regulation of intracellular iron regulation.  When iron levels are low, IRP-1 bind to regions of mRNA known as iron-responsive elements and by doing so, causes the levels of iron to rise by either up-regulating or down-regulating the production of proteins involved in iron transport and storage.  When the iron concentration is restored to normal levels, IRP-1 assumes its other identity – that of a cytoplasmic aconitase.  (Aconitase, an enzyme in the TCA cycle, is normally found in the mitochondria rather than the cytoplasm.)  As an aconitase, it can no longer bind to RNA and has a structural feature not found in IRP-1: a [4Fe-4S] iron-sulfur cluster. Cytoplasmic aconitase also catalyzes the isomerization of  citrate to isocitrate, just like its mitochondrial counterpart.

I am interested in studying how IRP-1/aconitase converts between its two active forms.  What is the relatively stability (free energy) of these two forms?  Can we learn about the conversion process by determining how these proteins unfold?  What is the effect of mRNA on the stability on IRP-1? Conversely, what is the effect of the iron-sulfur cluster and the natural substrate citrate on the stability of the cytoplasmic aconitase?  

 How can an enzyme detect and repair a single misplaced oxygen on a large protein?

An inevitable consequence of living in an aerobic environment is dealing with damage caused by reactive oxygen species, such as superoxide, hydrogen peroxide, the hydroxyl ion and water.  Modification of proteins, DNA and lipid molecules by reactive oxygen species has been shown to contribute to human disease and the aging process.  Although most of these covalent changes, such as DNA crosslinking and protein fragmentation, are irreversible, two types of protein modifications, cysteine and methionine oxidation, can be reversed enzymatically.

  The process by which protein disulfide isomerases reduce cysteine crosslinks (disulfide bridges) is fairly well-understood, but much less is known about methionine sulfoxide reductase (MsrA), the enzyme that reduces methionine sulfoxide back to methionine and restores function to the damaged protein.  Although MsrA is an essential protein – it has been found in the genome of every organism sequenced to date – we are just beginning to understand how it works.

Some of the unanswered questions that I plan to address are:  How does MsrA recognize and repair proteins that contain oxidized methionine residues?  How is MsrA able to interact with such a variety of proteins? What are the biophysical properties of MsrA? What are the thermodynamic parameters of the folding reaction of MsrA and how can we use them to help us understand the physical features that stabilize the protein?  I will use the experimental techniques described below to explore these and other issues.

What is happening in autumn leaves after they stop performing photosynthesis?

The bright colors of autumn leaves are a bittersweet reminder of the end of summer and the arrival of winter. As trees prepare for winter, they cease photosynthesis and break down the chlorophyll that gives leaves their characteristic green color. The loss of green account for some, but not all of the bright colors that emerge. But why does nature bother to have brightly colored leaves? The bright colors are not used to attract pollinators or animals that might disperse their seeds. So why not just shed leaves immediately at the end of the season?  The answer must be that the leaves still have some work to do before they drop. Because of their abundance and bright colors, I have chosen to study the gingko tree as a way to answer some of these questions.

The leaves of the Ginkgo biloba tree are known for their medicinal properties, which are primarily attributed to their antioxidant activity. We have performed direct analysis of total antioxidant activity on a full season's worth of leaves. And because metal ions are often involved in oxidation-reduction reactions, we investigated the levels of important metal ions, including magnesium and iron. It was found that the levels of magnesium rise sharply in the beginning of the season (April) when new leaves are put on and that levels remain high until leaf fall in December. By analyzing both total leaf extracts obtained from nitric acid and peroxide digests of leaf powder and the methanol-soluble fraction, which would presumably includes only membrane-bound magnesium chelated in chlorophyll, we were able to analyze the water-soluble and membrane-bound ions separately.  We have also looked at the levels of chlorophyll spectroscopically to determine a correlation with magnesium levels. Future studies on gingko leaves will include the use of EPR to examine levels of antioxidants and analysis of protein and RNA expression.

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