My projects focus on drug binding within cytochrome P450 enzymes, which metabolize environmental toxins and drug molecules, primarily in the liver.  Prediction of metabolism and potential drug-drug interactions is difficult, due in part to poorly understood behavior of the enzymes in vitro.  One mechanism of drug-drug interactions involves multiple drug molecules binding to the active site of the same P450 enzyme, where addition of a second molecule influences metabolism of the first.  The mechanism of interaction is poorly understood, and we propose to explore:

Where does the first ligand bind?

How does the second ligand alter the position of the first?

How is protein conformational change involved?

We can answer these questions using some of the following experiments. 

1)Measure the visible absorbance spectrum of the P450 heme iron and monitor how it changes when a drug ligand is added.  A plot of this binding titration can be used to calculate the affinity of the enzyme for that particular ligand.  The same experiment performed in the presence of a second drug molecule tells us if the second drug influences the metabolism of the first. 

2)Use NMR paramagnetic relaxation enhancement experiments (requires a series of 1D NMR experiments and some curve fitting of the intensities) to measure the distance between the paramagnetic heme iron atom and the protons on the bound drug molecule.  To convert the NMR PRE to a distance requires a known ligand-protein affinity (from UV/Vis titration) and a known iron spin state (calculate by simple deconvolution of a Visible absorbance spectrum).    Perform the same experiments again in the presence of a second drug ligand to determine if the second ligand alters the orientation of the first. 

3)Use Mass Spectrometry to identify P450 eryF labeled with the nitroxide spin label MTSL, including identifying the location of the label with tandem mass spectrometry.  Then use NMR spectroscopy (as in project 2) to measure the distance between the nitroxide spin label and the bound ligand molecule. 

4)Other related experiments are also possible, and some projects may be a subset of those listed above. 

Recent projects in my lab:

Students in my lab have identified the mass of P450 eryF in our LC-MS instrument, and identified a product of the chemical labeling reaction that matches the expected mass of the protein plus the chemical label.  

Students have performed visible absorbance spectroscopy to measure the affinity of the P450-ligand complex, along with curve fitting to the hyperbolic ligand binding equation.  This has also been done in the presence of a second ligand to show that addition of one equivalent of the second ligand has no effect on the affinity, but a second ligand interferes with binding of the first ligand. 

A student in my lab has also peformed the NMR PRE experiments to demonstrate that the ligands exchange fast enough to use the free ligand resonances to obtain information about the paramagnetic center on the protein.  These measurements need confirmation but are very exciting validation of our approach and future students will spend less time figuring out protocols and more time analyzing different ligand combinations.