Our Research

The goal of research in the Robia lab to understand how the heart muscle responds to the varying demands of exercise and rest, and how it becomes disordered in disease states. Our research program consists of several major areas:

The molecular physiology of cardiac signaling
Of particular interest are the dynamic interactions of membrane proteins with their regulatory partners. Such protein-protein interactions are the fundamental currency of cell signaling. Our goal is to quantify the kinetics and thermodynamics of these interactions to better understand how they transmit signals and regulate function in a living cell. Recently we have focused on several questions relating to the cardiac calcium pump “SERCA” and its regulatory partner, phospholamban (PLB). Unanswered questions include: How stable are these regulatory complexes? How rapidly do they bind and unbind? How do these parameters change when the proteins are phosphorylated or mutated? Phospholamban and SERCA have been shown to play an important role in several common diseases, including heart failure, so they are considered high-value targets for clinical treatments. Our hope is that by learning more about the biochemistry of these proteins, we can more effectively intervene in the disease process.

The structural biology of membrane proteins
Our experiments focus on the tertiary and quaternary structure of protein complexes. We are interested in static features of membrane protein structure (3D arrangement of domains in a protein) and dynamics (conformational changes). In a typical experiment, we evaluate structure models by measuring distances between site-directed fluorescent probes. Because these probes are genetically encoded, we can obtain such distance measurements by microscopy of live cells. This makes our experiments very complimentary to traditional structural biology methods such as X-ray crystallography and NMR. We take structural hypotheses generated from such in vitro experiments and test them in the natural environment of the living cell.

New directions
Building on ensemble measurements of populations of proteins, we will perform single molecule measurements in order to observe how protein structures change over time. Such reversible structure transitions may include 1) PLB pentamer conformation changes 2) SERCA enzymatic substate transitions and 3) PLB:SERCA complex structure fluctuations, depicted schematically below.

In addition to our ongoing experiments with phospholamban and SERCA, we are also beginning to look at other signaling proteins that are important for regulation of cardiac function. With our collaborators, we have investigated diverse proteins including calmodulin, Hax-1, phospholemman, G-protein coupled receptors, ubiquitin ligase, and protein kinases.

Fluorescence methods
Our lab uses a variety of fluorescence spectroscopy and imaging methods. Special techniques include Total Internal Reflection Fluorescence Microscopy (TIRF) and Fluorescence Resonance Energy Transfer (FRET).

We also use the Cell and Molecular Physiology Department's shared multiphoton microscope for fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS) experiments. A description of this resource can be found here.  This facility was constructed with the support of the McCormick Foundation.

Research in the Robia lab has been supported by the National Institutes of Health (National Heart, Lung, and Blood Institute, National Institute of Biomedical Imaging and Bioengineering), the American Heart Association