The Fessenden laboratory studies fundamental Ca2+ signaling mechanisms in skeletal muscle through structural analysis of the type 1 ryanodine receptor (RyR1), an enormous intracellular Ca2+ release channel which plays a pivotal role in skeletal muscle excitation contraction (EC) coupling.
In this process, muscle fiber depolarization detected by the Cav1.1 voltage-gated Ca2+ channel activates RyR1, thereby releasing Ca2+ stored in the sarcoplasmic reticulum (SR), which in turn activates muscle contraction. In humans, single missense mutations in RyR1 can cause the skeletal muscle disorders malignant hyperthermia (MH) and central core disease (CCD). Virtually nothing is known as to how these point mutations affect the structure of RyR1 nor how these structural changes in the protein lead to these debilitating skeletal muscle disorders.
We have developed a method to measure structural changes in RyR1 using fluorescence resonance energy transfer (FRET), a technique which has been used as a “molecular ruler” to measure distances within or between proteins. A prerequisite first step in these studies is to site-specifically label RyR1 with the fluorophores required for FRET measurements. The method we have adapted for these studies takes advantage of the strong interaction between Ni2+/nitrilotriacetic acid (NTA) complexes and polyhistidine “tags” used for protein purification. By coupling NTA to the fluorophore, Cy3 (Figure, top), the resulting compound (Cy3NTA) can be specifically targeted to His tags engineered into RyR1 containing a fused GFP to act as a fluorescence donor. FRET can then be measured as a decrease in GFP fluorescence when Cy3NTA is bound to a nearby His tag. Thus, both static FRET measurements, to provide low resolution topological information about RyR1, and changes in FRET after RyR modulation, to measure conformational changes in the protein (Figure, bottom), can potentially be conducted using this assay system.
- James D. Fessenden, Ph.D
- Mohana Mahlingam, Ph.D.
- Tanya Girgenrath, M.S.
- Fessenden, J.D. (2009). Forster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method. PLoS One. 4, pp. e7338.
- Raina, S. A., Tsai, J., Samso, M. and Fessenden, J.D. (2012) FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1. PLoS One. 7(6), pp. e38594.
- Geldart, A., Fessenden, J.D., Arons, E., Mitsialis, A., Kourembanas, S. and Christou, H. (2012) Extracellular acidosis alters the metabolic phenotype and susceptibility to apoptosis of pulmonary artery smooth muscle cells. Clin. and Exp. Med. Sci. vol. 1, pp. 141-160 (In press).