Protein expression and purification
The proteins that we use in our studies are produced recombinantly as this allows us to manipulate the protein sequence using site directed mutagenesis. These proteins are produced in bacteria grown in culture in a large format New Brunswick shaker incubator. Following expression, the proteins are purified using a GE Healthcare AKTA FPLC.Once purified the protein are lyophilized and stored at -20 for future use.
Stopped flow kinetic analysis
To determine the mechanism by which a change in protein sequence alters the calcium affinity of a troponin C mutant, stopped flow kinetic analysis is used to measure the rate of calcium disassociation from the protein (koff).This technique uses changes in fluorescence in real time to measure the rate. The fluorescence is produced by a fluorescent reporter that has been engineered into one of the component proteins, such as troponin C, or by a fluorescent chelator. For these measurements we use a Stopped Flow Kinetic Analyzer from Applied Photophysics. This instrument has a dead time of 1.2 msec.
Steady state measurement of troponin calcium affinity
To characterize how changes in protein sequence alter the ability of troponin C (isolated or in complex) to be activated by calcium, we monitor changes in protein confirmation using fluorescence. The fluorescence is produced by a reporter molecule that we have engineered into the protein. As the protein, or protein complex, is activated by Ca2+ itâ€™s confirmation changes, as a result the position of the fluorescent probe in the molecule changes influencing itâ€™s exposure to solvent. As a result, the fluorescent properties change. Fluorescence is measured using a Perkin Elmer LS 45 Luminescence Spectrometer.
Biomechanics of muscle contraction
To characterize how changes in the structure/function of contractile proteins influences the Ca2+ activation of cardiac tissue we utilize a custom mechanics instrument from Aurora Scientific. This instrument enables the active and passive properties of cardiac trabeculae and single skeletal muscle fibers to be characterized. This instrument, built on an inverted microscope consists of a force transducer and a high speed length controller in close proximity to a robotic 8 well aluminum bath plate. In an experiment, a muscle preparation is mounted to the force transducer and length controller and then submerged in one of the 8 wells. The remaining 7 wells are filled with physiological solutions of varying Ca2+ concentration. Following each serial submersion the force generated by the preparation is measured by the force transducer. In addition the rate of force generation is measured using quick changes in muscle length. This instrument utilizes high speed video and a laser diode to monitor sarcomere length during each experiment.
Images of a skinned trout cardiac trabeculae preparation mounted to the muscle mechanics system. (A) Trabeculae preparation attached to force transducer and servomotor using aluminium foil clips. (B) Image of sarcomere pattern through 40× objective. The yellow box represents a typical sarcomere pattern that was analyzed using fast Fourier transformation to measure sarcomere length (SL).
Sample force trace resulting from the Ca2+ activation of a trout cardiac trabeculae preparation at 15°C and SL 2.2 μm. The noise on the force trace is due to motor movements during rate of force redevelopment (ktr) measurements (*) and movement of preparation between solution wells (↓). Force is displayed as mN mm–2 and pCa values are indicated along the bottom of the trace.
2D Gel electrophoresis
To characterize how environmental stressors influence protein expression in the vertebrate heart we are utilizing a Ettan 2D Gel electrophoresis system from GE Healthcare. 2D gel electrophoresis first separates proteins based on their isoelectric point (IP) and then by their molecular weight. As IP is determined by the amino acid composition, 2 proteins that have the same mass can be separated if they have a different composition or if one has been phosphorylated as both of these change the IP. Our 2D system is composed of the Ettan IPGphor III Isoelectric Focusing Unit and the EttanDALTsix System. The isoelectric focusing system (1st D) allows for 12 IPG strips to be run simultaneously and the EttanDALTsix System (2nd D) runs gels that are 24 cm wide. This system enables 2D DIGE experiments where we directly compare the protein complement of 2 samples on the same gel. This technique uses different cye dyes to differentially label the proteins. These dyes are size and charged matched so that they do not differentially influence how the same protein from 2 samples runs on the gel. The gels are imaged using a Typhoon scanner and then the protein constellations compared. Proteins are identified on the gels using protein standards as well as mass spectrometry.