Unfolding RXRalpha Students: Nancy Doremus and Jessie Dodge-Gittins Mentors: Zeb Jones and Mike Stoops
Background Information • The way a protein is assembled and folded is very important in determining its purpose. It’s like a second genetic code. • RXRalpha is a receptor protein for retenoid (vitamin A derivatives) molecules. • These receptors are implicated in cell differentiation, mammalian development, and skin diseases like acne and psoriasis. Jessie weighing buffer materials
Purpose: • Understand the stability of RXRalpha • Comprehend dynamics of the protein unfolding and folding • Fit data to a theoretical folding model Jessie and Nancy getting ice
Process: • Purifying the RXRalpha protein • Checking for contamination • Testing concentration • Collecting data after exposure to a chemical denaturant Jessie using the pipette Nancy filling test tubes
Centrifuge The RXRalpha sample needed a higher concentration for the experiment, so the centrifuge was used to purify it. The centrifuge spun the protein at 4000 RPM’s for 40 minutes, which separated the protein from the filtrate. After testing the concentration of the protein, it was found that it wasn’t high enough, so the centrifuge was needed again. Looking down into the centrifuge Nancy and Jessie loading the centrifuge
Bradford Protein Assay The spectrophotometer measures the absorbency of light through a sample. It was used to find the concentration of the RXRalpha protein. Samples of known concentration ( Bovine )
Electrophoresis Electrophoresis gives a rough molecular weight range. SDS ( Sodium dodecyl sulfate ) in the gel acts as a surfactant. Surfactant denatures proteins allowing for band resolution as proteins migrate through an electric field. Multiple bands suggest contamination.
Fluorometer The fluorometer shines light through a sample which makes some amino acids fluoresce. The machine detects this and sends the data to a computer where it is compiled into a graph. A denaturant ( guanadine ) was added to the RXRalpha sample to divide the tetramers into monomers. This change is visible on the graphs.
Statistical Analysis • Import data into Excel • Calculate net fluorescence (subtracting buffer blank) • Calculate weighted mean wavelength < λ > : < λ > = Σ ( I * λ ) / Σ ( I ) λ = wavelength I = net fluorescence • Correlate calculated < λ > to known values.
Conclusions Weighted-mean average wavelengths correspond well with previous data. Each protein configuration has a concise average wavelength, distinguishable by several nanometers. The unfolding scheme appears to follow a progression from tetramer to dimer and finally, as the chemical denaturant (guanadine) concentration approached 5.5 M, monomer. Further experiments might assay the refolding of the protein as guanadine is diluted.