Poster abstracts
Poster number 23 submitted by Shaun Christie
SHARP RNA recognition motif optimizations, extensions, and mutations for use in 2D and 3D NMR experiments
Shaun Christie (University of Akron), Joel Caporoso, Louis Ray, Dr. Thomas Leeper (University of Akron)
Abstract:
SMRT/HDAC Associated Repressor Protein interacts with the long noncoding RNA, produced by SRA, by binding at the RRMs. Three projects were done to prepare the truncated proteins for use in 2D and 3D NMR experiments. The first focuses on RRM 3 and its optimiza- tion during the purification process. The second focuses on RRM 2-4, which was found to be missing two alpha helices that may be important for protein stability. These helices can also in- teract with RRM 3 as well due to the tight association of RRMs 3 and 4. The two step PCR ex- tension of RRM 2-4 was assumed to work based on sequencing and agarose gels. The final project focused on serine to cysteine mutations of RRM 2 to allow site directed spin labeling for use in NMR PRE experiments. Both mutants developed in this project do not affect the protein structure or the residues surrounding the mutation based on wild type and mutant HSQC spec- tra. The spectra of the spin labeled samples does show broadening some resonances, but may need reductant removed from the buffer sfter the labeling reaction.
Keywords: Protein-RNA interaction, NMR PRE