Poster abstracts

Poster number 3 submitted by David Bowles

Further Structural Elucidation of the Signaling Pheromone Er-23 using NMR Structural Techniques

David Bowles (OSBP), Alexandar Hansen (CCIC), Calvin Rhoads (OSU Chemistry and Biochemistry), Sidharth Mohan (Biophysics), Chunhua Yuan (CCIC)

Abstract:
Er-23 is a 51 residue signaling pheromone produced by the ciliated protozoan Euplotes raikovi that is structurally interesting due to a high glycine content (20%) and high cysteine content (20%). The ten cysteine residues form five disulfide bridges, resulting in a remarkable set of biophysical properties, such as extreme resistance to thermal denaturation. Previous work on Er-23 was performed on protein produced by homologous expression, preventing sample labeling with 15N or 13C. This limitation resulted in a solution structure from very limited constraints. The Magliery lab has been able to produce this protein using heterologous expression in Escherichia coli, originally intended for biophysical characterization and mutagenesis studies, but also for NMR studies utilizing 15N and 13C labeling. The Magliery lab was very interested in replacing disulfide bridges in Er-23 with alanine-valine pairs, but for this experiment to be meaningful, the locations of the disulfide bridges needed to be certain. 15N-H HSQC experiments previously inaccessible to the samples prepared with homologous expression show Er-23 to be a well-folded protein with a single primary conformation. Replication of a published COSY spectra shows that the E. coli heterologous expression product is the same fold as the homologous E. raikovi expression product. Biophysical characterization methods such as circular dichroism and IR spectroscopy show results not entirely consistent with the published NMR structure, and so 3-D NMR experiments such as HNCA and HNCOCACB were utilized to assign the backbone and side chains of the protein. After this, NOE data and residual dipolar coupling data was collected in order to provide three-dimensional constraints in order to determine a more accurate NMR structure so the true disulfide linkages can be determined.

Keywords: NMR Er-23 Disulfide NOE Structure