Mapping the Structural Dynamics of the SRP Protein Transport Pathway
Aileen R. Ariosa
Mentors: Dr. Shu-ou Shan, Dr. Vinh Q. Lam
The signal recognition particle (SRP) and its receptor (SR) are components of a universally conserved cellular pathway responsible for targeting nascent polypeptides bound for membrane integration or secretion. E.coli SRP is a ribonucleoprotein comprised of a 4.5S RNA, a GTPase (NG-domain), and a methionine-rich domain (M-domain). The M-domain is responsible for binding to the RNA subunit and for recognizing the signal sequence of the target protein. However, little information is available about how the M-domain interacts with the NG-domain and how information is communicated between them. The objective of this project is to characterize the mobilities of various residues in the M-domain throughout the translocation pathway using site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR). Several single cysteine mutations were introduced into SRP’s M-domain wherein a thio-specific EPR free radical spin probe can be attached. Translocation assays were performed to ensure that these alterations do not hinder proper activity. The mobilities were measured and acquired using continuous wave (cw) EPR at X-band frequency.