Monitoring the Global Conformation of SRP During Co-translational Protein Targeting
EMSL Project ID
38493
Abstract
The signal recognition particle (SRP) is the major cellular machinery that mediates co-translational targeting of membrane and secretory proteins to the eukaryotic endoplasmic reticulum (ER) membrane, or the bacterial plasma membrane, thereby ensuring proper localization of proteins to their correct subcellular destinations. The functional core of SRP is a SRP54 protein containing a cargo-binding M-domain and a GTPase NG-domain. During the protein targeting reaction, information about cargo loading and unloading in the M-domain is actively communicated to the GTPase domains and vice versa, ensuring an ordered series of cargo loading, delivery, and unloading processes that is driven by conformational rearrangements in the GTPase domain. A central unresolved question is how these communications are established between the two functional domains of the SRP. To address this question, I propose to use site-directed spin labeling (SDSL) and pulsed electron paramagnetic resonance (EPR) to determine the intramolecular distances between pairs of spin probes incorporated into the M- and NG-domains of the SRP54 protein. Using 8-10 distance constraints, we will be able to map the relative orientation between the M- and NG-domains, thus defining the global structure of SRP54. We will also repeat these measurements when SRP54 interacts with its binding partners during the protein targeting reaction progresses. This work will significantly advance our understanding of the structural and molecular mechanism of this universally conserved protein targeting machine, and will potentially generate publications in high quality journals such as Nature Structural and Molecular Biology and Proceedings of the National Academy of Sciences. This project requires pulsed EPR measurements that allow distances up to 70 D to be measured. There are no publicly accessible facilities for such measurements at or near the Caltech campus. Hence access to the pulsed EPR facility at EMSL is crucial to continue data collection and to ensure the progress and success of this project.
Project Details
Project type
Exploratory Research
Start Date
2009-12-29
End Date
2011-01-02
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members