The focus of this proposal is to investigate signal sequence recognition by the E. coli signal recognition particle homologue, FfH, and to develop a model for inter-domain regulation in this protein which will have broad ramifications for the eukaryotic SRP as well. Proteins which are now synthesized within the cell must be correctly targeted to the proper membrane for membrane integration or secretion. In eukaryotic cells, this task is performed by the SRP complex which is made up of six protein subunits and a 7S RNA. One particular subunit, SRP54, is responsible for binding the myriad N-terminal signal sequences of nascent proteins as well as docking with the membrane-bound SRP receptor. These functions have been previously localized to the M and NG domains of SRP54, respectively; each of these domains appears capable of regulating the other. Recent evidence, however, suggests the possibility that the NG domain also participates in signal sequence binding. The experiments outlined in this proposal will examine signal sequence binding by the E.coli SRP54 homologue in an effort to understand how diverse signal sequences are specifically recognized, as well as to gain insight into the inter-domain conformation consequences of signal sequence binding.
These aims will be accomplished using a combination of mutagenesis of signal peptides and Ffh, transfer NOE to determine the bound peptide conformation, methionine oxidation to map the peptide binding site, and chemical crosslinking of peptides to Ffh. The results from these experiments will be incorporate into a model for Ffh signal sequence recognition.
| Swain, J F; Sivendran, R; Gierasch, L M (2001) Defining the structure of the substrate-free state of the DnaK molecular chaperone. Biochem Soc Symp :69-82 |
| Swain, J F; Gierasch, L M (2001) Signal peptides bind and aggregate RNA. An alternative explanation for GTPase inhibition in the signal recognition particle. J Biol Chem 276:12222-7 |
