STRUCTURAL STUDIES OF THE GTPASE DOMAIN OF FFH, THE PROKARYOTIC SIGNAL SEQUENCE RECOGNITION PROTEIN.
Douglas M. Freymann*,
Thanh Lu & Yi Fan
Dept. of Molecular Pharmacology & Biological Chemistry, Northwestern
University Medical School, 303 E. Chicago Ave. Chicago, IL 60611
*Corresponding author: freymann@nwu.edu
Ffh is the prokaryotic homolog of the signal recognition particle protein, SRP54, which mediates recognition of the hydrophobic signal peptides of nascent secreted and membrane proteins, targeting them to the co-translational secretory pathway. The protein has three domains, the N-terminal and GTPase domains of which comprise a 30kD 'NG' domain which appears to form a structural and functional unit. The C-terminal M- domain mediates signal sequence recognition. The NG domain can be generated proteolytically from the intact protein. It has been crystallized, and its structure has been solved without bound nucleotide, and with bound GDP, at 2.0 Å resolution. Crystals of the apo- and GDP bound protein diffract to near 1.0 Å resolution, and our efforts are now directed towards obtaining complete data to that resolution from both crystal forms. Our goal is the refinement and analysis of these structures in order to improve our understanding of the protein structural elements which function in GTP binding and hydrolysis.
We have measured diffraction data to 1.10 Å using the MAR-CCD detector at DND-CAT 5-IDB. The crystals belong to space group C2, with unit cell dimensions a=99.85, b=53.63, c=58.10, b=119.5o. The figure shows a 1o oscillation frame collected using a 60s exposure, with a wavelength of 0.72 Å and detector distance of 70mm.
Figure: Diffraction to very high resolution from NG crystals.
The frozen crystals exhibit signs of decay at high resolution and we are focusing on this problem now.
We are also attempting to crystallize and solve the structure of the NG domain with various bound ligands. We have been able to obtain datasets from several candidate complexes and are now solving their structures using both molecular replacement and isomorphous replacement.
|
|
Resolution |
|
|
|
|
NGHIGH1 |
high resolution apo |
1.1 |
- |
processing data |
|
TESTGB2B |
high resolution GDP |
1.3 |
4.4 |
refinement started |
|
TLUVA2 |
experimental soak |
1.4 |
- |
processing data |
|
010A3 |
new NG complex |
1.7 |
4.6 |
molecular replacement |
|
TL007B4 |
new GDP crystal form |
2.0 |
6.0 |
difference map obtained |
|
TLUVA4 |
experimental soak |
2.0 |
- |
processing data |
Table: Representative datasets of Ffh NG crystals collected at DND-CAT 5ID-B