Structural characterization of free and fibronectin-bound anastellin
EMSL Project ID
3331
Abstract
Anastellin is a carboxy-terminal fragment of the 1st FN3 domain from human fibronectin. It is capable of polymerizing fibronectin in vitro and it displays anti-tumor, anti-metastatic and anti-angiogenic properties in vivo. We determined the structure of anastellin in the presence of the non-denaturing zwitterionic detergent CHAPS, which bound to the protein and stabilized it in a conformation suitable for NMR investigation. However, in the absence of CHAPS, anastellin exhibits conformation exchnge and dynamic fluctuations in solution that result in extensive line broadening and virtually no crosspeaks in 1H-15N HSQC spectra. We plan to characterize this partially ordered state and identify the features that are important for fibronectin polymerization. We will compare structural properties of free anastellin and anastellin bound to fibronectin fibrils. To accomplish the former, we will emply denaturant titration experiments. By tracking the appearance of individual resonances in 1H-15N HSQC spectra as a function of the concentration of urea, differential stability structural elements in the partially ordered state can be evaluated. Comparison of data collected for refolded recombinant anastellin, which induces fibronectin polymerization, and anastellin prepared without refolding, which is inactive, will enable us to correlate structural stability with the intriguing functional activity of this molecule. To characterize the bound state, we will map solvent accessibility of individual residues in anastellin when it is bound to fibronectin fibrils, and compare it with accessibility in the free protein. We will employ H/D exchange of the fibrils, followed by sissolution of the fibrils in DMSO and NMR analysis. For these experients, we request a total of three weeks of 600 MHz and two weeks of 750 MHz spectrometer time to perform the denaturant titrations and H/D exchange experiments, and to acquire double and triple resonance data needed to assign resonances of the denatured protein in the presence of urea and in DMSO. All the experiments for this challenging project will greatly benefit from data acquisition at higher field to relieve spectral overlap and improve resolution.
Project Details
Project type
Capability Research
Start Date
2003-04-15
End Date
2004-04-09
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members