We are currently co-crystallizing P-Rex1 PH with these small molecules to verify that they target the PIP 3-binding site. CONCLUSIONS: We have determined the structure of the P-Rex2 PH domain and have analyzed binding of small molecules to P-Rex1 PH, P-Rex2 PH, and Akt PH. Compound #13 (20 μM) was most notable in causing a 15.5 ☌ shift for P-Rex1, 22.5 ☌ for P-Rex2, and 13.5 ☌ for Akt PH. IP 4, a soluble analog of PIP 3, resulted in a 12.3 ☌ shift in T mfor P-Rex1 PH and a 15.4 ☌ shift for P-Rex2. I don't have any DSF files myself, so I have never tested this or looked deeply at it, but I strongly suspect that you are just seeing internal limits not that you. P-Rex2 and Akt PH (a kinase PH domain which has been successfully targeted with small molecule inhibitors) were used to further assess compound binding to a variety of PH domains. DaveBlake Wrote: With DSF format music files the tag reading is done by ffmpeg, and that is limited on what tags it processes compared to TagLib that is used for other file formats. Using DSF, we identified six small molecules from a high-throughput screen that bind the P-Rex1 PH domain. A comparison between the P-Rex1 PH and P-Rex2 PH structures shows divergence in the β3/β4 loop, shown by our lab to play a role in non-specific membrane localization, and the β5/β6 loop, a likely protein-protein interaction site. RESULTS: Crystallographic studies led to a high-resolution structure (1.90 Å) of the independent P-Rex2 PH domain. A DSF assay was used to analyze the binding interaction between compounds and PH domains. Final purity results from size exclusion chromatography. Recombinant protein was purified using Ni-NTA resin chromatography followed by a cleavage of the MBP tag with TEV protease. coli (Novagen) cells were used to overexpress P-Rex2 PH constructs as N-terminally 10xHis-tagged maltose binding protein (MBP)-fusion proteins, which increases yield and facilitates purification. My objective is to determine the structure of P-Rex2 PH and characterize the interaction of small molecules with different PH domains through qualitative analysis with differential scanning fluorimetry (DSF) and detailed assessment through X-ray crystallography.
However, the structural basis behind functional regulation with P-Rex is poorly understood.
#Taggig dsf with metadatics free#
The P-Rex family includes P-Rex1 and P-Rex2, which are activated by both the lipid PIP 3, generated by activated receptor tyrosine kinases, and free Gβγ subunits, generated by activated GPCRs. BACKGROUND: Phosphatidylinositol 3,4,5- trisphosphate (PIP 3)-dependent Rac exchanger 1 (P- Rex1) is a Rho guanine-nucleotide exchange factor (RhoGEF) that regulates cytoskeletal rearrangement and cell motility and is linked to tumor metastasis.