1 / 11

HTP Target protein TR8

HTP Target protein TR8. TR8. 133 a.a. + 22 a.a. in His-tag Correlation time is 10.3 ± 0.3 ns Sample concentration <1 mM Monomer (2D H-C planes of HNCO and TROSY-HNCO have equal sensitivity).

edana
Download Presentation

HTP Target protein TR8

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. HTP Target protein TR8

  2. TR8 • 133 a.a. + 22 a.a. in His-tag • Correlation time is 10.3±0.3 ns • Sample concentration <1 mM • Monomer (2D H-C planes of HNCO and TROSY-HNCO have equal sensitivity). MGTSHHHHHH SSGRENLYFQ GHMWNDLAVY IIRCSGPGTR VVEVGAGRFL YVSDYIRKHS KVDLVLTDIK PSHGGIVRDD ITSPRMEIYR GAALIYSIRP PAEIHSSLMR VADAVGARLI IKPLTGEDIV TERKMKLVNY GRTYFYEYIA EVRSR

  3. TR8 - AA composition

  4. TR8 – NC Samples • Sample 1 (March 7)- 450 mM salt buffer- salvaged after his-tag cleavage attempt; cleaved construct oligomerizes- 40% concentration of sample 2 • Sample 2 (March 15)- 300 mM salt buffer- still 17us 1H 90deg pulse- fine precipitation after NYSBC data collection • Both samples yield identical 15N HSQC spectra • Higher temperatures (up to 35 C) don’t have an effect on 15N HSQC • Higher temperatures make samples cloudy

  5. [15N,1H] HSQC • 197 peaks observed in HNCO • 148 peaks expected • SN ~18UBNMR > predict 0.7 18.3 9 RESULTS: For 95% T3d_hnncabca: 1006.24 T3d_cabcaconh: 292.657 T3d_habcabconhn: 728.519 For 85% T3d_hnncabca: 388.825 T3d_cabcaconh: 113.087 T3d_habcabconhn: 281.509 • Conventional experiments

  6. Spectra • 800 MHz BRUKER- 2D [15N,1H] HSQC- 2D [15N,1H] TROSY- 2D [13C,1H] HSQC ali- 3D HNCO (13 h)- 3D HNCACB (44 h)- 3D CBCA(CO)NH (21 h)- 3D HBHA(CBCACO)NH (24 h) - 3D simNOESY (48 h)- 3D HNCA (20 h) • 600 MHz Varian - 3D aliphatic (H)CCH-COSY (24 h)- 3D aromatic (H)CCH-COSY (3.5 h) - 3D aliphatic (H)CCH-TOCSY (19 h)- 3D His TROSY-HCN (18 h)

  7. BB assignment • 16/153 residues are not assigned4/133 in the protein proper MGSSHHHHHH SSGLVPRGSHMWNDLAVYII RCSGPGTRVV EVGAGRFLYVSDYIRKHSKV DLVLTDIKPS HGGIVRDDIT SPRMEIYRGA ALIYSIRPPA EIHSSLMRVA DAVGARLIIKPLTGEDIVTE RKMKLVNYGR TYFYEYIAEV RSR

  8. Minor component • Observed for ~40% of strips • Minor to major ratio is 1:2 • Exchange peaks can be seen in NOESY spectra • Minor component had to be assigned in order to complete the assignment of major

  9. Secondary Structure Prediction • PSIPRED MGSSHHHHHH SSGLVPRGSHMWNDLAVYII RCSGPGTRVV EVGAGRFLYVchhhhhhhhh hhccccceee eeecccchhh SDYIRKHSKV DLVLTDIKPS HGGIVRDDIT SPRMEIYRGA ALIYSIRPPAhhhhhhhccc eeeeeeeecc ccceeecccc ccchhhhccc ceeeeeccch EIHSSLMRVA DAVGARLIIKPLTGEDIVTE RKMKLVNYGR TYFYEYIAEVhhhhhhhhhh hhcccceeee cccccccccc cceeeccccc eeeeeeehhh RSRhcc

  10. Secondary Structure CSI • Residues with a distinct minor component in HN strips are underlined • Most are in secondary structure elements MGSSHHHHHH SSGLVPRGSHMWNDLAVYII RCSGPGTRVV EVGAGRFLYVhhhhhhhhhh hhccccceee eeecccccch SDYIRKHSKVDLVLTDIKPS HGGIVRDDIT SPRMEIYRGA ALIYSIRPPAhhhhhhcccc eeeeeccccc ccccccceee c cccccccc ceeec cc EIHSSLMRVA DAVGARLIIKPLTGEDIVTE RKMKLVNYGRTYFYEYIAEVcchhhhhhhh hhcceeeeee eeeee eeeeccc eeceeeehhh RSRhcc

  11. Outlook • Side-chain assignment

More Related