P50 GM073197 JCIMPT NMR Team ____________________ Dr. Reto Horst Dr. Pedro Serrano Dr. Pawel...

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Screening of New Detergents Dr. Reto Horst Dr. Qinhai Zhang Michael Geralt Financial Support through JCIMPT (PI Dr. Ray Stevens) by the NIH Roadmap Initiative and through JCSG (PI Dr. Ian Wilson) by the NIH PSI.

Transcript of P50 GM073197 JCIMPT NMR Team ____________________ Dr. Reto Horst Dr. Pedro Serrano Dr. Pawel...

P50 GM JCIMPT NMR Team ____________________ Dr. Reto Horst Dr. Pedro Serrano Dr. Pawel Stanczak __________________________________________________ Dr. Kurt Wthrich Screening of New Detergents Dr. Reto Horst Dr. Qinhai Zhang Michael Geralt Financial Support through JCIMPT (PI Dr. Ray Stevens) by the NIH Roadmap Initiative and through JCSG (PI Dr. Ian Wilson) by the NIH PSI. 600 L5 L Gilson Liquid Sample Handler Bruker Automated Sample Changer 1 mm diameter capillary tube vs 5 mm diameter tube NMR Screening Setup [ 15 N, 1 H]-TROSY spectra of refolded [ 2 H, 15 N]-labeled OmpW in: DHPCFos30 NMR BUFFER: 20 mM Na Phosphate pH = mM NaCl 0.3 % (v/v) NaN 3 10 % (v/v) D 2 O 2 % (w/w) DHPC NMR BUFFER: 5 mM Na Phosphate pH = mM NaCl 0.3 % (v/v) NaN 3 10 % (v/v) D 2 O 1 % (w/w) Detergent Reconstitution of OmpX from inclusion bodies Detergent DHPCNew Detergents NMR Sample Preparation of OmpX Micro-scale 1D 1 H NMR Measurement of Detergent Concentration NMR Sample Preparation of OmpX Monitoring Detergent Concentration Using Micro-coil 1D 1 H NMR Impact of Protein Concentration 1.7 % Fos-10 [OmpX] = 0.84 mM 3.3 % Fos-10 [OmpX] = 1.04 mM 28.5 % Fos-10 [OmpX] = 0.77 mM [ 15 N, 1 H]-TROSY-NMR 1.7 % Fos-10 [OmpX] = 0.84 mM 3.3 % Fos-10 [OmpX] = 1.04 mM 28.5 % Fos-10 [OmpX] = 0.77 mM [ 15 N, 1 H]-TROSY-NMR, cross-sections 1.7 % Fos-10 [OmpX] = 0.84 mM 3.3 % Fos-10 [OmpX] = 1.04 mM 28.5 % Fos-10 [OmpX] = 0.77 mM [ 15 N, 1 H]-TROSY-NMR, cross-sections