2-D NMR and more EPR CW-pulsed二维NMR与EPR CW脉冲.pptVIP

Practical Course in NMR and EPR(Part 2) 2006 Web-related stuff The powerpoints and handouts will be available at: Tons of web-related NMR resources can be found at: /labwiki/NEX/NEX1/nmr_resources.htm Web-related EPR resources can be found at: /labwiki/NEX/NEX1/epr_resources.htm Review Energy Levels Magnetic Spin Chemical Shift Splitting of Energy Levels (j-coupling) Different Nuclei gyromagnetic ratio natural abundance Relaxation T1 relaxation T2 relaxation Chemical Exchange Saturation Transfer NOE/ROE Spin Diffusion Why do 2D-experiments? Spectral crowding Through-bond interaction Through-space interaction Simplify spectra (Specific interaction) What do you need? 1 mM of your small molecule for 2D-proton 12 hours or more experiment Idealized System 1D-spectrum 2D-spectrum 2D NMR methods for determining small molecule NMR structure 2D-Correlation Spectroscopy (COSY) Through-bond Saturation transfer between J-coupled nuclear spins. 2D-Nuclear overhauser effect spectroscopy (NOESY) or 2D Rotating frame overhauser effect spectroscopy (ROESY) Through-space Saturation transfer between nuclear spins. COSY COSY COSY Can’t Differentiate NOESY NOESY What if your small molecule is ~1000 Daltons? NOE and Molecular Weight ROE and Molecular Weight ROESY Example of a ROESY So you want to solve a protein NMR structure Required Protein Properties 60 KD MW Soluble/monomeric Stable for days at room temperature 200 mM protein Time 2 days (2D-experiment) – 1 month (3D-experiment) per experiment Project: High throughput small protein (months) versus large protein (years) General Steps for Determining Protein Structure Step 1: Make amino acid assignments Step 2: Find NOE restraints Step 3: Run molecular dynamics (MD) simulation with NOE restraints Step 1: Protein NMR assignment 2D-NMR techniques for amino acid assignment 15N-1H Heteronuclear Single Quantum Correlation (HSQC) 3D-NMR techniques for amino acid assignment HNCO, HN(CA)CO, HNCA 2D-NMR for amino acid assignment 15N-