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Protein-ligand geometry

Protein-ligand geometry. Schaefer EPSP synthase-inhibitor Rotatonal echo double resonance (REDOR). Secondary Structure. ( 13 C  ), ( 13 C  )  local backbone conformation Internuclear distances  backbone dihedral angles Relative tensor orientations  backbone dihedral angles

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Protein-ligand geometry

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  1. Protein-ligand geometry • Schaefer EPSP synthase-inhibitor • Rotatonal echo double resonance (REDOR)

  2. Secondary Structure • (13C), (13C)  local backbone conformation • Internuclear distances  backbone dihedral angles • Relative tensor orientations  backbone dihedral angles • N-acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2 (A16-22) ppt’d fibrils CTDQFD (RFDR) both Chem synthesis Selective, [U-13C,15N] 1-5 mg 2D MAS exchange -strand C=O CSA tensors distances Balbach,… Tycko, 2000

  3. Anisotropic interactions & angular restraints C=O CSA tensors dipolar tensors: NH/NH, NH/CH, CH/NH Luca, Heise, Baldus, 2003

  4. Supramolecular Organization • MQ signal   m nearby nuclei • 13C-15N REDOR  distance  6 Å • N-acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2 (A16-22) In-register, parallel * * * antiparallel * * * Balbach,… Tycko, 2000

  5. Multiply labeled peptides & proteins: resonance assignments • [U-13C,15N] protein produced biosynthetically • Amino acid type: 1Q-1Q CC (1H-driven spin diffusion) or 1Q-2Q CC (e.g., POST-C7) • Intra-residue: NCACB • Sequential: NCOCACB • Sidechain: CXCYCZ • Thioredoxin example to follow… Ala-Gly-Gly. Luca, Heise, Baldus, 2003

  6. Multiply labeled peptides & proteins: internuclear distance restraints 1H-mediated (3D) CCHHC NHHCC Long-range distances 1H-mediated -selective recoupling of weak CC or CN interactions Spin dilution (1H/2H, 13C blocks) Ala-Gly-Gly. Luca, Heise, Baldus, 2003

  7. Multiply labeled peptides & proteins: de novo structures • [U-13C, 15N]blocks of Transthyretin (TTR105-115) in amyloid fibers • 13C linewidths  1 ppm  surprising long-range order (stability?) • 76 distance & dihedral angle restraints • Extended -strand backbone, precise side-chain conformation • No supramolecular structure Jaroniec,… Griffin, 2004

  8. Multiply labeled peptides & proteins: de novo structures • [U-13C, 15N] blocks of microcrystalline 62-aa -spectrin SH3 domain • Almost complete 13C & 15N assignments NCACB • XHHY for long-range distance restraints to 7Å • interresidue restraints: 286 CC, 6 NN Castellani,… Oschkinat, 2002

  9. References • JJ Balbach, Y Ishii, ON Antzutkin, RD Leapman, NW Rizzo, F Dyda, J Reed, R Tycko, Biochemistry,39, 13748-13759, 2000. • F Castellani, B van Rossum, A Diehl, M Schubert, K Rehbein, H Oschkinat. Nature. 420, 98-102, 2002. • S Luca, H Heise, M Baldus, Acc. Chem. Res.,36, 858-865, 2003. • M Bjerring, T Vosegaard, A Malmendal, NC Nielsen, Concepts Magn. Reson.18A, 111-129, 2003. • LM McDowell, B Poliks, DR Studelska, RD O'Connor, DD Beusen, JSchaefer,J Biomol NMR28, 11-29, 2004 • CP Jaroniec, CE MacPhee, VS Bajaj, MT McMahon, CM Dobson, RG Griffin, Proc Natl Acad Sci U S A,101, 711-6, 2004. • H Förster, Bruker Reports, Spring, 2004

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