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Insights into Protein-Ligand Interactions and Structural Dynamics via REDOR and MAS Techniques

This work explores the characterization of protein-ligand geometries, particularly focusing on the EPSP synthase inhibitor and its interactions with peptides such as N-acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2. Utilizing advanced techniques like Rotational Echo Double Resonance (REDOR) and 2D Magic Angle Spinning (MAS), the study analyzes secondary structures, internuclear distances, and backbone dihedral angles. The relative tensor orientations provide insights into the local backbone conformations and the dynamical behavior of multiply labeled peptides and proteins, revealing important structural information crucial for understanding amyloid fibril formation.

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Insights into Protein-Ligand Interactions and Structural Dynamics via REDOR and MAS Techniques

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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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