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Solid-state NMR of Oriented Biological Samples: Methods and Practice

Solid-state NMR of Oriented Biological Samples: Methods and Practice. Center for NMR Spectroscopy & Imaging of Proteins The Bubble @UCSD Tuesday, March 15, 2005 12:15 Sign-in, light lunch ** 1:00 What is the RCN for NMR of Biological Solids? (R. Stark)

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Solid-state NMR of Oriented Biological Samples: Methods and Practice

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  1. Solid-state NMR of Oriented Biological Samples: Methods and Practice Center for NMR Spectroscopy & Imaging of Proteins The Bubble @UCSD Tuesday, March 15, 2005 12:15 Sign-in, light lunch** 1:00 What is the RCN for NMR of Biological Solids? (R. Stark) 1:05 The NMR Research Resource at UCSD (S. Opella) 1:15 Tailoring NMR Experiments for Oriented Samples (A. Wu) 1:30 NMR Probe Design for Oriented Samples (C. Grant) 1:45 Sample Preparation for Bilayer Samples (F. Marassi) 2:00 Sample preparation for Bicelle Samples (A. de Angelis) 2:15 Laboratory tour and discussions (Hosts) **RSVP to brocato@mail.csi.cuny.edu and see http://nmrresource.ucsd.edu/index.html

  2. Why choose ssNMR for protein structure? • Can’t crystallize for x-ray crystallography • Can’t dissolve at 0.5 mM • Protein dissolves but aggregates or dissociates in solution • Motional averaging in solution is insufficient, T2 is too efficient for solution NMR (MW > 50 kDa, gels, membrane media) • Don’t want to dissolve in aqueous media (integral membrane proteins, fibrils, aggregates) • Want to study weak protein-protein or protein-ligand complexes

  3. What can we learn about the proteins? • Pairwise to distinguish proposed conformations, interactions • Secondary structure • De novo structures • (dynamics)

  4. Challenges: sample preparation Microcrystals, nanocrystals, glasses, PEG precipitants, lyophilates

  5. Challenges: sensitivity • Lower T • Observe 1H?

  6. Challenges: resolution • 0.5-1 ppm 13C in well ordered pp’s (Baldus) • 0.2-0.5 ppm 15N w/o microcrystals (Polenova)

  7. Strategies: nD CPMAS Broad lines (CSA, DD) Poor S/N (dilute spins, long T1(C)) Transfer polarization from 1H to 13C Repeat as permitted by short T1(H) Remove heteroDD interactions Average chem shifts to liquid- state values if (3 cos2 - 1) = 0 Combined advantages: high resolution spectra Note that D dominates Luca, Heise, Baldus, 2003

  8. N(CA)CX coherence transfer SIMPSON & sss for numerical simulation of pulse seqs: efficiency, pwr req, etc. Label atoms on coherence plot Bjerring,… Nielsen, 2003

  9. Challenges: hardware • Amplifier: power droop, T stability, etc. • Probe: arcing, capacitor burn-up, rf homogeneity • Spin rate: 20 kHz to avoid 13C sidebands; >40 kHz for 1H detection; stability for synchronized sequences • Sample heating (decoupling, pulse trains that suppress HH interactions) • Lossy samples (high salt) Vosegaard T, Nielsen NC, J Biomol NMR,22, 225-47 2002.

  10. Pairwise interactions & structural Q’s • What is the conformation of the retinal chromophore in dark-adapted bacteriorhodopsin (bR)? • RFDR (define) recouples 13C-13C pairs to identify proximal carbons, enhances resolution with MAS, requires laborious specific labeling • bR555: r = 3.1 Å  syn +  • bR568: r = 3.9 Å  anti * • 2D spectrum here Structures Go over reasoning! Ref: Griffin… 1994 NOT protein!! Included in Tycko fibril…

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