1 / 10

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)

giza
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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…

More Related