Backbone resonance assignment in large protonated proteins using a combination of new 3D TROSY-HN(CA)HA, 4D TROSY-HACANH and 13C-detected HACACO experiments

J Biomol NMR. 2003 May;26(1):69-77. doi: 10.1023/a:1023008719248.

Abstract

A new method for backbone resonance assignment suitable for large proteins with the natural (1)H isotope content is proposed based on a combination of the most sensitive TROSY-type triple-resonance experiments. These techniques include TROSY-HNCO, (13)C'-detected 3D multiple-quantum HACACO and the newly developed 3D TROSY multiple-quantum-HN(CA)HA and 4D TROSY multiple-quantum-HACANH experiments. The favorable relaxation properties of the multiple-quantum coherences, signal detection using the (13)C' antiphase coherences, and the use of TROSY optimize the performance of the proposed set of experiments for application to large protonated proteins. The method is demonstrated with the 44 kDa uniformly (15)N,(13)C-labeled and fractionally (35%) deuterated trimeric B. Subtilis Chorismate Mutase and is suitable for proteins with large correlation times but a relatively small number of residues, such as membrane proteins embedded in micelles or oligomeric proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbon Isotopes
  • Magnetic Resonance Spectroscopy / methods*
  • Models, Theoretical
  • Nitrogen Isotopes
  • Protein Conformation
  • Proteins / chemistry*
  • Protons
  • Quantum Theory
  • Sensitivity and Specificity

Substances

  • Carbon Isotopes
  • Nitrogen Isotopes
  • Proteins
  • Protons