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Journal Article
Research Support, Non-U.S. Gov't
Review
Detection of intermolecular NOE interactions in large protein complexes.
Progress in Nuclear Magnetic Resonance Spectroscopy 2016 November
Intermolecular NOE interactions are invaluable for structure determination of biomolecular complexes by NMR and they represent the "gold-standard" amongst NMR measurements for characterizing interfaces. These NOEs constitute only a small fraction of the observed NOEs in a complex and are usually weaker than many of the intramolecular NOEs. A number of methods have been developed to remove the intramolecular NOEs that interfere with the identification of intermolecular NOEs. NMR experiments used to observe intermolecular NOE interactions in large protein complexes must cope with the short T2 relaxation time of the protons and heteronuclei in these complexes because they result in severe losses in sensitivity. The isotope-edited/isotope-filtered experiment is a powerful method for extraction of intermolecular NOEs in biomolecular complexes. Its application to large protein complexes is limited because of severe losses in signal-to-noise ratio caused by delays in the pulse sequence necessary for the multiple magnetization transfer steps between protons and heteronuclei. Isotope-edited/isotope-edited experiments, in which one protein is usually labeled with (13)C and the other is labeled with (15)N, reduce possible artifacts in the filtering experiments and improve somewhat the sensitivity of these experiments. Sensitivity can also be improved by deuteration of the components of the complex in order to replace either or both of the filtering or editing steps. Asymmetric deuteration, where aromatic residues in one protein and non-aromatic amino acids in the other are reverse protonated, can eliminate the editing and the filtering steps altogether, thus maintaining high sensitivity even for large proteins complexes. Difference spectroscopy and the use of 2D NOESY experiments without using editing or filtering steps can significantly increase the signal-to-noise ratio in experiments aimed at observing intermolecular NOEs. The measurement of NOESY spectra of three different preparations of a heterodimeric complex under investigation in which one or neither of the components is uniformly deuterated, and calculation of a double difference spectrum provides information on all intermolecular NOEs of non-exchangeable protons. Recent studies indicate that many protein-protein interactions are actually between a protein and a linear peptide recognition motif of the second protein, and determinants represented by linear peptides contribute significantly to the binding energy. NMR is a very versatile method to study peptide-protein interactions over a wide range of binding affinities and binding kinetics. Protein-peptide interactions in complexes exhibiting tight binding can be studied using single and/or multiple deuteration of the peptide residues and measuring a difference NOESY spectrum. This difference spectrum will show exclusively intra- and intermolecular interactions of the peptide protons that were deuterated. Transferred nuclear Overhauser spectroscopy (TRNOE) extends NMR to determine interactions within and between a weakly-bound rapidly-exchanging peptide and its protein target. TRNOE, together with asymmetric deuteration, is applicable to complexes up to ∼100KDa and is highly sensitive, taking advantage of the long average T2 of the peptide protons. Among the methods described in this review, TRNOE has the best potential to determine intermolecular NOEs for the upper molecular weight limit of proteins that can be studied in detail by NMR.
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