Resonance Assignment/Principles and concepts

Introduction

This section is intended to introduce a few definitions and concepts in resonance assignment protocols. For in depth description of the process see e.g. Kurt Wütrich's book NMR of Proteins and Nucleic Acids (Wiley, 1986) and John Cavangh's et al. textbook Protein NMR Spectroscopy. Principles and Practice (2^nd edition, Elsevier, 2007). 

Stable isotope labeling schemes

Through the NESG consortium, the most prevalent isotope labeling schemes are as follows:

• 100% ¹⁵N, 100%¹³C-labeled (or doubly-labeled) samples are the main category, to which the majority of the information on this site applies. They are used for complete resonance assignments and structure calculation.
  
• 100% ¹⁵N-labeled samples are used for screening with 15N-HSQC. They can also find limited use in collecting RDC-type data.
  
• 100% ¹⁵N, 5-7% ¹³C-labeled samples are used to obtain stereospecific assignment of Val and Leu side chain methyl groups, usually important for proper packing of hydrophobic core.
• 100% 14N, 100% 12C (or unlabeled) or alternatively natural abundance samples can be used in 50%-50% mixtures for homodimer structure determination.
  

In addition to these labeling schemes, one can find it useful, especially larger proteins to have selectively labeled samples, such as SAIL NMR (http://www.sailnmr.org/). To reduce signal broadening due to spin-spin relaxation, it may be advantageous to deuterate the protein to a certain level.

NMR experiments

This section describes the types of connectivities that can be established between nuclei by given experiments. For specifics and experimental setup, please refer to the NMR Data Collection section of this site.

Through bond

• ¹⁵N-HSQC - correlates a ¹⁵N nucleus and a 1H directly attached to it. Mainly used to identify backbone amide groups.
  
• ¹³C-HSQC - correlates a ¹³C nucleus and a 1H directly attached to it. Can cover aliphatic and/or aromatic range of ¹³C chemical shifts
  
  • Constant time - enhances resolution. Peak sign gives information on the number of ¹³C neighbors a given ¹³C nucleus has
• HNCO, 3D - correlates a backbone amide with the C' of preceding residue or a Cγ/Cδ of Asn/Gln with NH₂ of the side chain
• HNCA, 3D - correlates a backbone amide with the Cα of the same and preceding residues
• HNCACB, 3D - correlates a backbone amide with the Cα and Cβ of the same and preceding residues. Usually has Cβ and Cα peaks of opposite signs
• CBCA(CO)NH, 3D - correlates a backbone amide with the Cα and Cβ of the preceding residue. Together with HNCACB, HNCA and HNCO it is used to assign the backbone chemical shifts.
  
• HBHA(CBCACO)NH, 3D - correlates a backbone amide with the Hα and Hβ resonances of the preceding residue. Used together with HCCH-type experiments and ¹³C-HSQC to assign the side chain resonances.
  
• H(C)CH-COSY, 3D - correlates an aliphatic C-H pair with an adjacent aliphatic ¹H of the same side chain. Closely related experiment is H(C)CH-TOCSY, 3D, which correlates an aliphatic C-H pair with all aliphatic ¹H nuclei of the same side chain.
• (H)CCH-COSY, 3D, and (H)CCH-TOCSY, 3D, are very similar to above, except the third correlated nucleus is ¹³C
• All HCCH-type spectra can also be taylored for the aromatic region of 13C range. Correlations would be detected only within an individual aromatic ring.

(HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE, 2D - correlate Hδ and H<math>\epsilon</math> aromatic protons with Cβ of the same residue. Useful for assigning chemical shifts of nuclei in aromatic rings.

Long range 15N-HSQC, 2D - correlates Hδ2 and H<math>\epsilon</math>1 protons on His rings with Nδ1 and N