NMR experiments for protein sequence assignment

The very first experiment that one should record is a 1D-¹H NMR experiment to check for the quality of the protein. This basic experiment presents a fingerprint of all the protons present in the protein. If the protein is well folded, there should be well dispersed signals from the methyl protons present around 0 ppm. Another indication is the extent of spread of the signals in the 1D spectrum. In a folded protein, the chemical shift dispersion is much more than that in an unstructured protein where for example, the proton signals of the backbone amides will be clustered between 6-8 ppm as opposed to 6-10 ppm for a well folded protein. One thing to consider here is that if the protein is majorly alpha helical, then also the chemical shift dispersion could be minimal like that in case of unstructured protein.

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With increasing size of the protein, the complexity of the 1D-¹H NMR spectrum increases due to extensive signal overlap. Therefore, if the protein looks well folded, one can proceed with 2D-NMR experiments. If the correlation is measured between same nuclei, it is known as homonuclear 2D experiment while if measured between different nuclei, it is known as heteronuclear 2D experiment (for example, ¹H-¹⁵N or ¹H-¹³C). A 2D ¹H-¹⁵N HSQC (Heteronuclear Single Quantum Coherence) is a simple experiment to measure the correlation between ¹H and ¹⁵N nuclei of each amide bond. It represents the fingerprint of the protein as each backbone amide is represented as a single peak in the spectrum (except for proline residues) and is usually the first heteronuclear experiment that is recorded. Additionally, correlations between tryptophan side chain N-Hand asparagine/glutamine side chain N-H2/ N-H are also visible. The arginine N-H cross-peaks are also visible but as folded signals as the chemical shift of N falls outside of the region usually recorded. At low pH, arginine Nη -Hηand lysine N- are also visible, but again as folded signals.

For large proteins, the application of 2D-¹H-¹⁵N HSQC becomes limited due to spectral crowding especially in the central region, and due to transverse relaxation (T2) effects leading to broad linewidths that decrease the quality of the NMR spectrum. The T2 relaxation rates for high molecular weight proteins are high, which leads to rapid decay of the NMR signal.

Replacing protons (major source of T2 relaxation) with deuterons can help achieve better signal to noise (Gardner and Kay 1998). About two decades ago, 2D ¹H,¹⁵N-TROSY experiment (Transverse Relaxation Optimized Spectroscopy) was introduced (Pervushin, Riek et al. 1997, Salzmann, Pervushin et al. 1998). It correlates the same nuclei as ¹H,¹⁵N-HSQC but decreases relaxation effects to attain better linewidths, spectral resolution and sensitivity by selecting the coherence component where cancellation of relaxation due to dipolar coupling and chemical shift anisotropy occurs. It therefore extends the protein size limitation which could be studied by NMR (Fernandez and Wider 2003).

Next, to assign the correlations observed in the ¹H-¹⁵N HSQC spectrum, sequential protein assignment is done using triple resonance experiments whereby the backbone resonances of the protein are assigned sequentially. These include HNCA, HNCACB and CBCA(CO)NH (Shan, Gardner et al. 1996, Sattler, Schleucher et al. 1999) triple resonance experiments. The HNCA experiment is the most sensitive followed by CBCA(CO)NH and lastly HNCACB. In these experiments, correlation between backbone amide with Cand Cresonances is achieved. In the HNCACB for every amide visible in the ¹H-¹⁵N HSQC, two

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sets of Cand Cresonances are observed, one belonging to the current residue (i) while the other to the previous residue (i-1). Therefore, a sequential walk of the protein sequence is possible by connecting and matching the peak positions of residue i and i-1 between the different experiments (for example, HNCACB and CBCA(CO)NH) via backbone amides as shown in Figure 13.

Figure 13 Representation of protein backbone assignment

Sections of CBCA(CO)NH and HNCACB experiments used for backbone assignments for RBM5 RRM1-Zf1-RRM2. Sequential walk linking (i-1) and (i) residues between the two experiments is shown.

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After the sequential connectivity is achieved, it is necessary to identify which resonance corresponds to which residue in the protein. For this, certain residues which have very typical Cor Cchemical shifts can be used as starting points. For example, a Glycine residue is easily identifiable as it contains only Cresonance which typically appears ~ 45 ppm or Alanine residue which has a characteristic Cchemical shift ~15-20 ppm. Similarly, unambiguous assignments can be achieved by looking at neighbouring residues as well which might possibly yield a single solution.

Preliminary information reagrding the secondary structure elements of the protein can be already extracted from the backbone assignments. The Cα and Cβ chemical shifts are sensitive indicators of the secondary structure elements in the protein including α-helix, β-sheet and loops (Spera and Bax 1991). For this purpose, the random coil shifts for each amino acid are subtracted from the actual chemical shift observed for the protein. The random coil chemical shifts can be extracted from previously published databases (Wishart, Sykes et al.

1992). The secondary chemical shifts in the structured parts of the protein differ significantly from random coil chemical shifts with positive deviations for α-helical regions and negative deviations for β-strands.

Next, side chain assignment experiments like HccH-TOCSY and hCCH-TOCSY are recorded to assign all side chain carbon and hydrogen atoms for each residue. Additionally, CC(CO)NH and H(CCO)NH side chain assignment experiments can be recorded which help connect carbon and hydrogen atoms of residue i-1 to the backbone amide of residue i. It is a very helpful experiment as the correlations are directly made to the backbone amides present in ¹H-¹⁵N HSQC, the only negative point being that Proline residues which do not have an amide resonance in the ¹H-¹⁵N HSQC would not be visible in these experiments.

After achieving complete or near complete assignment of the protein, one can proceed to structure determination using a variety of experiments including the traditional NOE (Nuclear Overhauser Effect) based experiments as well as experiments used to obtain long-range distance restraints like PRE (Paramagnetic Relaxation Enhancement) or orientation restratints like RDC (Residual Dipolar Coupling) measurements.