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Automating NMR Structures

SBKB [doi:10.1038/sbkb.2015.9]
Technical Highlight - April 2015
Short description: An automated NMR protocol is used to solve the structure of a 25-kDa protein.

Structure of NP_346487.1 as solved by NMR (green) and crystallography (black). 20 NMR conformers and X-ray structure were superimposed for best fit of the core domain (left) or cap domain (right). Figure from ref. 1, with kind permission from Springer Science and Business Media.

One of the oft-cited disadvantages of NMR is that it is limited to small proteins. Extending the size limit can entail labor-intensive data collection and processing, and special isotope-labeling techniques.

In a recent study, Wüthrich and colleagues (PSI JCSG) showed that a largely automated NMR protocol routinely used to solve the structures of moderately sized proteins (∼150 residues) can also be used for larger proteins. Using the J-UNIO protocol previously developed in their lab, the authors solved the NMR structure of the 25-kDa protein NP_346487.1 from Streptococcus pneumoniae.

J-UNIO uses automated projection spectroscopy (APSY) NMR data to automatically assign polypeptide backbone chemical shifts and three-dimensional 13C- and 15N-resolved 1H-1H nuclear Overhauser effect spectroscopy (NOESY) to provide amino acid side-chain chemical shift assignments and distance restraints. The software UNIO is used to process the data. Importantly, the authors were able to generate a structure-quality solution, achieving the protein concentration (1.5 mM) necessary to acquire high-quality NMR spectra using conventional pulse sequences.

The software was able to automatically assign backbone chemical shifts for 77% of the residues. The authors then manually validated and increased the number of assignments to 97% of the residues. Automated side-chain assignments were achieved for 77% of the side-chain chemical shifts; manual validation increased this value to 88%. The quality of the NMR structure of NP_346487.1 (PDB 2MSN) was similar to that obtained for smaller proteins by J-UNIO and consistent with a previously solved crystal structure (PDB 2GO7).

One of the values of NMR is the ability to gain insight into structural flexibility. NP_346487.1 consists of two domains joined by two flexible linkers. In the crystal structure, the orientation of the two domains does not significantly change between crystal units. The NMR structure, however, revealed that the orientation of the two domains varies widely in solution, with the double-linker region acting as a hinge. NOEs between the two linkers, however, constrained the range of motion.

This study shows that, given a structure-quality protein sample, the automated protocol J-UNIO can be used to determine the structure of a protein of approximately 200 residues using conventional pulse sequences, without the need for unique labeling techniques.

Jennifer Cable

References

  1. K. Jaudzems et al. J-UNIO protocol used for NMR structure determination of the 206-residue protein NP_346487.1 from Streptococcus pneumoniae TIGR4.
    J Biomol NMR. 61, 65-72 (2015). doi:10.1007/s10858-014-9886-3

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