Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment

Scott A. Robson, Koh Takeuchi, Andras Boeszoermenyi, Paul W. Coote, Abhinav Dubey, Sven Hyberts, Gerhard Wagner and Haribabu Arthanari ()
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Scott A. Robson: Harvard Medical School
Koh Takeuchi: National Institute of Advanced Industrial Science and Technology
Andras Boeszoermenyi: Harvard Medical School
Paul W. Coote: Harvard Medical School
Abhinav Dubey: Harvard Medical School
Sven Hyberts: Harvard Medical School
Gerhard Wagner: Harvard Medical School
Haribabu Arthanari: Harvard Medical School

Nature Communications, 2018, vol. 9, issue 1, 1-11

Abstract: Abstract Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations. However, this experiment suffers from chemical shift degeneracy problems during the assignment procedure. We present a strategy that increases the effective resolution of HNCA and enables near-complete resonance assignment using this single HNCA experiment. We utilize a combination of 2-13C and 3-13C pyruvate as the carbon source for isotope labeling, which suppresses the one bond (1Jαβ) coupling providing enhanced resolution for the Cα resonance and amino acid-specific peak shapes that arise from the residual coupling. Using this approach, we can obtain near-complete (>85%) backbone resonance assignment of a 42 kDa protein using a single HNCA experiment.

Date: 2018
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DOI: 10.1038/s41467-017-02767-8

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