Poster Presentation 2014 International Biophysics Congress

Segmental labeling of multidomain proteins for structural studies by multidimensional NMR spectroscopy (#629)

Venkatraman Ramanujam 1 , Glenn F. King 2 , Mehdi Mobli 1
  1. Centre for Advanced Imaging, The University of Queensland, Brisbane, QLD, Australia
  2. Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia

Nuclear Magnetic Resonance is a powerful method for studying the three dimensional structures of proteins as well as their interactions and dynamics. The slower molecular tumbling of large proteins and their overlapping NMR signals makes it hard and time consuming to interpret their NMR spectra. A number of approaches have been developed to overcome difficulties due to overlapping signals of large proteins. An alternative and efficient way is segmental labeling, in which a region of the protein is isotopically labeled1 .

The double knot tarantula toxin  (DkTx) has been shown to be the most avid binder of TRPV1 channel and has shown to consist of two inhibitory cysteine-knot (ICK) motifs2. The absence of structural details and occurrence of homologous domains presents a technical challenge for structure determination by NMR. Here, we employed segmental isotope labeling of ICK motif thereby studying the nuclear resonance signal from one domain (internal domain) in context of the full-length protein. Data is acquired using non-uniform sampling and processed using maximum entropy reconstruction significantly reducing the overall data acquisition time and producing high-resolution data for facile analysis3

  1. Zuger S, Iwai H (2005) Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies. Nature Biotechnology 23: 736-740.
  2. Bohlen CJ, Priel A, Zhou S, King D, Siemens J, et al. (2010) A bivalent tarantula toxin activates the capsaicin receptor, TRPV1, by targeting the outer pore domain. Cell 141: 834-845
  3. Hoch JC, Maciejewski MW, Mobli M, Schuyler AD, Stern AS (2014) Nonuniform sampling and maximum entropy reconstruction in multidimensional NMR. Acc Chem Res 47: 708-717