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NVoy Polymer is an Ideal Protein Solubilisation Agent for NMR.

 
Summary
NVoy technology can be used to protect, stabilise and improve the solubility of proteins by masking areas of surface exposed hydrophobicity. NVoy allows proteins to be concentrated and protected in solution, and is compatible with key NMR spectroscopy techniques using 13C and 15N labelled amino acids.
   
Introduction
NMR spectroscopy is a powerful technique which enables the study of the structure, dynamics and function of proteins in solution. This technique requires maintaining high concentrations of protein over a period of several hours, and possibly at elevated temperatures, which is problematic for proteins prone to aggregation or with limited stability. Even a moderate tendency for aggregation can severely impede complex NMR projects, giving poor chemical shift dispersion and extreme line broadening of NMR resonances which can preclude the application of state-of-the-art 2D and 3D NMR techniques.
   
Methodology

 

NV10 can be prepared as a concentrated stock and added to a protein solution immediately prior to use.
   NV10 will co-concentrate with the protein of interest. As such the chosen protein-NV10 ratio is maintained during concentration and buffer exchange.

   
Results
15N-HSQC NMR spectra of CyaY

CyaY is a small iron binding protein which is a bacterial ortholog of the human mitochondrial protein Frataxin.  The loss of Frataxin is associated with the neurodegenerative disorder Friedreich’s ataxia. Functional studies of this protein have utilised 15N-HSQC NMR spectroscopy to clarify details of the iron-binding sites.  15NHSQC is a two dimensional NMR technique which detects correlations between the nitrogen (15N) atom of an NHx group with the directly attached proton (1H).  Each amino acid residue, except proline, gives one signal that corresponds to the N-H of the amide group, with additional contributions from NHx sidegroups depending on the conditions used. 15N-HSQC NMR spectra of CyaY were recorded in the absence or presence of NV10.

 

 
   
Conclusion
Expedeon’s NV10 polymer is invisible in 15N-HSQC NMR and shows no effect on the secondary structure of CyaY.  NVoy technology enables the preparation of stable, concentrated protein solutions suitable for NMR applications using 15N and 13C labelled amino acids.
   
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Application Notes