Methods

Cloning and construction of an NOR expression plasmid

• Using a G.stearothermophilus genomic DNA, amplified it by PCR and cloned it into a vector.
• The cloned qNOR gene was sequenced, and a DNA fragment encoding the ORF of qNOR was amplified by PCR and then ligated between.

Purification of recombinant qNOR

• The E.coli strain Rosetta3 was induced to express the recombinant enzyme, qNOR.
• The purified protein was formed through many steps of centrifugation and concentrations, and was stored at -80°C.

 Crystallization, data collection and structure determination

·         Recombinant qNOR was crystallised using the sitting-drop vapour-diffusion method.


                                         Diagram of the sitting drop vapour-diffusion method

     http://hamptonresearch.com/documents/growth_101/4.pdf·      

·          

      The crystals of HQNO-qNOR were obtained by co-crystallising the enzyme in the presence of HQNO, following the same crystallisation method as for the recombinant enzyme.

• Crystals were cryoprotected in buffer.
• Initial phases were calculated by the MAD method using SHARP and SOLOMON.
• Model building was carried out by COOT.
• A Ramachandran analysis was carried out

Enzymatic activity measurements

·         NOR activity was assayed using a Clark-type electrode.

o   The clark electrode is an electrode that measures oxygen on a catalytic platinum surface using the net reaction.

Molecular dynamics simulation

·         The molecular dynamics simulation was carried out in NAMD.