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Re: [ccp4bb] Crystallographic Experiment with NADH and a Flavoprotein |
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CCP4bb navigationCCP4bb <-- 2007 <-- December 2007 <-- 31 December 2007Subject: Re: Crystallographic Experiment with NADH and a Flavoprotein From: Juan Sanchez Weatherby weatherby {- at -} EMBL {- dot -} FR Date: 2007-12-31 I may be wrong in this but I think there's no difference between NADH and NAD2H and there are lots of structures on the PDB databank for both structures with either NAD+ or NADH (upto about 600) and also several with NADP+ or NADPH. A redox reaction involving NAD+ is a 2 electron and 2 proton loss or gain. That is you go from A-H2 + NAD+ to B + NADH + H+ and vice versa. The cofactor takes two electrons and a proton and there is an extra proton. This is why I think it may be accepted to write NAD2H or NADH2 instead of NADH + H+. I have attached a figure that illustrates the chemical conversion. In the case of FAD, when the full reaction happens, there are also two electrons and two protons being transfered but FAD takes both protons and thus is called FADH2. So, in summary, I think that Oxidized NAD is called NAD+ (and sometimes NAD) and reduced NAD is called NADH (and sometimes NADH2) but I don't think there is a different redox state called NAD2H. The fact that you cannot find structures with NADH is because from the crystallographic point of view, as you cannot see the hydrogens, then both NAD+ and NADH are the same structure and are thus labeled as "NAD" in a PDB. Therefore if you are looking for a NADH structure you need to look for the structure of a NAD containing enzyme in the reduced state. And this will also be the way to find examples of ways of getting your enzyme in the correct redox state prior to freezing. The easier way tends to be to add artificial electron acceptors or donors to keep your enzyme in the desired redox state but you may need to use anaerobic cambers in some cases. What is normally more difficult to achieve is to get the conversion and see the substrate or product in the active site too as the binding affinity tends to be very low if you are getting turnover. You can try different substrate or cofactor analogs that will not get converted and thus capture the reaction complex in the crystal. The redox state of NAD is, in general, easy to follow by spectral changes thus you should be able to check the redox state of your crystals using a UV/VIS microspec. Hope this helps and I'm not wrong about NAD2H but I'm sure, if I am, once people get back from holidays we'll get the right answer right away. Good luck and Happy New Year, Juan Buz Barstow wrote: > Dear All, > > I'm planning an experiment to study the oxidation of NADH by a > flavoprotein at cryogenic temperatures to facilitate collection of > X-ray diffraction data. > > In planning this experiment, I have seen a few obstacles that I am > looking for help in overcoming. > > 1. There are no structures in the PDB that are complexed with NADH or > NAD2H. > Has anyone ever attempted to solve a structure complexed with NADH or > NAD2H, especially at cryogenic temperatures, and if so, what are the > difficulties? Does NAD+ de-bind from the protein too fast to permit > data collection? > > > 2. NADH oxidation typically takes less than a second by a flavoprotein > at room temperature. > Is there an NADH or NAD2H analog that has a much longer half time for > oxidation by a flavoprotein, for example tens of minutes, rather than > tenths of a second, and can this analog still be oxidized at cryogenic > temperatures, with a reasonable half time, of several hours or so? > > > Thanks! and all the best, > > --Buz > > > -- Juan Sanchez-WEATHERBY Tel:33 (0) 47620 7266 Website: http://www.embl.fr *********************************** Postal address: EMBL Grenoble 6 rue Jules Horowitz, BP 181 38042 Grenoble Cedex 9 FRANCE *********************************** Delivery address: EMBL c/o ILL, Polygone Scientifique 6 Rue Jules Horowitz 38042 Grenoble Cedex 9 France *********************************** CCP4bb navigationCCP4bb <-- 2007 <-- December 2007 <-- 31 December 2007 |
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