[Ifeffit] Beryllium XAFS in proteins

Gina - measuring Be in proteins is extremely difficult and probably unprecedented, but it may be possible to substitute another element that is chemically similar and probe that instead. For example, often metalloenzymes remain functional under such substitutions, e.g. if you substitute a Mn or a Co in a Ca site. The covalent radius of Be is very small, so substituting Ca might not be feasible without excessively distorting the site, but Aluminum isn't far off, and might work. Of course Aluminum itself isn't easy because of its low k-edge, but it may be feasible. If a heavier element (Ca or heavier) could be made to substitute and retain the functionality/properties you are studying, it might be a suitable probe. good luck! Grant Bunker On Dec 18, 2012, at 3:19 PM, ifeffit-request@millenia.cars.aps.anl.gov wrote:

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Today's Topics:

1. Re: Beryllium and EXAFS (Erik Farquhar) 2. Larch 0.9.17 available for download and testing (Matt Newville)

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Message: 1 Date: Tue, 18 Dec 2012 12:50:30 -0800 (PST) From: Erik Farquhar To: XAFS Analysis using Ifeffit Subject: Re: [Ifeffit] Beryllium and EXAFS Message-ID: <1355863830.4845.YahooMailNeo@web141002.mail.bf1.yahoo.com> Content-Type: text/plain; charset="iso-8859-1"

A bit unrelated to the question at hand, but regarding #5, yes, people do neutron PX.? The focus?is almost entirely on locating hydrogen atoms (e.g. for protonation state), so I wonder what?the neutron PX guys?would say about looking for a Be atom. The downside is that you usually need?quite large crystals compared to X-ray PX and long data collection times. ? Erik

________________________________ From: Matthew Marcus To: Erik Farquhar ; XAFS Analysis using Ifeffit Sent: Tuesday, December 18, 2012 1:39 PM Subject: Re: [Ifeffit] Beryllium and EXAFS

Just to stir the pot a little more: 1.??? ALS has some beamlines which can go down as far as Li.? Work has been done on Li, but only on concentrated stuff. 2.??? The sample is probably really dilute in Be.? The signal will be pathetic. 3.??? Radiation damage will be a major issue, especially with X-ray Raman, which is a highly inefficient technique.? Even LHe cooling doesn't solve that problem. 4.??? Are there model compounds available for proposed local structures of the Be center?? You'd need that to have any hope of making sense out of Be XANES. 5.??? Is there such a thing as neutron PX?? That might see the Be. ??? mam ? ----- Original Message -----

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From: Erik Farquhar To: XAFS Analysis using Ifeffit Sent: Tuesday, December 18, 2012 5:32 AM Subject: Re: [Ifeffit] Beryllium and EXAFS

To add to Matt's last paragraph, you might also consider beamline 6-2 at SSRL (http://www-ssrl.slac.stanford.edu/beamlines/bl6-2/), as they do a fair amount of work with proteins and have an X-ray Raman setup. I think the beamline scientist occasionally?posts to this list.

Good luck, Erik

-- Erik Farquhar, Ph.D. Case Center for Synchrotron Biosciences Brookhaven National Lab NSLS, Building 725A-X3 Upton, NY? 11973 efarquhar@bnl.gov +1-631-344-8174

________________________________ From: Matt Newville To: XAFS Analysis using Ifeffit Sent: Tuesday, December 18, 2012 7:52 AM Subject: Re: [Ifeffit] Beryllium and EXAFS

Hi Gina,

On Mon, Dec 17, 2012 at 5:23 PM, Clayton, Gina wrote:

...

Hi there

I am trying to find out more about EXAFS. I am a protein crystallographer.

I want to identify the environment of a beryllium ion in the protein, I work with,? of which I have a crystal structure.? EXAFS was recommended as a good technique for identification of the beryllium site and species. The beryllium is an acidic pocket of the protein surrounded by oxygens from the protein, likely covalently bound. However I have had? zero result finding a beam line that can tune their setups to the Be edge at 111 eV. Also some EXAFS users say that EXAFS technique would be good for working out the environment of the Be but some say it would not . In particular? I have been advised that? Be EXAFS would not be useful since? the Be? EXAFS signal would have unacceptable interference from the carbon K-edge at 285 eV and the oxygen K edge signal at 535 eV.

Would EXAFS work or something in a similar vein? Or, for instance,? could the acidic pocket be identified using EXAFS, by measuring the oxygens (instead of the Beryllium) using the protein with and without beryllium?

Thank you so much for any advice.

Gina

As you and as the person who advised you suspect, you face a number of challenges:

1.? The extremely low energy (111 eV).? You might be able to find a UV source or x-ray beamline that can reach this energy , but often such work is done in vacuo, and so may not be suitable for proteins.? Using electrons (from a TEM, say) might be possible too -- these can sometimes do C and N edges either directly or looking at Electron Energy Loss (send in an electron of high energy E1, and look for electrons of energy E1 - E, and scan the analyzer for the loss energy E through the absorption edge).? Again, electron probes may not be what you want for your proteins.

2. The C K-edge will limit your signal to "only" 175 eV.? I would say to not worry so much about this limitation.? You'll really be measuring NEXAFS (near-edge X-ray absorption ....)? but that's OK. People measure the B, C, N, and O K edges, all facing the same problem, especially for the O K edge: our planet is more or less covered with the stuff ;).? These spectra tend to be very rich and sensitive to ligands.

3. Not much (zero?? I can almost believe that!) literature on Be XAFS. This is unfortunate because most NEXAFS is measure and compare to known spectra.? I think that might mean you have to spend effort to building up a spectral library for Be in organic phases.

I think trying to measure the O K edge with and without Be is nearly hopeless.? All the X-ray spectroscopies average equally over all the atoms of the absorbing species.? In addition all the spectra are grossly the same (a step function from 0 below the edge to 1 above the edge).? These make it difficult to see changes below about 1%.? ? So I think you would just have too high of an O::Be ratio to see anything useful.

One option to consider is using X-ray Raman scattering (http://en.wikipedia.org/wiki/X-ray_Raman_scattering).? Here, one hits the sample with high-energy X-rays (say, 10 keV), which are highly penetrating for reasonably sized protein sample.? One analyzes an energy loss (again, E1 - E, where here E1 is around 10 keV) to get the edge of interest.? I'm familiar with (been involved with) studies on B, C, and O using this technique (all in solids, mostly at high pressure).? See, for example,? S. K. Lee, et al "Probing of bonding changes in B2O3 glasses at high pressure with inelastic X-ray scattering",? Nature Materials 4, pp 851-854 (2005), and also the work of Simo Huotari.? ? I'm not aware of anyone doing Be K-edge this way, but it should definitely be possible. For the work I've been involved with, Be would be challenging because there are other excitations in solids that appear around 100 eV.? I believe that effect would be far suppressed in a protein, though I'm not sure.

That's somewhat more exotic than straightfoward XAFS,, but it might be the best chance for measuring Be in a protein.? ? I'm not sure I know any beamlines that do X-ray Raman on proteins, but I'd suggest APS beamline 20-ID or ESRF beamline as places to start.? ? The next questions would be "how many Be atoms are in your protein?", and "how much material do you have?"....

Hope that helps,

--Matt Newville

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