1. Structural characterization of non- crystalline systems Biophysics and Synchrotron Radiation James E. Penner-Hahn Department of Chemistry & Biophysics Research Division The University of Michigan

2. Outline Motivation – why do we want to do this Experimental – spectrometer details Theory – physical background System – biological background Results

3. Zn Penner-Hahn group’s view of protein structure The good stuff The chicken fat

4. Techniques for studying metal sites in proteins UV-visible spectroscopy EPR spectroscopy Magnetic susceptibility MCD Require open d shell Requires I=1/2 nucleus Requires crystals X-ray crystallography NMR spectroscopy

5. Outline Motivation – why do we want to do this Experimental – spectrometer details Theory – physical background System – biological background Results

15. Outline Motivation – why do we want to do this Experimental – spectrometer details Theory – physical background System – biological background Results

16. X-ray absorption spectroscopy S A A S

17.  coordination number  bondlength  scatterer  -10 -5 0 5 10 02468 12 EXAFSk 3 k (Å ) Amplitude Frequency Phase Shape

18. where and -10 -5 0 5 10 02468 12 EXAFSk 3 k (Å ) Amplitude  coordination number Frequency  bondlength Phase  scatterer Shape  scatterer  k   N s A s k  S 0 2 kR as 2  exp  2k 2  as 2  exp  2R as   sin2kR as  k    k  2m e E  E o   2  k    E   o E   o E    E   s E   b E 

19. X-ray fluorescence spectra give element sensitivity

20. Sensitivity of XRF Wide dynamic range

21. Sensitivity of XRF femtomole detection limits < 1 nL irradiated volume

22. Kirkpatrick-Baez focusing optics ~ 1  m spot size Zone plate ~ 30 nm spot size

23. Outline Motivation – why do we want to do this Experimental – spectrometer details Theory – physical background System – biological background Results

24. Hippocampus Located within the temporal lobe Important for converting short term memory into permanent memory Early target of Alzheimer’s disease Hippocampus

25. CA3 Dentate gyrus Hippocampus is rich in “chelatable” Zn, as determined using TIMM’s staining (sulfide/silver)

26. Outline Motivation – why do we want to do this Experimental – spectrometer details Theory – physical background System – biological background Results

27. X-ray microprobe allows determination of total Zn (and other metals) on the  m scale

29. Cu may be localized in myelin in older rats Fe: 0.18 mM average conc. Cu: 0.24 mM average conc. Zn: 0.23 mM average conc.

31. Fits as 3.8 O/N at 1.945 Angstroms Debye-Waller=0.006