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

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

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

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

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

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

X-ray absorption spectroscopy S A A S

 coordination number  bondlength  scatterer  EXAFSk 3 k (Å ) Amplitude Frequency Phase Shape

where and 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 

X-ray fluorescence spectra give element sensitivity

Sensitivity of XRF Wide dynamic range

Sensitivity of XRF femtomole detection limits < 1 nL irradiated volume

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

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

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

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

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

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

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.

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