Structural characterization of non-crystalline systems

Biophysics and Synchrotron Radiation

James E. Penner-HahnDepartment of Chemistry &

Biophysics Research DivisionThe 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

SA

A S

E 2E 1

coordinationnumber

bondlength

scatterer

scatterer

-10

-5

0

5

10

0 2 4 6 8 10 12

EXA

FS•k

3

k (Å -1)

Amplitude

Frequency

Phase

Shape

where

and

-10

-5

0

5

10

0 2 4 6 8 10 12

EXA

FS•k

3

k (Å -1 )

Amplitude

coordinationnumber

Frequency

bondlength

Phase

scatterer

Shape

scatterer

k N sAs k S02

kRas2 exp 2k 2as

2 exp 2Ras sin 2kRas as k

k 2me E Eo 2

k E o E o E

E s E

b E

X-ray fluorescence spectra give element sensitivity

0

10000

20000

30000

40000

50000

0 200 400 600 800 1000

Cou

nts

[Zn(II)] (M)

Sensitivity of XRFWide dynamic range

0

1000

2000

3000

4000

5000

0 20 40 60 80 100

Cou

nts

[Zn(II)] (M)

Detection limit (S/N=3)ca. 8 M

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

CA3Dentategyrus

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.

Zn EXAFS for Hilus of Dentate Gyrus

k0 2 4 6 8 10 12

Frac

tiona

l Mod

ulat

ion

-10-8-6-4-202468

Zn EXAFSFit

Fourier Transform and Fit

R+0 1 2 3 4 5

FT m

agni

tude

0

2

4

6

8

10

FTFit Fits as 3.8 O/N at

1.945 Angstroms

Debye-Waller=0.006