Transcript NEXAFS SPECTROSCOPY OF BIOLOGICAL MOLECULES: …

SOFT X-RAY SPECTROSCOPY OF
BIOORGANIC MATERIALS WITH 3rd
GENERATION SYNCHROTRON
SOURCES
Yan Zubavichus
Kurchatov Centre for Synchrotron Radiation and Nanotechnology
Russian-Italian seminar
11 December 2009
Biologically relevant materials:
• Biomimicking artificially designed nanostructured
materials and devices (sensors, biochips, etc.)
Russian-Italian seminar
11 December 2009
Complexity
• Small molecules: amino acids, purine and pyrimidine
bases, simple carbohydrates, fatty acids, nucleoside
phosphates, etc.
• Macromolecules: proteins, nucleic acids, polysacharides,
lipids, etc.
• Supramolecular complexes: lipoproteins, ribosomes, etc.
• Organelles: membranes, nuclei, mitochondria, etc.
• Cells
• Tissues
Soft X-ray synchrotron
spectroscopy
(Photon Energy Range h < 1500 eV)
Technique
PES
Photoemission
Spectroscopy
Information
Elemental composition, the
number of chemically
nonequivalent groups, film
thicknesses, energy diagram
of occupied electronic states
in the VB
“Fingerprints” of specific
NEXAFS
functional groups, energy
(XAS)
Near-edge X- diagram of unoccupied
ray
electronic states in the VB
Absorption
with element and orbital
Fine Structure momentum specificity
XES
X-ray
Emission
Spectroscopy
Possible modes/modifications
Core-levels/Valence band, angleresolved, spin-resolved, resonant,
photoelectron microscopy
Detection modes: transmission,
total electron yield, partial
electron yield, Auger electron
yield, fluorescence yield, ion
yield, etc.; linear/circular
dichroism, edge-enhanced X-ray
microscopy
Energy diagram of occupied Non-resonant / resonant (RIXS),
electronic states in the VB
linear/circular dichroism
with element and orbital
momentum specificity
Russian-Italian seminar
11 December 2009
Limitations/Drawbacks
Non-applicable to dielectric
samples (powdered organics)
For C,N,O K, the quantum
yield is very low (core holes
predominantly decay through
Auger)  long data
acquisition times & exposure
doses are required
Measurements: - BESSY II, Berlin, Germany (HESGM, PM1, UE52 PGM, U41 PGM)
- ALS, Berkeley, USA (8.0)
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11 December 2009
Severe experimental problem: Radiation damage
Fast changes in PES & NEXAFS spectra
N K-edge
O K-edge
C K-edge
Fluorescence yield (a.u.)
14 min.
30 min.
9 min.
17 min.
20 min.
11 min.
4 min.
5 min.
pristine
10 min.
pristine
pristine
pristine
a
284 285 286 287 288 289 290 291
530
Photon energy (eV)
535
540
545
395
400
405
410
Photon energy (eV)
Photon energy (eV)
Gaseous decomposition products in the residual vacuum
-10
Ion current, A
3.0x10
-10
H2
NH2
-8
p=1.50x10
-10
2.0x10
2 (H2)
6.0x10
CO2
16 (NH2)
mbar
-10
5.0x10
CO
18 (H2O)
-10
1.0x10
a
28 (N2, CO)
-10
4.0x10
44 (CO2)
0.0
0
5
10
15
20
25
30
35
40
45
50
55
60
-10
3.0x10
M/z
-10
2.0x10
Soft X-ray induced decomposition of amino acids:
an XPS, mass-spectrometry, and NEXAFS study, Y.
Zubavichus, et al., Rad. Res. 161 (2004) 346.
-10
1.0x10
Russian-Italian seminar
11 December 2009
0.0
0
60
120
Exposure time (min.)
180
Experimental considerations to suppress
or avoid damage
• Light source: BM is easier than undulator
• Fastest possible data collection
• Strict control of damage effects by
successive scans
• New series – new spot on a sample (or
continuous shift of the sample position
during scan)
• Cooling or efficient heat sink
Russian-Italian seminar
11 December 2009
Combined PES + NEXAFS study:
Amino acid Histidine (His) on Au(111)
O

H2N
CH
b
C
CH2
 Exists in a number of charge forms (mediator of electron/proton
transfer in proteins)
4
3
5
N
2
OH
 Coordinatively active amino acid residue (often responsible for metalbinding in proteins)
NH
1
Preparation:
Molecular Beam deposition
with a Knudsen effusion cell
Surface chemistry of ultrathin films of histidine on gold as
probed by high-resolution synchrotron XPS
Y. Zubavichus et al. JPCB 109 (2005) 884
Evaparator
temperature, ºC
Substrate temperature
Deposition time, min
Film thickness, Å
ML
Russian-Italian seminar
11 December 2009
Film 1
105
Film 2 Film 3
115
115
RT
40
5.2
1
RT
40
18.5
~3
RT
80
38.8
~6
PES data
Absolute Intensity [counts]
Au 4f
90
88
86
84
82
C 1s
290
288
286
284
282
O 1s
534
532
530
528
Film 1: Monolayer
Film 2: 3 Molecular layers
Film 3: 6 Molecular layers
Russian-Italian seminar
11 December 2009
N 1s
404
402
400
398
Valence
band
25
20
15
10
5
0
Linear Dichroism in NEXAFS
Multilayer film
C K-edge
3.5
3.0
2.5
90°
55°
20°
2.0
1.5
1.0
0.5
4.0
N K-edge
3.5
3.0
2.5
2.0
1.5
90°
55°
20°
1.0
0.5
0.0
290
300
310
320
390
Photon energy (eV)
400
410
3.5
O K-edge
3.0
2.5
2.0
1.5
1.0
90°
55°
20°
0.5
0.0
0.0
280
420
-0.5
520
430
Photon energy (eV)
530
540
550
560
Photon energy (eV)
1.5
1.0
90°
55°
20°
0.5
Intensity (arb. units)
C K-edge
2.0
1.0
N K-edge
0.5
90°
55°
20°
Intensity (arb. units)
Monolayer film
2.5
Intensity (arb. units)
4.0
Intensity (arb. units)
4.5
Intensity (arb. units)
Intensity (arb. units)
4.0
O K-edge
1.0
0.5
90°
55°
20°
0.0
0.0
280
290
300
310
Photon energy (eV)
320
0.0
400
420
Photon energy (eV)
520
540
Photon energy (eV)
No distinct polarization dependence  no preferred molecular orientation
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11 December 2009
560
Multilayer film
+
-
-
-
+
-
+
-
-
+
-
-
+
+
-
-
+
+
ZI His
+
+
-
+
+
+
+
-
-
-
+
Weak van der
Waals interactions
Au
Monolayer film
+
Strong ioniccovalent bonding
-
+
+
+
+
+
His- anions
+
Au
+
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11 December 2009
+
+
+
+
C,N,O NEXAFS library for
native amino acids
Sample preparation:
“as is” powders pressed into
In foil
NEXAFS data acquisition in the
partial electron yield mode Innershell Absorption Spectroscopy of Amino
Acids at All Relevant Absorption Edges, Y.
Zubavichus, et al. J. Phys. Chem. A 109 (2005)
6998.
Russian-Italian seminar
11 December 2009
Amino acids: NEXAFS fingerprints of specific functional groups
Phenylalanine (Phe) vs. Tyrosine (Tyr)
Aspartic acid (Asp) vs. Asparagine (Asn)
H2N
CH
C
OH
H2N
CH
C
O
O
O
O
H2N
OH
CH
C
OH
H2N
CH
C
OH
CH2
CH2
CH2
C
CH2
C
O
O
NH2
OH
OH
Partial Electron Yield [a.u.]
C K-edge
285
285
290
295
286
300
287
305
288
310
Photon Energy [eV]
289
O K-edge
N K-edge
290
315
530
535
540
545
550
555
Photon Energy [eV]
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560
400
405
410
415
420
425
Photon Energy [eV]
430
Effects of the charge state of an amino acid residue in NEXAFS
O
O
O
O
+
H3N
CH
C
OH
H2N
CH
R
C
OH
+
H3N
CH
R
cation
Partial Electron Yield, a.u.
+
295
300
305
310
Photon Energy, eV
Solid-state NEXAFS spectra of glycine in various
charge states, Y. Zubavichus, et al., J. Phys. Chem.
B, 110 (2006) 3420
R
anion
ZI-Gly
(Gly1)
ZI-Gly
(Gly1)
+
Gly Cl
-
+
Gly Cl
+
Neutral
Gly (gas)
400 405 410 415 420 425 430
Photon Energy, eV
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11 December 2009
-
-
Na Gly
-
Na Gly
-
Neutral
Gly (gas)
290
C
O K-edge
Na Gly
285
CH
N K-edge
+
+
H2N
zwitter ion
ZI-Gly
(Gly1)
Gly Cl
O
R
neutral molecule
C K-edge
C
Neutral
Gly (gas)
530
535
540
545
550
Photon Energy, eV
555
O
Amino acids  peptides: compositional effects
LeuTyr
LeuLeuTyr
Normalized Absorbance [a.u.]
His-Phe
GlyGlyTyrArg
His
Gly
Leu
Tyr
Tyr
Phe
285
290
295
300
Photon Energy [eV]
305
Arg
280
290
300
310
Photon Energy [eV]
280
290
300
Photon Energy [eV]
The ‘building block” approach is valid to a certain extent
NEXAFS spectroscopy of biological molecules: from
amino acids to functional proteins, Y. Zubavichus, et al.,
NIMA 603 (2009), 111
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11 December 2009
310
Amino acids  peptides: NEXAFS fingerprints of the peptide bonds
glycine
diglycine
H
CH
C
NH 2
NH 2
NH 2
H
tryglycine
C
O
H
CH
H
H
N
O
H2C
NH
NH
OH
CH
C
O
2,5-diketopiperazine DKP
(glycine anhydride – cyclic dipeptide)
H
CH
CH
O
C O
C O
O
CH 2
N
H
NH
OH
H
CH
C O
OH
C1s
N1s
O1s
C
A
Intensity (arb. units)
B
C
287
D E
288
289
D
DKP
DKP
Trigly
Trigly
Digly
Digly
290
F
DKP
B
A
Trigly
Digly
Gly
Gly
Gly
290
300
310
NEXAFS study of glycine and glycine-based
oligopeptides, Y. Zubavichus, et al. J. Electr. Spectr.
Relat. Phenom. 134 (2004) 25
400
410
420
430
Photon Energy, eV
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11 December 2009
530 535 540 545 550 555 560 565
One more step ahead: homopolypeptides (designed polymers
composed of one sort of amino acids)
[
O
H
N
CH
CH
CH2
CH3
C
]
CH3
[
O
H
N
CH
CH
CH2
H
N
C
O
H
N
CH
CH2
C
]
O
H
N
CH
H
N
C
O
HN
CH2
CH
Polytyrosine
CH3
CH2
N
CH
C
CH2
N
NH
OH
*
*N-CH2 N-CO
N K-edge
OH
*O=C
*O=C
O K-edge
DKP
FEFF8
model
*N-CO
DKP
FEFF8
model
DKP
DKP
Polyhis
Polytyr
Polyile
Polyile
400
410
NEXAFS spectroscopy of homopolypeptides at
all relevant absorption edges: polyisoleucine,
polytyrosine, and polyhistidine, Y. Zubavichus,
et al., J. Phys. Chem. B, (2007) 111 9803
[
O
H
N
]
Polyhistidine
Polyisoleucine
CH3
Partial Electron Yield [a.u.]
C
H
N
420
430
530
540
Photon Energy [eV]
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11 December 2009
550
560
NH
Collagen (300 kDa)
Towards functional proteins
C K-edge
C
BSA (66 kDa)
Lysozyme
BSA
Ovalbumin
Collagen
E
D
Lysozyme (14.4 kDa)
B
A
285
290
295
D
B
305
N K-edge
E
Fractions of amino acid residues
C
Lysozyme
BSA
Ovalbumin
Collagen
30
Fraction, %
A
10
Russian-Italian seminar
11 December 2009
Val
Tyr
Trp
Thr
Ser
Pro
Phe
Lys
Met
Ile
Leu
His
Gly
Glu
0
Gln
Is X-ray absorption spectroscopy sensitive to the amino
acid composition of functional proteins?, Y.Zubavichus,
et al., J. Phys. Chem. B 112 (2008) 4478.
420
Cys
415
Asp
410
Asn
405
Ala
400
20
Arg
Intensity (arb. units)
300
C,N,O NEXAFS library for nucleotide bases
Cytosine (C)
7
Uracil (U)
7
NH2
1
6
Thymine (T)
8
O
N
6
NH
3
8
O
N
NH
H
10
NH2
1
9
2
N
7
5
4N
8
Guanine (G)
N
O
1
8
2
9
NH
7
3
5
H
2
3
5
4N
1
7
2
6
10
O
1
2
Adenine (A)
O
4N
8
6N
9O
H
C K-edge
H
5
3
N
4
6 N
H
5
4
N
3
11
NH2
O K-edge
N K-edge
Normalized Intensity (arb. units)
A
G
A
G
T
G
T
T
U
U
290
295
300
305
310
315
C
C
C
285
U
400
405
410
415
420
425
Photon Energy (eV)
X-ray absorption spectroscopy of the nucleotide bases at the carbon, nitrogen, and
oxygen K-edges, Y. Zubavichus, et al., J. Phys. Chem. B 112(44) (2008) 1371113716.
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seminar
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530
535
540
545
550
555
560
Soft X-ray spectroscopic studies of
solutions: 2 approaches
Microfluidic liquid cell with
a thin window semitransparent to soft X-rays
Microjet
of a solution + efficient
differential pumping
Window
material: Si3N4,
SiC (50-100 nm)
Investigation of volatile liquid surfaces by synchrotron X-ray
spectroscopy of liquid microjets, K.R. Wilson, et al., Rev. Sci. Instr. 75
(2004) 725
All techniques are available
A liquid flow cell to study the electronic structure of liquids
with soft X-rays, O. Fuchs, et al., NIMA 585 (2008) 172
Only photon in – photon out
techniques (XES, FlY NEXAFS, RIXS)
Russian-Italian seminar
11 December 2009
Full O1s RIXS map of liquid water
Si3N4 Membrane
(75 nm)
Two states : -Н-bonded intact water molecules
-Ultrafast proton transfer induced by
the oxygen core hole
Isotope and Temperature Effects in Liquid Water Probed
by X-Ray Absorption and Resonant X-Ray Emission
Spectroscopy,
Russian-Italian seminar
O. Fuchs et al., PRL 100 (2008) 027801 & 249802
11 December 2009
Aqueous glycine: pH effects
SiC Membrane
(75 nm)
N K-edge
NEXAFS
NEXAFS
N K
XESXES
NH3+ NH2
Soft x-ray spectroscopy
of organic molecules and liquids, O. Fuchs
PhD thesis (2009)
Russian-Italian seminar
11 December 2009
Conclusions
• Synchrotron-based PES, NEXAFS, XES, and RIXS
are a highly efficient and informative set of
experimental techniques to elucidate organic
materials of biological significance in different forms,
including bulk powders, thin evaporated films,
adsorbates, aqueous solutions, etc.
• Radiation damage problems are serious but can be
avoided
• The results presented above could be a good starting
point for research projects with novel soft X-ray
instrumentation at the KCSRNT
Russian-Italian seminar
11 December 2009
Acknowledgements
Prof. M.Grunze
Dr. M.Zharnikov
University of Heidelberg, Germany
Prof. E. Umbach
Dr. O. Fuchs
Dr. L. Weinhardt
University of Würzburg, Germany
Prof. C. Heske
Prof. F. Netzer
Dr. G. Tzvetkov
University of Nevada, Las Vegas, USA
University of Graz, Austria
Russian-Italian seminar
11 December 2009