The document discusses the use of Langmuir-Blodgett (LB) thin protein films for protein crystal growth using LB nanotemplates and robotics. Key points: - LB technique is used to create organized thin films of proteins layer-by-layer at the air-water interface for use as nanotemplates in protein crystal growth. - Various proteins including lysozyme, thaumatin, proteinase K, and ribonuclease A were tested using both classical and LB nanotemplate crystallization methods. - LB nanotemplates were found to facilitate the nucleation and growth steps in crystallization, resulting in earlier formation and larger crystals compared to classical methods. - Protein crystals

1. The role of LB thin protein film in protein crystal
growth by LB nanotemlate and robot
Prof. Eugenia Pechkova
Laboratories of Biophysics and
Nanobiotechnology
University of Genova
Italy

2. Thin Film Technologies
Langmuir-Blodgett
Layer-by-Layer self assembly
Barrier position (mm)
Surface
pressure
(mN/m)
Gas phase
Liquid phase
Solid phase
Protective plate
S P S P
P
S
P
P S
S
P
S
S P
S P
S P
P
S
air
water
S
air
water
water
P
S water P
S water P
after washing
S
An example of
deposited structure
protein molecules
S
P
BA1
BA2
M2
M5
M3
A1 A2
W
B2
T2
T1
B1
M4
M1 P
S
MB
from Alternate-Monolayer
Deposition System (M6)
from
Kelvin
probe
from
tempe-
rature
probes
to Analog-to-Digital
Converter
to Motor Drivers
M1-M6 - stepper motors; BA1, BA2 - Wilhelmy balances; B1,B2 - barriers; T1,T2 –
Langmuir troughs; A1, A2 - compartments for adsorption; W - compartment for
washing; S - substrate; MB-mobile block

3. The Langmuir-Blodgett Technique
SURFACE PRESSURE (mN/m)
AREA PER MOLECULE (A)
SOLID PHASE
LIQUID PHASE
GAS PHASE
°
Displacement
Transducer
Spring
Wilhelmy
plate
Spreading
Compression
p - isotherm
p - measurements

4. LB nanotemplate protein crystallization method.
Pechkova E, Nicolini C., Protein nanocrystallography: a new approach to structural
proteomics, Trends in Biotechnology 22, 117-122, 2004.
LB nanotemplate protein crystallization method

5. Isotherm and Atomic Force Microscopy of Lysozyme
Langmuir-Blodgett film.

6. AFM images (in air):
LB film (2 monolayers) of thaumatin (24 kDa) at 200 nm scale

7. Nanogravimetric measuarments
y = 3,1207x
R
2
= 0,9955
0
5
10
15
20
25
30
35
40
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Number of protein monolayers
Surface
density,
ng/mm^2
y = 3,6833x
R
2
= 0,9716
0
5
10
15
20
25
30
35
40
45
50
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Number of protein monolayers
Surface
density,
ng/mm^2
Protein surface density
Human protein kinase CK2
(MW 45 kDa)
Cytochrome P450 scc
(MW 56 kDa)
Film packing (area per molecule)
29,52 nm2
20,36 nm2
17,8 nm2 30 nm2
Experimental
Close parking system

8. Test proteins for classical vs LB
nanotemplate crystallization

9. Dependence of the surface density of deposited ribonuclease A,
thaumatin, proteinase K and lysozyme nanostructured films upon the
number of transferred protein mono-layers, at 20 mN/m of pressure.

10. Vacuum
grease
Protein solution with precipitant
I
II
VDrop
CSalt
CProtein
Precipitant
Protein
solution
Non-specific
aggregates
Specific
aggregates
Critical nuclei
Crystals
Time
Energy ΔG
Precipitant
Specific aggregates
Pechkova E, Nicolini C, From art to science in protein crystallization by means of
thin film technology, Nanotechnology 13, 460-464, 2002.
Classical hanging drop
LB nanotemplate

11. Protein Protein
solution
Reservoir solution Drop Cryoprotectant
Proteinase K 20 mg/ml in
25 mM
HEPES
buffer pH 7
400 mM Na/K-
tartrate in 25 mM
HEPES buffer pH 7
1:1 4M TMAO
Ribonuclease
A
10 mg/ml in
50 mM Na-
acetate pH
5.5
1.75 M (NH4)2SO 4
2M NaCl in 100mM
Na-acetate pH 5.5
1:1 Mother liquid
Thaumatin 15 mg/ml in
100 mM
ADA
buffer pH
6.5
1 M Na/K in 100
mM ADA buffer
tartrate pH 6.5
1:1 30% Glycerol,
0.7M Na/K in
100 mM ADA
buffer tartrate
pH 6.5
Thermolysin 100 mg/ml
in 50 Mm
MES pH 6.0
35% saturated
(NH4)2SO 4
1:1 with
1M
NaCl in
50mM
MES pH
6.0
Dry Paraton-N
(0.5h in vacuum
centrifuge)
Insulin
(Zn-free)
18 mg/ml in
50 mM
Na2HPO4 pH
10.4
1 mM
EDTA
400 mM Na2HPO4
pH 10.4
10 mM EDTA
1:1 30% Glycerol
in400 mM
Na2HPO4 pH
10.4
10 mM EDTA
Lysozyme 40 mg/ml in
50 mM
NaAc buffer
pH 4.5
0.9 M NaCl 1:1 20% Glycerol in
0.9 M NaCl
Protein crystallization conditions used for both LB and classical hanging drop
vapour diffusion method. The cryo-protectant used during the X ray
diffraction data collection is also indicated

12. Classical Proteinase K crystal LB Proteinase K crystal

13. Screening test for thaumatin “critical” concentration. A: screening test for ribonuclease
A “critical” concentration. B: screening test for proteinase K “critical” concentration

14. A: critical concentration for proteinase K with classical vapor diffusion
method. B: critical concentration for thaumatin with classical vapor
diffusion method. C: critical concentration for ribonuclease A with
classical vapor diffusion method.

15. RESULTS Standard protocol
concentration
Critical concentration
Lysozyme 40 mg/ml 5 mg/ml
Proteinasi K 20 mg/ml 9.5 mg/ml
Thaumatin 15 mg/ml 2 mg/ml
Ribonucleasi A 10 mg/ml 4 mg/ml

16. Effects of LB template in crystal growth (in brackets number of crystals), Effect A:
template facilitates nucleation step. LB crystals grow earlier than classical, Effect B:
template facilitates crystal growth. LB crystals grow larger than classical.

17. Phase Diagrammes of Proteinase K, Ribonuclease K, Thaumatin and Lysozyme (clockvise)

18. European Synchrotron Radiation Facility
(ESRF), Grenoble, France

19. Beamline
at ESRF
Protein crystal
and method
Of preparation
Number
of
crystal
being
studied
Crystal size
(μm)
Beam size
(μm)
Total
radiation
dose (MGy)
ID13 Lysozyme
classical
2 400×200×100 20×20 10 (500s
exposure)
ID13 Lysozyme LB 2 750×375×190 20×20 100 (4800s
exposure)
Lysozyme LB 2 Less than 1 μm 0.5×0.5
ID29 Thaumatin
classical
1 100×200×150 100×100 9.5
ID29 Thaumatin LB 3 100×300×150 100×100 9.5
ID23–1 Proteinase K
classical
1 200×100×150 80×60 44,1
ID23–1 Proteinase K LB 1 200×100×150 80×60 44,1
ID14–2 Proteinase K
classical
1 200×300×100 160×200 2.4
ID14–2 Proteinase K LB 3 160×200×50
300×200×200
350×300×300
160×200
100×100
100×100
2.43
2.22
2.16
ID14–2 Ribonuclease
classical
1 140×140×50 140×140 4.06
ID14–2 Ribonuclease
LB
1 140×140×50 140×140 4.96
LB and classical protein crystals analyzed at the different
ESRF beamlines.

20. High throughput protein
crystallization

21. Electron density of crystals grown using robotic technique for LB-based nanotemplate method and classical method.. A: comparison of electron
density map for carboxilic acid group of aspartic acid at 55 position contoured at 2.01sigma. B: comparison of electron density map of disulphide
bond of cystein residue at 71 position contoured at 2.5 sigma. C: comparison of electron density map of disulphide bond of cystein residue at 149
position contoured at 2.5 sigma. D: comparison of electron density map of proline residue at 135 position of classical and LB crystal contoured at
2.5 sigma. E: comparison of electron density map of cysteine residue at 159 position of classical and LB crystal contoured at 2.5 sigma. From left
to right and from top down.

22. Protein nanotemplate
X ray beam
i
f
microGISAX in situ
Detector
MAR CCD
Kapton window
The tubes connected with pamps fo the salt solution exchange:
Cs=Cd “stop solution” for aliement procedure
Cs»Cd for accelerated nucleation
Cs=2Cd for controlled grouth
Protein and salt solution, Cd
Salt solution , Cs
T1
T2
Pump1
Pump 2
Sealed camera

23. Setup of GISAXS setup at
ID13 beamline/ESRF. The
flow through crystallisation
cell is tilted by y to adjust a
fixed angle of incidence (i).
As typical features the
Yoneda Peak (Y) at c and
the specular peak at f=i
are shown in the 2d GISAXS
pattern.

24. Figure 3. Actual experimental configuration

25. MODEL for reaction pathways on LB-film
Pechkova, Gebhardt, Riekel, Nicolini, Biophysical Journal, Part I, 2010
Gebhardt, Pechkova, Rielel, Nicolini, Biophysical Journal, Part II, 2010