The document summarizes Giovanna Fragneto's presentation on using neutron scattering techniques like diffraction, SANS, and reflectometry to study soft matter and biological structures. It provides examples of using these techniques to determine the structure of RNA complexes, membrane proteins, lipid bilayers, and protein adsorption on surfaces. Neutron scattering is well-suited for these applications because it is non-destructive and hydrogen and deuterium have similar scattering lengths, allowing selective deuteration for contrast variation.

1. Italian Roadshow - June 2014
Neutron Scattering structural characterisation:
Examples from Biology and Soft Matter
Giovanna Fragneto
Institut Laue-Langevin, Grenoble
PARTNERSHIP FOR SOFT MATTER
PARTNERSHIP FOR STRUCTURAL BIOLOGY

2. LOOKING AT STRUCTURE: soft matter and biology
represent ~30% of research activities at the ILL
REFLECTOMETRY

3. LADI-III
ILL instrumentation for bio work:
Crystallography

4. Q-range 0.02 – 2.5 Å-1
Times ~min
ILL instrumentation for soft/bio work:
Diffraction
D16

5. ILL instrumentation for soft/bio work:
SANS
Q-range 10-3 – 1 Å-1
Times <sec - min
D11 D33
D22

6. ILL instrumentation for soft/bio work:
reflectometry
λ=2-30Å
Δλ/λ 1.2-10%
Beam strikes both sides of interfaces
D17
Q-range 0.001 – 2 Å-1
Times ~sec - min

7. structure and dynamics on length scales ranging from the nearest-neighbor distances
of lipid molecules to length scales of more than 100 nm
covering time scales from about 0.1 ps to almost 1 µs
http://www.rheinstaedter.de/maikel/
Inelastic, Backscattering, Spin-Echo:
!
!
!
▪ Time of Flight : IN5 and IN6
!
▪ Spin echo: IN11 IN15 WASP
!
▪ Cold neutron backscattering :
IN10 and IN16
!
▪ Thermal neutron backscattering : IN13

8. Neutron Diffraction
✤ Neutrons are scattered by atomic nuclei rather
than by the electrons (highly penetrating, non-
destructive)
✤ Scattering lengths for H and D of similar
magnitude to other common elements in soft
and bio-material
✤ Large incoherent scattering for H
✤ Traditionally protein crystals are soaked in
D2O buffers to exchange labile H-atoms for D-
atoms (perdeuteration leads now to crystals
one order of magnitude smaller)
!
H & D positions can be located & distinguished
at resolutions of ~1.5 and 2.5Å, respectively.
Atom
type
Coherent
scattering
length (fm)
Incoherent
scattering cross-
section (barn)
H -3.74 80.27
D ( 6.67 2.05
C 6.65 0
N 9.37 0.5
O 5.81 0
P 5.13 0.01
S 2.80 0
Fe 10.10 0

9. Neutron Macromolecular
Crystallography
Neutrons very useful for the study of:
!
✤ Enzyme mechanisms: protonation
states of AA’s (His, Asp, Glu, Lys,
Arg), ligands, inhibitors;
✤ Solvent structure: position and
orientation of D2O water molecules.
Distinguish D3O+
or OD-
✤ Metalloproteins: absence of photo-
reduction of metal cofactors.
✤ Drug-binding interactions: H-
bonding (distances, angles),
protonation states.
Type-III Antifreeze
protein
Asp
Asp
His
His
His
Cytochrome c
peroxidase
HIV-1 PR/APV

10. LADI – first results…
! Pyrophosphatase (C2, 106 x 96 x 114Å)
data to 2.2Å from perdeuterated crystal
of volume 0.32 mm3 (Ng/Garcia-Ruiz)
! Largest unit-cell volume studied thus
far in neutron protein crystallography
! Fatty Acid Binding Protein (P212121, 35 x 55
x 71Å) data to 2.0Å from perdeuterated
crystal of volume 0.05 mm3 (Podjarny/
Howard)
! Smallest crystal volume used to
collect high-resolution neutron data
! HIV-1 Protease (P21212, 59 x 87 x 46Å)
data to 2.0Å from perdeuterated crystal of
volume 0.20 mm3 (Kovalevsky/Langan)
! Galectin (P212121, 37 x 59 x 64Å) data to
2.1Å from perdeuterated crystal of
volume 0.16 mm3 (Logan)
Inorganic
pyrophosphatase
Courtesy M. Blakeley

11. RNA shape within the complex
from SANS data
Con$nuous'shape'of'both'
RNAs'in'the'apo0complex,'
in'contrast'to'EM'models'
FIBs'are'not'in'contact'with'the'
guide- RNA' (in' an' “off”'
posi$on);'only'2'FIB'copies'can'
reach'RNA'
dRNA-SANS-data--
at-42%D2O-
SANS lecture Gabel (HERCULES 2014)
Lapinaite, A., Simon, B., Skjaerven, L., Rakwalska-Bange, M., Gabel, F. and Carlomagno T. (2013)
The structure of the box C/D enzyme reveals regulation of RNA methylation. Nature 502(7472), 519-523.
SANS: bio-molecules in physiologically relevant
environment, large complexes, low resolution
RNA shape within a complex (c.F.Gabel)

12. SANS contrast variation to highlight the
interesting parts of the system
✤ 14h
Membrane proteins structural
characterisation by using
nanodiscs
Maric et al. Acta Cryst. 2013

13. ContrastVariation
Courtesy Selma Maric
D2O
H2O

14. SANS signal
expected from
membrane proteins
✤ SAXS from
Sensoryrhodopsin-II
Courtesy Selma Maric

15. h-lipid
d-lipid
thickness
Scattering length density profile
extracted from data analysis
Solid Si-SiO2
z
Liquid D2O
Reflectometry: looking at surfaces

16. Density Profiles of Proteins
in Polymer brushes
biocompatible surface functionalization
!
“brush failure” via protein adsorption
!
modes of protein adsorption:
primary, secondary, ternary
!
structural characterization
for “rational design” of protein resistant functionalization
(role of grafting density and polymer length)

17. Adsorption of deuterated myoglobin to PEG brushes
grafted on hydrophobic polystyrene surfaces
✤ Significant adsorption for all
brush parameters!
✤ only primary adsorption
Schneck, Schollier et al., Langmuir 2013

18. Adsorption of deuterated myoglobin to PEG brushes
grafted on hydrophobic polystyrene surfaces
✤ inner-layer: protein amount decreases
with grafting density!
✤ anchoring points obstacles adsorption!
✤ outer protein layer depends on overall
PEG amount and protein-protein
interactions are altered by the presence
of PEG!
✤ Information only accessible with
neutron reflection combined with
protein perdeuteration
Schneck, Schollier et al., Langmuir 2013

19. Specific adsorption: PEG antibodies
✤ Classically PEG purely repellent, in fact it is
antigenic!
✤ PEG antibodies produces in animals (0.1% - 25%
in humans)!
✤ Implications on brush functioning - failure?!
✤ IgG AB bind specifically to end segments of PEG
Brushes grafted to
hydrophilic phospholipid
surface to prevent primary
adsorption

20. Specific adsorption: PEG antibodies
Neutron reflectometry measurements
Brushes grafted to
hydrophilic phospholipid
surface to prevent primary
adsorption !

21. Specific adsorption: PEG antibodies
Schneck et al., submitted
Antibodies adsorbs at brush periphery!
No primary adsorption!
Amount increases with grafting density!
Saturation - molecular crowding!
!
Antibodies become the dominant surface:!
Brush no more functional!
foreign-body reaction

22. Interface Structure of Hyaluronan to Physical
Properties of Future Biomaterials
What is the gel?
Immobilized HA
Implant Surface
Berts et al., 2013
Ismall
Ilarge
SANS

23. HA bisphosphonate (BP)
BMP-2
HA gel
with BP
HA gel
without
BP
Titanium
Silicon wafer
Bare
titanium
oxide
surface
HA without
bisphosphonate
Bare substrate
HABP coating
Dilute BMP-2
Concentrated BMP-2
BMP-2 rinsed with Ca2+
Berts et al., 2013

24. Adsorption and triggered release
✤ The coating containing bisphosphonates (drugs
in the treatment of various bone diseases)
anchors strongly to the titanium by chemically
reacting with the oxide.
✤ The adsorption of a protein that naturally
promotes bone growth, bone morphogenetic
protein-2, onto the oxide surface is reversed by
the presence of Ca ions when BP is present, but
not from the uncoated surface.
✤ BP prefers to bind with calcium, which then
weakens the binding of the BMP-2 to the coated
surface.
These results demonstrate that this HA–BP coating system is
thus an effective option for ensuring the effectiveness of
implants, and should be explored further clinically.

25. Looking at fully hydrated single
bilayers with few Å resolution

26. Peptide induced thinning of
lipid bilayers
Bobone et al., University of Rome Tor Vergata (2013)
26
The short antimicrobial peptide peptaibol trichogin
GA IV forms pores and kills bacteria by causing the
thinning of the bilayer

27. Cholesterol modulates the fusogenic activity of a
membranotropic domain of the FIV glycoprotein gp36†
Vitiello et al., University of Naples (Soft Matter 2013)
✤ The molecular mechanism of the C8–
membrane interactions is studied by
combining Neutron Reflectivity and
Electron Spin Resonance experiments,
and molecular dynamics simulations. !
!
!
!
✤ Our findings suggest that cholesterol
rules, by an indirect mechanism, the
activity of viral fusion glycoproteins.

28. Dynamics of lipid rafts favours membrane fusion operated by a
membranotropic domain of the HSV-type I glycoprotein gH
Vitiello et al., University of Naples (under revision)
✤ The lipid composition is fundamental to drive the membrane-peptide
interaction.
✤ The presence of lipid ordered domains, mimicking the lipid rafts, influence the
mechanism of action of the gH625 peptide, favouring its insertion in the bilayer.

29. Towards complex biomimetic membranes
Rondelli et al. ,University of Milan (BBA Biomembranes 2012, EPJE 2013)
✤ The presence of ganglioside
forces asymmetry in cholesterol
distribution, opposite to what
happens for a ganglioside-free
membrane where a full
symmetrisation of cholesterol
distribution is observed.
✤ A preferential asymmetric
distribution of ganglioside and
cholesterol is attained revealing
that a true coupling between the
two molecules occurs.
SLD(10-4
nm-2
)
Z(nm)
0
2
4
6
0
5 10 15

30. Understanding effects of pollutants on our lungs
• we need to understand the mechanism of how pollutants cause us breathing difficulties
- lung surfactant is a complex mixture of lipids, proteins and other biomolecules
- monolayers of lipids were exposed to ozone & the loss of material was monitored
• the unsaturated lipids are destroyed by ozone which impairs lung function
- high flux & expert technical support on FIGARO allow this complex experiment to work
Langmuir, 2013, 29, 4594

31. Neutron tools for Soft Matter and Biology
!
!
✓more and more popular thanks to
deuteriation, improved sample
preparation, in-situ complementary
characterisation, and instrument
optimization
!
✓enormous potential to solve a great
number of problems (adhesion,
transport through membranes,
structure and dynamics of real
systems, drug delivery, effects of
nanoparticles, …)
COMPLEXITY
LIQUID/LIQUID interfaces
REDUCED SAMPLE
VOLUMES and SURFACES
DEUTERIATION
FAST KINETICS