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

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

REFLECTOMETRY

LADI-III

ILL instrumentation for bio work:

Crystallography

Q-range 0.02 – 2.5 Å-1 Times ~min

ILL instrumentation for soft/bio work:

Diffraction D16

ILL instrumentation for soft/bio work:

SANSQ-range 10-3 – 1 Å-1 Times <sec - min

D11 D33

D22

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

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

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

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

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

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'gu ide- RNA' ( i n' 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)

SANS contrast variation to highlight the interesting parts of the system

✤ 14hMembrane proteins structural

characterisation by using nanodiscs

Maric et al. Acta Cryst. 2013

Contrast Variation

Courtesy Selma Maric

D2O

H2O

SANS signal expected from membrane proteins

✤ SAXS from Sensoryrhodopsin-II

Courtesy Selma Maric

h-lipid

d-lipid

⇒thickness

Scattering length density profile extracted from data analysis

Solid Si-SiO2

z

Liquid D2O

Reflectometry: looking at surfaces

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)

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

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

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

Specific adsorption: PEG antibodies

Neutron reflectometry measurements

Brushes grafted to hydrophilic phospholipid surface to prevent primary

adsorption !

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

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

HA bisphosphonate (BP)

BMP-2

Sam

ple

C

Sam

ple

A

HA gel with BP

HA gel without BP

Sam

ple

B

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

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.

Looking at fully hydrated single bilayers with few Å resolution

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

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.

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.

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

05 10 15

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

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