Norbert Kučerka

Katedra Fyzikálnej Chémie Liečiv, FaFUK v Bratislavea

Frankovo Laboratórium Neutrónovej Fyziky, SÚJV v Dubne

predmet: BiofyzikaFarmaceutická fakulta UK v Bratislave: Október 10, 2019

Norbert Kučerka

Katedra Fyzikálnej Chémie Liečiv, FaFUK v Bratislavea

Frankovo Laboratórium Neutrónovej Fyziky, SÚJV v Dubne

predmet: BiofyzikaFarmaceutická fakulta UK v Bratislave: Október 10, 2019

Neutron Sources

Properties of Neutrons

Fluid Biomembranes

Structure-Function Correlations

Structural Significance of Lipid Diversity

Platforms for Studying the Alzheimer Disease

• 1895 – Wilhelm Röntgen – discovery of X-rays

• 1932 – James Chadwick - discovery of neutron

• 1933 – Leó Szilárd – the idea of nuclear chain reaction

• 1937 – Glenn T. Seaborg – concept of nuclear spallation

• 1942 – Enrico Fermi – the first artificial nuclear reactor Chicago Pile-1

• 1947 – Elder, Gurewitsch, Langmuir and Pollock – observation of synchrotron radiation

• 1947-1993 & 1957 – National Research eXperimental & National Research Universal reactors

• 1950-1954 – Ernest O. Lawrence – the first spallation source Materials Testing Accelerator

• 1955 – Dimitry I. Blokhintsev – the idea of pulsed reactor

• 1960-1968-2001 – Ilya M. Frank & Fyodor L. Shapiro – the pulsed reactor IBR, IBR-30

• 1961 – Synchrotron Ultraviolet Radiation Facility at NIST – the first generation synchrotron

• 1970 – Synchrotron Radiation Source at Daresbury, UK – the second generation: dedicated source

• 1971 – Institute Laue Langevin – the most intense continuous neutron flux reactor

• 1984-2006/2010-2037 – IBR-2, IBR-2M

• 1994 – European Synchrotron Radiation Facility – the third generation: optimized for brightness

• 2006-present – Spallation Neutron Source operational

• 2017-future – X-ray Free Electron Laser – the fourth generation synchrotron

• 2018-future – PIK reactor

• 2019-future – European Spallation Source constructing

Historical Overview

p.4

Neutrons(1932) through Fission(1933) vs. Spallation(1937)

• fissile material required

• low energy needed

• low efficiency outcome

• no “dangerous goods”

• high energy particles needed

• high efficiency outcome

p.5

Neutron Sources based on Reactors(1942) vs. Accelerators(1951)

Continuous vs. Pulsed

National Research Universal (1957) reactor at Chalk River, ON

Spallation Neutron Source at Oak Ridge, TN (2006)

Inefficient use of neutrons produced in stationary reactors due to

• monochromators (acceptance~1%)

• choppers in Time-of-Flight methods

Complex systems at spallation sources

p.6

Idea of IBR (Импульсный Быстрый Реактор) in 1955

Institute of Physics and Power Engineering in Obninsk, Russia

The idea of the pulsed fast reactor of neutrons belongs to russian scientist Dimitry Ivanovich Blokhintsev

“Why not fix a part of the reactor active zone on a rim of the disk so that at each revolution this part passes near the stationary zone and creates a supercritical mass for a short time?”

p.7

Frank Laboratory of Neutron Physics in 1956-1960

1958 – Nobel Prize in physics toI.M.Frank and I.Ye.Tamm for a theoretical explanation of the Vavilov-Cherenkov radiation

World’s first reactor of a new type built in 3 years at the FLNPdirected by Nobel Laureate Ilya Mikhailovich Frank and under the guidance of Fyodor Lvovich Shapiro

• June 23, 1960 - start-up

• operation power of 1kW

• upgraded to 6kW

p.8

High Flux Pulsed Reactor IBR(1960)-2M(2010)

p.9

Advantages of Neutrons

• Charge:

• Magnetic Moment:

• Scattering Power:

==> deep penetration into matter

==> the world smallest magnetometer

==> sensitivity to light elements

==> sensitivity to isotope exchange

p.10

What Neutrons Can Do

Structure

C.G. Shull - The Nobel Prize in Physics 1994 - B.N. Brockhouse

Dynamics

“It might well be said that, if the neutron did not exist, it would need to be invented!”

p.11

Small-Angle Diffraction

The two planes will scatter in phase ifthe path difference 2 d sin(q) is awhole number of wavelengths ‘nl’:

nl = 2d sin(q) Bragg’s Law

=

+=

1h

h0D

2ππhcosF

D

2F

D

1(z)Δρ

0 2 4 6 8 10 12 14 16 180.01

0.1

1

10

100

Inte

nsi

ty

2theta

-30 -20 -10 0 10 20 30-1.0

-0.5

0.0

0.5

1.0

Neu

tron

Sca

tterin

g Le

ngth

Den

sity

[10

-6 Å

-2]

Distance form Bilayer Centre [Å]

scattering angle ~ 1 / distance

inverse space: Q=2π/d :real space

p.12

Neutron Scattering – Simple Models

Kučerka,N., J.F.Nagle, S.E.Feller and P.Balgavý, Physical Review E vol. 69 (2004) 051903

• neutron scattering data are inherently featureless

• however, the mid-q region provides stronginformation on bilayer overall structure, reflecting the large scattering contrast between the lipid bilayer (a lot of H) and solvent (D2O)

0.01 0.110

-5

10-3

10-1

101

103

I(q

) [c

m-1]

q [Å-1]

bilayer overall

structure

bilayer inner

structure

p.13

X-ray Scattering – Complex Models

N.Kučerka, S.Tristram-Nagle, J.F.Nagle, Biophysical Journal Letters 90 (2006) L83-85L

• high resolution X-ray scattering data allows

for using complex models, thus enhancing the

spatial resolution of the bilayer structure

D'B

2DC

Total

MethylCG

P

CH2

Water +

Choline

-30 -20 -10 0 10 20 300.0

0.1

0.2

0.3

0.4

ele

ctr

on d

ensity

[e/Å

3]

z [Å]

p.14

Joint Refinement – Advanced Models

N.Kučerka, J.F.Nagle, J.N.Sachs, S.E.Feller, J.Pencer A.J.Jackson, and J.Katsaras, Biophys. J (2008)

Scattering Density Profile analysis• Each of the component groups has nearly

the same functional form for all of the different contrast conditions (X-rays and neutron contrast variation)

• Volume distributions satisfy a spatial conservation principle

The SDP model is fit (with only one set of parameters) simultaneously to the set of scattering data obtained at different contrast conditions.

p.15

MD Simulations – beyond advancedMD Simulations + Experiment = beyond advanced

detailed structural information

N. Kučerka, J. Katsaras, and J.F. Nagle, Journal of Membrane Biology 235 (2010) 43-50

SIMulation to EXPeriment analysis• MD simulations aid the analysis of experiment

• experimental results help to improve simulations

• direct comparison reconciles the simulation and experiment

hydrogen bonding interactionslipid – cholesterol - water

p.16

Lipid Property-Structure Correlations

Chain Length Dependence Temperature Dependence

Double Bond Position Dependence

p.17

Biomimetic Membranes

Biological membranes deliver• Protection (separate cells)

• Signaling (transport of information)

• Selective permeability (transport of matter)

• Active functions are mainly provided byproteins

• Functionality depends strongly on thestructure and dynamics of a lipid matrix

• Lipid matrix is a 2D liquid, where lipids andproteins diffuse almost freely

Escribá et al., JCMM (2008)

Engelman, Nature (2005)

Singer and Nicolson, Science (1972)

p.18

p.18

0 5 10 15 20 25 30 35 40 45

12

14

16

18

20

diC14:1PC

diC22:1PC

diC18:1PC

hydrocarbon region thickness

cholesterol fraction [mol %]

dC

[Å]

Cholesterol’s effect on Biomembrane

N Kučerka, et al., Eur Phys J E 23 (2007) & N Kučerka, et al., Biophys. J (2008)

diC14:1PCdiC18:1PC cholesterol

diC22:1PC

Cholesterol acts as a spacer in the bilayer’s hydrophobic core.

1. The hydrocarbon ordering effect of cholesterol dominates over its ability to reduce the hydrophobic mismatch.

2. Cholesterol is covered by the headgroups of its two neighboring lipids, thus avoiding contact with water

--> umbrella model

3. Bilayer curvature may result in an asymmetric distribution of cholesterol.

p.19

Structure-Function Correlations of Melatonin

E.Drolle,N.Kučerka,M.I.Hoopes,Y.Choi,J.Katsaras,M.Karttunen,Z.Leonenko, BBA 1828 (2013)

05

1015

2025

30

30

31

32

33

34

35

36

37

38

0

5

10

15

2025

30

DH

H [

Å]

mel

aton

in [

mol

%]

cholesterol [mol %]

Proposed location of cholesterol resulting in increased order

The location of melatonin introduces disorderwithin the membrane

• cholesterol condensates membrane enabling amyloid

fibrils formation associated with Alzheimer Disease

• melatonin counteracts the effect of cholesterol

p.20

-30 -20 -10 0 10 20 30

-0.4

0.0

0.4

0.8

1.2

0.0

0.2

0.4

0.6

0.8

1.0

NSL

D

z [Å]

Prob

abili

ty

Cholesterol Melatonin

Amyloid-Beta

Location – Function Correlations

p.21

Contrast Variation in Neutron Scattering

Scattered intensity is proportional to

“the square of the difference between scattering length density of studied material and medium”

low contrast

2 ways to increase contrast

Neutron contrast variation can be done without changing the chemical properties of the system, because the neutron scattering lengths of isotopes can be very different.

p.22

Water Distribution

)()()( 11 zPzPz BBWW += )()()( 22 zPzPz BBWW +=

)()()()( 2121 zPzz WWW −=−

-30 -20 -10 0 10 20 30

0

2

4

6

NS

LD

[x

10

-6 Å

-2]

Distance from Bilayer Centre [Å]-30 -20 -10 0 10 20 30

0

2

4

6

NS

LD

[x

10

-6 Å

-2]

Distance from Bilayer Centre [Å]

-30 -20 -10 0 10 20 30

0.0

0.2

0.4

0.6

0.8

1.0

pro

ba

bili

ty

Distance from Bilayer Centre [Å]

The difference profiles of contrast varied scattering density profilesprovide distributions of membrane/water probability.

p.23

Lipid Rafts by Neutron Scattering

Heberle, Petruzielo, Pan, Drazba, Kučerka, Standaert, Feigenson, Katsaras, JACS 135 (2013)

Neutron scattering (via selective deuteration) is sensitive to nanometer-sized domains, previously not accessible by other methods.

The study of complex membrane models, including functional domains,helps to furtherour understandingof how the cell mayregulate criticalbiological processvia the compositionof lipid bilayers.

p.24

Label Distribution

-20 -10 0 10 20

0

1

2

NS

LD

[x

10

-6 Å

-2]

Distance from Bilayer Centre [Å]-20 -10 0 10 20

0

1

2

NS

LD

[x

10

-6 Å

-2]

Distance from Bilayer Centre [Å]

-20 -10 0 10 20

0

1

2

NS

LD

[x

10

-6 Å

-2]

Distance from Bilayer Centre [Å]

)()()()( zPzPzPz LLDWWBBD ++= )()()()( zPzPzPz LLHWWBBH ++=)()()()( zPzz LLHLDHD −=−

p.25

In contrast to ordered bilayers composed of saturated lipids (DMPC) or those with a small

degree of unsaturation (POPC),

more disordered bilayers containing polyunsaturated lipids (PUFA) orient cholesterol molecules parallel to the bilayer plane.

Lipid’s Effect on Cholesterol

Kučerka, Marquardt, Harroun, Nieh, Wassall, Katsaras, JACS (2009)

p.26

Cholesterol in Biomembranes

Cholesterol is essential to life.

•modifies the fluidity of biological membrane

• is important in the development of memory

• is necessary for the uptake of hormones in the brain

• is the main organic molecule in the brain, making up half the dry weight of the brain

•functions as a powerful antioxidant, protecting us against cancer and aging

• is a precursor to Vitamin D

• is a precursor to hormones that regulate blood sugar levels

• is a precursor to the sex hormones

• is a precursor to the bile salts

Cholesterol – Bad ?

p.27

Neutron Scattering is able to support bio-relevant studies with data not accessible to other techniques

Specific deuteration and contrast variation enhances neutron scattering by revealing subtle details

Structural studies help to further our understanding of critical biological process taking place in biomembranes