DYNAMICS IN BIOLOGICAL SYSTEMS

Proton dynamics in biology: a sophisticated scenario revealed by neutron scattering

ILL- June 19th 2007

F. NATALI

CNR-IOM - OGG c/o ILL – Grenoble -FRANCE

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10-8-10-7 m

10-6-10-5 m

10-10-10-8 m

E. coli

RBC Thermophilic bacteria

10-3 m

… a long way from physics to biomedicine

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Light micrograph of a section

cut through human nervous

tissue

10-3 m

IS IT POSSIBLE TO INVESTIGATE, AT ATOMIC RESOLUTION,

PROTON DYNAMICS IN SUCH A COMPLEX SYSTEM ?

?

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Elastic intensity binned over Q as a function of T for hydrated powders (h=0.2) of myoglobin, polyglycine and polyalanine. The arrows mark the temperatures at which breaks in the temperature dependence are observed.

The results provide direct experimental evidence that the first anharmonic activation, around 150 K, is largely due to the activation of methyl groups rotations, but that contributions on the order of 10-20 % of motions of other groups (e.g. the phenolic ring and the methylene groups) are present. Our data also indicate that the dynamical transition occurring around 230 K can be attributed, at least at the hydration level investigated, mainly to motions involving backbone fluctuations.

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•associated with widely diffused pathologies

• the role of aggregate species in toxicity is unclear

• a major problem in developing protein delivery systems and protein based drugs

•fibril structural features: cross- structures in which - strands run perpendicularly to the long

axis of the fibril

G. Schiro’, V. Vetri, C. Andersen, Natali F. and A. Cupane. To be submitted

Insights into amyloid fibrils dynamics

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solvent-coupled motions are enhanced in the

amyloid state and reduced in the amorphous

one

proteins in the fibril state are

characterized by increased diffusive-like

motions, responsible for quasi-elastic

signal

enhanced solvent-coupled atomic fluctuations in ConA amyloid fibrils

amyloid formation bring proteins to expose side-chains to the fibril external and internal surfaces: lower steric

hindrance from other amino acids and larger interaction with hydration water

conversely, amorphous aggregation, via nonspecific interactions, hides protein surfaces from hydration water

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THERMAL FLUCTUATIONS OF HEMOGLOBIN FROM DIFFERENT SPECIES: ADAPTATION TO TEMPERATURE VIA MOLECULAR DYNAMICS

Tm

Specie TB/ºC Tm/ºC

Platypus Hb 33.0 60.2

Human Hb 36.6 63.1

Crocodile Hb 25.0-

34.0

66.9

Chicken Hb 41.0 68.3

The amino acid sequence of Hb seems to have evolved to permit an optimal flexibility of the protein at body

temperature.

A. M. Stadler, C. J. Garvey, A. Bocahut, S. Sacquin-Mora, I. Digel, G. J. Schneider,

F. Natali, G. M. Artmann, and G. Zaccai. 7

EMBEDDED PROTEINS IN SILICA HYDROGELS MAINTAIN ALMOST UNCHANGED THEIR FUNCTIONAL PROPERTIES, AND SHOWN INCREASED CONFORMATIONAL STABILITY AND RESISTANCE AGAINST

DENATURATION. 8

2- BASIC RESEARCH

HOMOGENEOUS AND OPTICAL TRANSPARENCY DOWN TO 5 K.

THE PROTEIN IS TRAPPED IN CAGES. ROTATIONS AND GLOBAL CONFORMATIONAL CHANGES ARE RESTRICTED, BUT LOCAL MOTIONS REQUIRED FOR LIGAND BINDING ARE STILL PERMITTED. THE PROTEIN CAN BE TRAPPED IN A GIVEN QUATERNARY STATE (T, R) IN THE GEL, AND THE MATRIX SUPPRESS THE R-T INTERCONVERSION.

OPTICAL ABSORPTION MEASUREMENTS ALLOWED AT

CRYOGENIC TEMPERATURES.

THE EQUILIBRIUM AND THE KINETICS OF LIGAND BINDING TO THE PROTEIN CAN BE STUDIED IN A GIVEN QUATERNARY CONFORMATION. UNFOLDING & REFOLDING OF ENCAPSULATED PROTEIN MAY BE FOLLOWED.

1- INDUSTRIAL APPLICATIONS: BIOSENSOR

DEVICE THAT COMBINE THE RECOGNITION OF BIOLOGICAL MOLECULES WITH ADVANCED TRANSDUCERS WHICH CONVERT THE BIOLOGICAL SIGNAL INTO A MEASURABLE OUTPUT.

FERRIC HEMOGLOBIN I (HbI) from Lucina pectinata: PHYSIOLOGICAL RECEPTOR FOR HYDROGEN SULFIDE – A GAS WHICH MAY PROVOKE LOSS OF CONSCIOUSNESS AND DEATH.

MYOGLOBIN: RECOGNITION ELEMENT FOR THE DETECTION OF NITRIC OXIDE (NO), CYANIDE (CN), CARBON MONOXIDE (CO).

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MYOGLOBIN GIRATION RADIUS: ~ 30 ANGS.

TRIDIMENSIONAL SiO2 MATRIX: DISTRIBUTION PORES SIZES CENTERED AT 50 ANGS

ONE OR NONE MYOGLOBIN MOLECULE TRAPPED IN EACH CAGE + SURROUNDING WATER MOLECULES.

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Pharmaceutical applications: flexibility

and drug release

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1) Beta-cyclodextrin inclusion complexes

H. Bordallo, Copenhague 13

Gerelli Y., Di Bari M.T., Barbieri S. Sonvico F. Colombo P., Natali F. and Deriu A. , SOFT MATTER (2010), 6, 685

Nanoparticles: lecithin/chitosan particles loaded with isopropyl

myristate and cetyl-stearyl alcohol, two lipophilic molecules with

different melting temperatures which are commonly added to improve

drug loading efficiency. At physiological temperatures they both

improve the particle flexibility.

AIM: the effect of lipophilic additives (excipients and drugs) on the behavior of lipid/saccharide

nanoparticles

2) lipid/saccharide nanoparticles

Effect of tamoxifen citrate, a lipophilic drug frequently used in breast cancer therapy: addition of tamoxifen

leads to stiffer structures and to lower amounts of released drug.

Temperature dependence of the MSD for nanoparticles (panel a) and for lecithin vesicles (panel b). The effect of the stiffer structure of the nanoparticle is clearly visible at high hydration h.

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Living BHK 21 cells

Cells are the most complex systems conclusions can be drawn by comparing various

systems: living and dead cells !

J. Zaccai, T. Hauss, N. Dencher, M. Frenzel, D. Skoufias, F. Lacroix

Characterization of functional molecular dynamics in cells in vivo under different ageing states

Effective force constants are very similar for two living control samples and softer than for dead cells, which seems

to be stiffer.

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Halobacterium salinarum

RBC E. coli

T. kodakaraensis T. barophilus

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MOLECULAR DYNAMICS RESPONSE OF HALOBACTERIUM SALINARUM TO ENVIRONMENTAL STRESS

V. Marty, M. Jasnin, E. Fabiani, F. Gabel, M. Trapp, J. Peters, G. Zaccai and B. Franzetti (2012) Submitted on SCIENCE.

In vivo molecular dynamics of Halobacterium salinarum proteome and cytosol under hyposaline stress conditions. Molecular dynamics variations detected under either hyposaline are consistent with a softening of structures due to protein unfolding.

Effective force constants <k’>, corresponding to resilience for unstressed cells (4.2M external NaCl), for cells exposed to 2.5M external NaCl, for cells exposed to 2.0M NaCl and values from control experiments in which cells were lysed in salt-free buffer.

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pressure High pressure

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• Membrane has to be in the liquid phase in the given environment.

• Pressure shifts main phase transition to higher temperatures same effect as decreased hydration.

• 75% of the biosphere lie below 1000m sea-level, corresponding to 100 bar or more.

• With increasing pressure, more unsaturated lipids in the membrane can be found in order to maintain the membrane fluidity.

• Decontamination and preservation of food products

• Elimination of microbial pathogens

• Keeping intact most of the organoleptic properties of food products

• Preservation of vitamin and flavour compounds

• ….and more

Why high pressure

Biological applications of HP to food products

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Deep Sea: study of model marine micro-organisms • Comparison of E.coli, as a mesophilic control; T. kodakaraensis and T. barophilus as hyperthermophilic cells as function of high pressure and temperature.

0 200 400 600 800 1000 1200

0,025

0,030

0,035

0,040

0,045

Su

mm

ed

in

ten

sity (

a.u

.)

p (bar)

Ecoli

Kodakarensis

Barophilus

275 280 285 290 295 300 305 310 315 320 325 330 335

0,004

0,005

0,006

0,007

0,008

0,009

0,010

Su

mm

ed

in

ten

sity (

a.u

.)

T (K)

Ecoli

Kodakarensis

Pressure and temperature seem to have inverse effects on the summed intensities. Kodakarensis undergoes a phase transition around 100 bar, Barophilus seems to be more stable against pressure than Ecoli.

(B. Franzetti, IBS Grenoble, M. Jebbar, IFRMER Brest, P. Oger, ENS Lyon, G. Zaccai and J. Peters, ILL, IBS and UJF Grenoble) 20

Where physics meets medicine to reveal brain pathologies

Anomalous proton dynamics of water in neural tissue. Impact on medical imaging techniques

The role of myelin in degenerative diseases

Model membranes

Cerebral tissue

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THE ROLE OF MYELIN IN DEGENERATIVE DISEASES

DEGENERATIVE DISEASES

The myelin sheath is a lipid-rich tightly packed multi-layered membrane structure surrounding and insulating the nerve axons in the

central nervous system (CNS) and the peripheral nervous system (PNS). It enables the fast transfer of nerve impulses. Myelin is

destroyed by autoimmune processes in inflammatory demyelinating diseases such as multiple sclerosis (MS) in the CNS and the

Guillan-Barré syndrome in the PNS.

THE MYELIN

SCIENTIFIC APPROACHES

• Investigation of dynamics in reconstituted model membranes simulating the myelin fibers

• Investigation of more complex scenario: the biological tissues in Central Nervous System 22

THE MODEL MEMBRANE in collaboration with the University of Basilicata and Genova, IT

Myelin proteins : primarily for biogenesis and structural stability.

In Central Nervous System (CNS): MBP: up to 30% w of total proteins

In Peripherical Nervous System (PNS): MBP: from 5 to 15%; P2: from 1 to 15%

MEMBRANE RECONSTITUTION and SAMPLE CHARACTERIZATION

Liposomes made of artificial lipids, DMPA, spread on silicon wafers to promote

membrane orientation. MBP is added to study its effect on the membrane dynamics.

Gel-to-liquid lipid phase transitions: T~ 320 K.

Prior to neutron scattering experiments all samples are firstly characterized using

standard lab techniques including Light Scattering, sucrose gradient process to

check liposome sizes, polidispersity and protein-liposome binding.

First studies of the influence of the Myelin Basic Protein (MBP) in CNS:

Natali, et al. (2002) Applied Physics A. 74, 1582-1584.

Natali, et al. (2003) Chem. Phys. 292 (2-3), 455-464.

Natali, et al. (2004) Physica B, 350 (1-3), E623-E626.

We address now the CNS !

THE MYELIN COMPOSITION

STRUCTURAL PROPERTIES

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DYNAMICAL PROPERTIES – FIRST RESULTS

Neutron scattering data acquired on

IN13 and IN16 at ILL (FR, 8 meV

FWHM and 0.8 meV FWHM),

respectively.

MEMBRANE RECONSTITUTION and SAMPLE CHARACTERIZATION

135°

45

°

n

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THE MODEL MEMBRANE in collaboration with the University of Oulu, Oulu, Finland

Myelin proteins : primarily for biogenesis and structural stability.

In Central Nervous System (CNS): MBP: up to 30% w of total proteins

In Peripherical Nervous System (PNS): MBP: from 5 to 15%; P2: from 1 to 15%

MEMBRANE RECONSTITUTION

Liposomes made of artificial lipids, DOPC and DOPS at 50% w/w.

The major myelin proteins, MBP and P2 are added one by one to study

their effect on the membrane dynamics. Gel-to-liquid lipid phase

transitions: TDOPC ~ 253 K and TDOPS ~ 262K.

First studies of the influence of the Myelin Basic Protein (MBP) in CNS:

Natali, et al. (2002) Applied Physics A. 74, 1582-1584.

Natali, et al. (2003) Chem. Phys. 292 (2-3), 455-464.

Natali, et al. (2004) Physica B, 350 (1-3), E623-E626.

We address now the CNS !

STRUCTURAL PROPERTIES

and SOON …

DYNAMICAL PROPERTIES – FIRST RESULTS

Diffraction images taken on

D16 at ILL in function of the

temperature for the protein free

lipids (upper panel) and MBP-

lipid complex (lower panel).

The lipid phase transitions are

clear visible, suggesting a shift

of at least one (or both) of the

transition temperature towards

higher value.

0

0.03

0.06

-0.5 0 0.5

Free lipidsLipids + MBPLipids + P2Lipids + MBP + P2

dE / meV

300K

45 deg

QE

NS

Neutron scattering data acquired at IN5 at ILL (10 meV FWHM).

Strong out-of-plane membrane stabilization is induced by the mixed MBP+P2

proteins at relative concentration typically found in physiology (MBP: 5% - P2:

2.5%). Effect less pronounced in the in plane dynamics and in the gel phase (230K).

Complementary data acquired on NEAT (DE), OSIRIS (UK) and IN13 (ILL).

DATA ANALYSIS UNDER PROGRESS.

To investigate the role of the mutant C8 of MBP, detected e.g. in

aggressive cases of multiple sclerosis, on the membrane stability.

THE MYELIN COMPOSITION

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At microscopic scale, water diffusion using MRI provide information on the tissue architecture and heterogenic structure. Changes in cellular water diffusion allow to highlight ischemic region induced by acute stroke.. But MRI averages all tissue heterogeneity!

Spin relaxation of hydrogen nuclei of water molecules magnetized by a strong external field.

26 F. Natali, J. Peters, G. Leduc, C. Stelletta, E. Barbier

Anomalous proton dynamics of water in neural tissue. Impact on medical imaging techniques

Nuclear Magnetic Resonance Imaging (RMI)

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First incoherent neutron scattering experiment of bovine cerebral tissue

IN5, 15 meV (FWHM) 30

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0.04

0.08

0.12

0.16

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0.000

0.004

0.008

(a)

w(meV)

(b)

Q2(Å-2)

tfast= 1.8 ps

Dfast= 2.3 *10-5 cm2/s

pfast= 73 %

DRfast= 2.3 *10-5 cm2/s

tslow= 6.7 ps

Dslow= 0.20 *10-5 cm2/s

pslow= 12 %

DRslow= 1.1 *10-5 cm2/s

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Checking data reproducibility

IN5, 15 meV (FWHM)

1- Tissue degradation 10 minutes scans were compared

2 –Dependence upon the specimen

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Effect of different preserving methods: cryogenic towards formalin addition.

Formalin: CH2O (37% w) in H2O

From MRI studies: formalin keeps unchanged the anisotropy of water diffusion, while it reduces the absolute value of water diffusion coef. D

Formalin maintain the tissue structure unchanged while it affects the membrane permeability.

IN5, 15 meV (FWHM) 34

~ 80% water, Mainly grey matter. INTERNAL Part of cerebrum. Intelligence, memory, emotion, speech

~80% water , Mainly grey matter. INTERNAL Posture, motor coordination Talking…

~ 74% water, Mainly white matter (fibers Connecting brain and spinal cord). EXTERNAL Respiration, heart rate, blood pressure …

~ 77% water, Mainly white matter. EXTERNAL Transmission of visual Information from the Retina to the brain.

DOWN TO ATOMIC SCALE: NEUTRONS PROBE THE BRAIN

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tslow= 6.7 – 18 ps

Dslow= 0.20 – 0.07 *10-5 cm2/s

pslow= 12 – 18 %

(RH - > MO)

DRslow= 1.1 *10-5 cm2/s

tfast= 1.8 – 3.7 ps

Dfast= 2.3-2.5 *10-5 cm2/s

pfast= 73 – 59 %

(RH - > MO)

DRfast= 2.3 *10-5 cm2/s

Neutron scattering is able to describe at atomic scale water diffusion in intracellular and extracellular compartments of cerebral tissue pinpointing:

RESULTS

Both pools do not show pure gaussian-like behavior higher residence time.

Strong dependence of water diffusion properties upon the microstructure of the tissue and grey-white matter ratio of the cerebral tissue perspectives for neurodegenerative disease involving myelin degradation.

Neutrons reveal 20% contribution arising from protein and membranes.

The existence of 2 waters pools characterized by slow and fast diffusion coefficients.

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Glioma: aggressive primary brain malignant tumor

It arises from the glial tissue. occurring predominantly in the cerebral hemispheres is highly malignant and rapidly progressing. It is distinguished pathologically from lower grade tumors by central necrosis (areas of dead cells) and microvascular hyperplasia (providing new nutrients refurbishment via, required for tumor expansion) .

Proton dynamics in malignant glioma tumors

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9-L Gliosarcome, at day 45 after implantation

Neutrons: cellular water diffusion is affected by the

presence of glioma. In particular, occurrence of necrosis,

typically associated to high tumor grade, speeds up water

diffusion.

dMRI (Castillo et al., 2001): The average water diffusion

constants (ADC) are enhanced in glioma when compared to

healthy tissue.

9-L Gliosarcome At day 21 after implantation

Bar graph of ADC values (s/mm2) for tumor, contralateral normal tissue, ipsilateral normal tissue, and edema for the group of 15 patients with high-grade cerebral gliomas.

M. Castillo et al., Am. J. Neuroradiol, 22, 60-64, 2001

The experiment clearly demonstrated that cellular water diffusion is affected by the presence of glioma. In particular, occurrence of necrosis, typically associated to high tumor grade, speeds up water diffusion.

QENS results

The average water diffusion constants (ADC) are enhanced in glioma when compared to healthy tissue.

DMRI results

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Anomalous water diffusion has been observed using QENS in malignant tumors extension to other tumors type.

PERSPECTIVES

Mice brain

Proton dynamics in malignant 9-L Gliosarcome tumors after radiation therapy

ROI The rat was sacrificed when moribund i.e. at D35 after implantation and irradiation in MRT configuration.

Exploring the brain with neutrons and dMRI

Healthy Cerebral tissues from different: - Organs - animal species - animal ages -Animal sex

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Proton dynamics of water at different degrees of confinement. Comparison of MRI and QENS on phantoms.

Proton dynamics in 9-L Gliosarcome tumors at different degrees of malignancy.

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Thank you !

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