Pourquoi soumettre des protéines à des hautes pressions?

Transcription

1 Pourquoi soumettre des protéines à des hautes pressions? Reinhard Lange o Évolution personnelle, possibilités expérimentales o Repliement et agrégation des protéines problématique o Atouts des hautes pressions o exemple: les prions o Généralisation o Applications possibles IATE 2013

2 Pourquoi soumettre des protéines à des hautes pressions? Reinhard Lange o Évolution personnelle, possibilités expérimentales o Repliement et agrégation des protéines problématique o Atouts des hautes pressions o exemple: les prions o Généralisation o Applications possibles IATE 2013

3 DR1 INSERM, Carrière interface Physique Chimie Biologie: Dynamique structurale et fonctionnelle des protéines Les basses températures > cryoenzymologie (Paris, Brighton) Les hautes pressions > repliement des protéines (Montpellier) Depuis 1 er Juin 2013: mis à disposition à l INRA > IATE > axe 2 > équipe BTA

4 Possibilités expérimentales: Spectroscopie UV/Vis s/s pression Spectroscopie de fluorescence s/s pression Cinétique rapide stopped flow Cinétique rapide saut de pression

5 Bi directional p jump device Absorbance 3 4 Fluorescence

6 Pressure jumps Total fluorescence (a.u.) downward p jump upward p jump Pressure (MPa)

7 Example p jump relaxation kinetics 1.6 Fluorescence Intensity at 350 nm upward jump downward jump Time, times Time, second k obs = k 1 + k 1 K eq = k 1 / k 1

8 Pourquoi soumettre des protéines à des hautes pressions? Reinhard Lange o Évolution personnelle, possibilités expérimentales o Repliement et agrégation des protéines problématique o Atouts des hautes pressions o exemple: les prions o Généralisation o Applications possibles IATE 2013

9 DNA transcription RNA translation Primary protein structure folding Stable tertiary and quaternary structure

10 DNA transcription RNA translation Primary protein structure folding Stable tertiary and quaternary structure

11 Post translational reactions Native dissociated unfolded aggregated misfolded

12 Native dissociated unfolded aggregated misfolded Complex interplay between different structural states Biological relevance of the different states

13 Native dissociated unfolded aggregated misfolded Stability of proteins = important for biotechnological applications

14 Pressure temperature perturbation : theory Hawley equation: ΔG = f(p,t) ΔG = ΔG 0 ΔS 0 (T T 0 ) + ΔC p (T T 0 ) Tln(T/T 0 ) + ΔV 0 (p p 0 ) (Δβ/2)(p p 0 ) 2 + Δ (T T 0 )(p p 0 ) = Mathematical expression of an ellipse Unfolded p III Folded ΔS = 0 ΔG > 0 ΔG < 0 Unfolded II ΔV = 0 I IV 4 stability domains T

15 Simple energy landscape is unlikely Unfolded 2 Unfolded 1 Unfolded 3 Energy native state T P

16 Complexity of multidimensional structural landscapes Reaction goes to different attractors Particular final structure X

17 Confirmation of attractors from kinetics From Marchal et al., Accounts Chem. Res. 2008

18 Complexity questions: Predictability? Structural basis? Sequence of states? Time scale? Answers important for biomedical applications Approach Pressure temperature perturbation

19 Pourquoi soumettre des protéines à des hautes pressions? Reinhard Lange o Évolution personnelle, possibilités expérimentales o Repliement et agrégation des protéines problématique o Atouts des hautes pressions o exemple: les prions o Généralisation o Applications possibles IATE 2013

20 Pressure gives information about weak chemical interactions -Ionic interactions: dissociation (electrostriction) -Hydrophobic interaction: dissociation, hydration -Aromatic interactions: strengthening (aromatic stacking)

21 Information drawn from pressure experiments Thermodynamics Kinetics

22 Pressure and thermodynamics Useful parameters: ΔG 0 = RT ln(k) p ΔV ΔV = RT ln(k)/ p stability at reference pressure steepness of p dependent structural transition presence of voids, hydration Δβ = ΔV/ p) compressibility flexibility Δα = ΔV/ T) expansibility flexibility p 1/2 = ΔG 0 / ΔV mid point of p dependent transition characteristic pressure of structural transition

23 Pressure and kinetics Parameters of the kinetic transition state such as V useful for determining which kinetic path a reaction will take indication of kinetic traps on the energy surface Inform about reaction mechanism

24 Transition state - analogy to activation energy initial V - Activation volume V - change of rate limiting V Final state step E-S - reveal intermediates reaction

25 Pourquoi soumettre des protéines à des hautes pressions? Reinhard Lange o Évolution personnelle, possibilités expérimentales o Repliement et agrégation des protéines problématique o Atouts des hautes pressions o exemple: les prions o Généralisation o Applications possibles IATE 2013

26 SHaPrP Model of PrPC structure Recombinant prion protein from Syrian hamster Expanding from residues (142 aa) Sequence = resistant core of the Sc form after PK digestion PrP C = soluble, mainly alpha helix PrP Sc = insoluble amyloid fibrils, mainly beta sheet Question: what is the mechanism of interconversion? Approach: p/t perturbation thermodynamics

27 Prion protein topologies T Native Structure Partially unfolded structure T >57 C Non amyloid aggregate ( sheet rich)

28 Prion protein topologies T (+P) T = 80 C P = 200 MPa Native Structure Partially unfolded structure Unfolded structure T >57 C Non amyloid aggregate ( sheet rich)

29 Prion protein topologies T (+P) T = 80 C P = 200 MPa Native Structure Partially unfolded structure Unfolded structure T >57 C Non amyloid aggregate ( sheet rich)

30 Prion protein topologies P P T = 80 C P = 200 MPa Native Structure Partially unfolded structure Unfolded structure T >57 C P Non amyloid aggregate ( sheet rich)

31 Aggregation T / disaggregation P C th derivative UV absorbance d 4 A / d C 200 MPa C Wavelength (nm)

32 Changes in secondary structure accompanying protein aggregation & disaggregation 2 nd derivative FTIR Band 1655 cm -1 : -helix Band 1620 cm -1 : -sheet 2 nd derivative aggregated disaggregated pressure Wavenumber (cm -1 ) 1500

33 Prion protein topologies P P = 400 MPa T = 80 C P = 200 MPa Native Structure Partially unfolded structure Unfolded structure T >57 C P Non amyloid aggregate ( sheet rich)

34 Prion protein topologies P P = 400 MPa T = 80 C P = 200 MPa Native Structure Partially unfolded structure Unfolded structure P T >57 C P P = 600 MPa Non amyloid aggregate ( sheet rich) Metastable oligomeric structure

35 Prion protein topologies P P = 400 MPa T = 80 C P = 200 MPa Native Structure Partially unfolded structure Unfolded structure P T >57 C P Amyloid like aggregate O/N [PrP] P = 600 MPa Non amyloid aggregate ( sheet rich) Aggregated Amyloidogenic intermediate Metastable oligomeric structure

36 Time Neutral ph Cu 2+ Alkaline ph Agg HP Time Incubation at high pressure Fibrils Amorphous aggregates Time Time HS Cu 2+ Native PrP Amorphous aggregates Native PrP Spherical particles Fibrils Atmospheric pressure Time HS I HP Time Incubation at high pressure Neutral ph Neutral ph & denaturants

37 Negative stained transmission electron micrographs of mprp aggregates. (A) Amorphous aggregates formed after transient treatment at 600 MPa at ph 8.5. (B) Spherical particles of about 20 nm in diameter formed on prolonged incubation (18 h) at 600 MPa at ph 8.5. (C) Ordered bundles of laterally associated PrP fibrils generated after 8 days of incubation at 470 MPa at ph 7.4 From El Moustaine et al., FEBS J 2008

38 Pourquoi soumettre des protéines à des hautes pressions? Reinhard Lange o Évolution personnelle, possibilités expérimentales o Repliement et agrégation des protéines problématique o Atouts des hautes pressions o exemple: les prions o Généralisation o Applications possibles IATE 2013

39 Insulin Jansen et al., Biophys J 2005

40 Ataxine Distinct morphological aggregates are detected in Q26- AT3 aggregates resulting from pressure (A) and heat (B) treatment. Bars: 200 nm

41 Pourquoi soumettre des protéines à des hautes pressions? Reinhard Lange o Évolution personnelle, possibilités expérimentales o Repliement et agrégation des protéines problématique o Atouts des hautes pressions o exemple: les prions o Généralisation o Applications possibles IATE 2013

42 Domaine des biomatériaux Différentes conditions extrêmes > différentes formes d agrégation Coopérations internationales Coopérations au sein de l IATE?