Major role of water on protein structure and dynamics Impact of processes and food proteins

Transcription

1 Major role of water on protein structure and dynamics Impact of processes and food proteins Camille Loupiac, Food Biochemistry Lecturer at AgroSup Dijon (ex ENSBANA) EMMA team (Eau, Molécules actives, Macromolécules, Activités) Burgundy University, AgroSup Dijon, France

2 Structure Proteins: Structure and dynamics Function in foods Dynamics - O + H 2N NH 2 - O O - O P O O NH NH 3 + H O Proteins function in food science: Hydration Emulsion Gel Impact of temperature, high pressure, salt, ph

3 Milk and milk proteins ion water lactose nm 0.3 g/l Humain Bovin Total amount 10,0 33,0 Casein(s) Whey proteins Lactoglobulin - 3 -Lactalbumin Lactoferrin Immunoglobulin casein random coil SAB lysozyme lactoglobulin (BLG) globular

4 Heat flow (mw/g) flux de chaleur (mw) Whey protein powders Impact of drying process Agregation-unfolding Native endo Native 2 Unfolded Temperature température ( C) ( C) Setaram DSC 3 Whey in suspension 20g/l «NATIVE» Gelation after heating at 80 C? Electrophoresis

5 Whey proteins powders Impact of formulation and storage Formulation Time of complete suspension as function of formulation Whey Whey + Lactose Whey + Lactose + Minerals Milk powders microstructure as function of storage conditions Agglomeration after High RH storage conditions Lost of solubility Lost of functionalities Role of water?

6 g H2O/g -lactoglobulin -Lactoglobulin hydration Second, third shells Freezable water Different from Bulk water View of the crystal structure 3BLG of β-lactoglobulin, Qin et al. Biochemsitry 1998 Qvist et al., PNAS Monolayer and Unfreezable water first shells protein bulk % relative humidity Autosorb, Biosystems, France Automatic sorption balance Or equilibration against saturated salts (red) In agreement with Ruegg et al., 1975 Beta-lactoglobulin : structure and dynamics :Water? Calorimetry: denaturation and glass transition temperature Neutron Scattering: picosecond dynamics

7 Heat flow (mw/g) Hydration and thermal denaturation Heat flow (mw/g) Water and denaturation temperature (calorimetry) Impact of water content Impact of ionic strength (Kpi) DSC 3 setaram 0.1 C / min High temperature flux de chaleur (mw) aw aw rehydratée eau Suspension 50g H20 / 100g powder 11g H20 / 100g powder température Temperature ( C) Td = 10 C Water content (RH) Td Stability Temperature ( C) Ionic Strength aggregation

8 Heat flow (mw/g) Hydration and glass transition Water and glass transition temperature (calorimetry) Low temperature DSC measurements at various level of protein hydration a) 8 b) 11 c) 50 g of water by 100 g of powder -0.2 a) 8 g water /100g powder b) 11 g water / 100g powder K cp= J/gK 220K c) 50 g water / 100g powder Temperature (K) DSC Q100 TA Instruments 10 C / min What is the origin of these two thermal events T 1 and T 2? Associated to water or protein dynamics?

9 Water and picosecond dynamics Elastic Incoherent neutron scattering Mean Square Displacements, picosecond dynamics Backscattering spectrometer,in13, ILL, Grenoble, D. Russo protein dynamics BLG (H/D) in D 2 O D 2 0 D D H D H D H D 2 0 D 2 0 D D D H H D 2 0 D 2 0 water dynamics BLG (H) in H 2 O <integrated intensity> BLG (H/D) / D 2 O BLG H / H 2 0 BLG (H/D) / D 2 O 180K 220K T (K) 50 g water / 100 g powder 50 g water / 100 g powder 8 g water / 100 g powder With freezable water BLG (H/D) in D20 (protein dynamics) dynamical transition temperature = 220 K BLG (H) in H20 (water dynamics) Dynamical transition temperature 180 K H 2 0 H H H H H H H H 2 0 H H 2 0 H H H H H 2 0 H 2 0 <integrated intensity> No dynamical transition without freezable water (in agreement with DSC results) T (K)

10 g( ), arbitrary units g( ), arbitrary unit Water and dynamical transition Inelastic neutron scattering, time of flight spectrometer Mibémol, LLB, Saclay, Jean-Marc Zanotti 2000 BLG (H) in 0.50g H K 190K 230K 250K 270K Energy transfer, mev g( ), arbitrary units K 190K 230K 250K 270K Energy transfer, mev BLG (H/D) in 0.50 g D20 160K 190K 220K 270K 300K Water vibrational density of states Change after 190 K= dynamical transition 0.1 No strong change in protein vibrational modes (mev)

11 Whey Proteins and high pressure High pressure for food science Novel technology: cheaper in energy to destroy microorganism Novel application: texturation, freezing, novel food To increase protein digestibility????? Whey protein powders Resuspended Tris buffer ph7 Solubility change Turbidity various protein concentration Various whey protein powders Unfolding aggregation????? 400 MPa, 10 min

12 Heat flow (mw) BLG / HP / biophysics BLG,20g/l, tris, ph7 Under Various HP 10 min, and after HP DSC III, setaram 0.1 C min 400 MPa 250 MPa 150 MPa ref HP Peak endo Peak exo unfolding, aggregation Temperature C BLG,0.4g/l, tris, ph7?max max (nm) nm Fluorescence enligne de blg sur haute pression à 25 C Trp fluorescence spectroscopy 50MPa HP max (10 nm) Trp more polar surrounding =>Reversible unfolding J. OF BIOL CHEM. Vol. 278, No. 47, pp , 2003, Yagi et al before pressure Under pressure After pressure

13 SANS and protein Neutron /2 q = (4 / ) sin( /2) Protein size Protein/protein interaction Protein partial volume Guinier law valid for qrg(c) < 1.3 I(q,c) = I (0,c) exp[-q 2 Rg 2 (c)/3] Rg A 2 v p isothermal compressibility c(p)[k(p)] 2 I(0,P,c) (P) = = [ N A Mp P Calmettes, LLB, Saclay D 2 0 D D H D H D H D 2 0 D 2 0 D D D H H D 2 0 D 2 0 [1+2A 2 (P)M P c(p)+ ] b P N A Mp - b(p) P (P)] BLG (H/D) in D 2 O 30 g/l

14 Crucial role of water HP unfolding? Studied proteins Myoglobin = helices Cavity + heam C. Loupiac et al., Eur. J. Biochem. 269, 4731 (2002). -Lactoglobulin= -sheet large central hydrophobic cavity Water hindrance with HP: Unfolding? C. Loupiac, et al., BBA (2006) 1764,

15 Perspectives: Protein stability / denaturation during drying or in the dry stated Dynamic in dry state in relationship with powder evolution during storage: Understanding the ageing process at a molecular level? Organisation of matrix during drying and its impact on stability Protein structure and dynamics in a complex matrices or in confined systems From milk proteins to vegetable proteins: new source of proteins How to use Neutron Scattering Experiments for these topics?

16 Coworkers: Dominique Champion and Philippe Cayot (Equipe EMMA, Dijon) Patrick Calmettes and Jean-Marc Zanotti (LLB, Saclay) Marco Bonetti (CEA, Saclay) Marie-Sousai Appavou (JCNS, München) Daniela Russo (Institut Laue Langevin, Grenoble) Thank you..questions?