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Protein Stability

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Protein Stability

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  1. Protein Stability Willem J.H. van Berkel Laboratory of Biochemistry Wageningen University The Netherlands

  2. Why use enzymes ? Advantages • Enzymes are efficient and selective • Enzymes act under mild conditions • Enzymes are environmentally acceptable • Enzymes are not restricted to their natural role • Enzymes catalyse a broad spectrum of reactions • Enzymes can be modified • Enzymes can be produced by fermentation

  3. Why use enzymes ? Disadvantages • Enzymes require narrow operation parameters • Enzymes display their highest activity in water • Enzymes occur in one enantiomeric form • Enzymes are prone to inhibition phenomena • Enzymes can cause allergies • Pure enzymes are expensive • Enzyme recovery can be difficult • Some enzymes need cofactors

  4. Enzyme Applications • Foods juice, cheese, beer, meat • Detergents washing performance • Fine chemicals amino acids, antibiotics • Therapeutic agents removal of toxins • Molecular biology restriction enzymes • Analytical tools clinical analysis, foods • Stability requirements depend on the application

  5. Enzyme Stability Long term stability • Production, storage, shipment • Enzyme purification • pH, ionic strength, temperature • Frozen, liquid, powder • Presence of additives

  6. Enzyme Stability Operational stability • Medicine • frequency of administrating a new dose • reduce costs, and inconvenience for patient • Laundry • presence of surface active compounds • high temperatures, alkaline conditions • resistance of lipases to proteases

  7. Enzyme Stability Operational stability • Industrial synthetic applications • Process conditions • pH, organic solvents, denaturants etc. • Reusage of biocatalyst

  8. Enzyme Stability Topics of this chapter • Factors determining protein folding and activity • Causes of inactivation • Methods to determine stability • Strategies to prevent inactivation

  9. Enzyme Stability Factors affecting protein folding and activity • Hydrogen bonds • Ionic bonds • Van der Waals forces

  10. Folding of a polypeptide chain Non-covalent amino acid interactions • Hydrogen bonds C=O …. HN • C=O : Glu, Asp, Gln, Asn • NH : Lys, Arg, Gln, Asn, His • OH : Ser, Thr, Glu, Asp, Tyr

  11. Folding of a polypeptide chain Non-covalent amino acid interactions • Ionic bonds COO-….+H3N • COO- : Glu, Asp pKa < 5 • NH3 + : Lys, Arg pKa > 10

  12. Folding of a polypeptide chain Non-covalent amino acid interactions • Van der Waals forces • electrostatic in nature, short ranges • dipole-dipole, ion-dipole etc. • Table 2.1

  13. Folding of a polypeptide chain Non-covalent amino acid interactions • Strength of interaction Table 2.1 • 0.4 - 400 kJ/mol • charge, dipole moment • distance, dielectric constant medium • D = 80 (water) D = 2 - 4 (protein interior)

  14. Folding of a polypeptide chain Levels of protein folding • Primary structure (unfolded state) • Secondary structure (-helix, -sheet) • Tertiary structure (domains, subunit) • Quaternary structure (several pp chains) • Intra- and intermolecular disulfide bonds

  15. Protein Folding Folding pathway • Spontaneous process ? Yes and No • In vitro folding is slow • Many folding intermediates • Prevention of misfolding or aggregation by molecular chaperones

  16. Posttranslational modifications Chemical alterations after protein synthesis • May alter activity, life span or cellular location • Chemical modification • Acetylation, phosphorylation, glycosylation • Processing • Proteolytic (in)activation, selfsplicing

  17. Protein Folding Fold classification • Three-dimensional structures (X-ray, NMR) • Sequence comparisons • Protein homology modeling • Structure prediction • No simple relation with protein stability

  18. Enzyme catalysis Active site topology • Spatial arrangement of catalytically active groups • Recognition of substrates and cofactors • Conserved mechanisms • Substrate specificity • Enzyme families

  19. Enzyme catalysis Reduction of activation energy barrier • Thermodynamically favourable reactions • Proximity effects (effective concentration) • Orientation and strain effects • Acid-base catalysis (substrate activation) • Covalent catalysis (covalent intermediates)

  20. Protein Inactivation What factors may cause inactivation or unfolding? • Proteases Surfactants, detergents • Temperature Extremes of pH • Oxidation Unfolding agents • Heavy metals Chelating agents • Radiation Mechanical forces

  21. Protein Inactivation Irreversible inactivation • Proteolysis • Partial unfolding may increase proteolytic susceptibility (surface loops) • Integrity protein can be studied by (limited) proteolysis

  22. Protein Inactivation High temperature • Increase of mobility of protein segments • Exposure of hydrophobic groups • Formation of non-native disulfide bridges • Precipitation, scrambled structures • Aggregation, denaturation

  23. Protein Inactivation High temperature • Chemical modification • Deamidation of Asn or Gln • Hydrolysis of peptide bonds (Asp) • Destruction of disulfide bonds • Chemical reactions between proteins and other compounds: carbohydrates, polyphenolics

  24. Protein Inactivation Thermostable enzymes • Hyperthermophilic microorganisms • Comparison with mesophilic counterparts • Many different structural reasons for increased thermostability • Compact (multimeric) proteins • Increase of number of salt bridges

  25. Protein Inactivation Low temperature • Freezing • Concentration of solutes • Changes in pH and ionic strength • Increase in oxygen sensitivity • Storage in liquid nitrogen

  26. Protein Inactivation Extremes of pH • Repulsion of charged amino acid residues • Chemical modification (deamidation) • Hydrolysis of Asp-Pro linkages • High pH: destruction of disulfide bonds

  27. Protein Inactivation Surfactants and detergents • Hydrophilic head, hydrophobic tail • Form micelles above CMC • Monomers interact with proteins • Exposure of buried hydrophobic residues • Anionic detergents SDS • Cationic detergents CTAB • Non-ionic detergents Triton

  28. Protein Inactivation Denaturing agents • reversible unfolding • urea, guanidinium hydrochloride • diminish intramolecular hydrophobic interactions • chaotropic salts • polar organic solvents • chelating agents • heavy metals and thiol reagents

  29. Protein Inactivation Oxidation • Oxygen, hydrogen peroxide, oxygen radicals • Tyr, Phe, Trp, Cys, Met UV-radiation • Cys, Trp, His

  30. Protein Inactivation Mechanical forces • Stirring and mixing: shear forces • Ultrasound, high pressure, shaking • Deformation and exposure of hydrophobic residues  aggregation • Adsorption to wall of reaction vessel

  31. Protein Inactivation Generalmechanisms • Disturbance of balance of stabilising and destabilising interactions by weakening or strengthening charge or hydrophobic interactions • Covalent modifications • Breaking disulfide bonds

  32. Monitoring protein stability Stages of enzyme inactivation Reversible inactivation • Partial unfolding • Chemical alteration Irreversible inactivation • Complete unfolding, aggregation • Chemical modification, proteolysis

  33. Monitoring protein stability How do we measure protein stability? • Thermodynamically G unfolding Conformational stability • Biochemically Enzyme activity Storage stability Operational stability

  34. Monitoring protein stability Thermodynamic approach • Conformational stability • Suitable for model systems • Information about folding intermediates • Urea or GdnHCl unfolding (Fig. 2.2) • Trp fluorescence, circular dichroism • Differential scanning calorimetry (temperature)

  35. Monitoring protein stability Thermodynamic chemical approach • Two state model N  U (K = [N]/[U]) • GU= - RT ln K GU= GN- GU • Ratio folded / unfolded protein as function of unfolding agent (Fig. 2.2)

  36. Monitoring protein stability Thermal inactivation • G= H - TS ln K = - H / RT + S / R • Ratio folded / unfolded protein as a function of temperature (Fig. 2.3) • H and S increase with T • Gopt for most proteins between 20 and 40 ºC

  37. Monitoring protein stability Thermodynamic approach • Proteins are only marginally stable in the folded active form • Globular proteins: GU= 40 - 80 kJ / mol • Optimum for most proteins between 20 and 40 ºC

  38. Monitoring protein stability Biochemical approach • Storage stability as function of pH, temp, salt etc. • Useful information for applications • Useful for insights into enzyme action • Incubation of resting enzyme • Measurement of residual activity with time • Kinetics of enzyme inactivation (Fig. 2.4)

  39. Monitoring protein stability Operational stability • Stability of catalytically active enzyme • Highly relevant for applications • Difficult to measure on a laboratory scale • Influence of substrates (Fig. 2.5) • Mimicking of reactor conditions • Product yield with time

  40. Monitoring protein stability Optimal stability vs. optimal activity • pH dependence of thermostability (Fig. 2.6) • pH dependence of enzyme activity (Fig. 2.7) • Temperature dependence of enzyme activity • Absence or presence of substrates or cofactors • Optimum conditions for maximum conversion • Cost aspects (reusage of biocatalyst)

  41. Prevention of inactivation Avoid harmful conditions • pH, temp, protein concentration • Addition of stabilisers • Use of thermophilic enzymes Fig. 2.8 • Enzyme immobilisation Chapter 3 • Protein engineering • Chemical modification • Apolar organic solvents Chapter 5