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Protein Stability

Protein Stability. Willem J.H. van Berkel Laboratory of Biochemistry Wageningen University The Netherlands. Why use enzymes ?. Advantages Enzymes are efficient and selective Enzymes act under mild conditions Enzymes are environmentally acceptable

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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

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