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
Protein Stability
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Presentation Transcript
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 • 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
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
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
Enzyme Stability Long term stability • Production, storage, shipment • Enzyme purification • pH, ionic strength, temperature • Frozen, liquid, powder • Presence of additives
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
Enzyme Stability Operational stability • Industrial synthetic applications • Process conditions • pH, organic solvents, denaturants etc. • Reusage of biocatalyst
Enzyme Stability Topics of this chapter • Factors determining protein folding and activity • Causes of inactivation • Methods to determine stability • Strategies to prevent inactivation
Enzyme Stability Factors affecting protein folding and activity • Hydrogen bonds • Ionic bonds • Van der Waals forces
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
Folding of a polypeptide chain Non-covalent amino acid interactions • Ionic bonds COO-….+H3N • COO- : Glu, Asp pKa < 5 • NH3 + : Lys, Arg pKa > 10
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
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)
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
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
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
Protein Folding Fold classification • Three-dimensional structures (X-ray, NMR) • Sequence comparisons • Protein homology modeling • Structure prediction • No simple relation with protein stability
Enzyme catalysis Active site topology • Spatial arrangement of catalytically active groups • Recognition of substrates and cofactors • Conserved mechanisms • Substrate specificity • Enzyme families
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)
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
Protein Inactivation Irreversible inactivation • Proteolysis • Partial unfolding may increase proteolytic susceptibility (surface loops) • Integrity protein can be studied by (limited) proteolysis
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
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
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
Protein Inactivation Low temperature • Freezing • Concentration of solutes • Changes in pH and ionic strength • Increase in oxygen sensitivity • Storage in liquid nitrogen
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
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
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
Protein Inactivation Oxidation • Oxygen, hydrogen peroxide, oxygen radicals • Tyr, Phe, Trp, Cys, Met UV-radiation • Cys, Trp, His
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
Protein Inactivation Generalmechanisms • Disturbance of balance of stabilising and destabilising interactions by weakening or strengthening charge or hydrophobic interactions • Covalent modifications • Breaking disulfide bonds
Monitoring protein stability Stages of enzyme inactivation Reversible inactivation • Partial unfolding • Chemical alteration Irreversible inactivation • Complete unfolding, aggregation • Chemical modification, proteolysis
Monitoring protein stability How do we measure protein stability? • Thermodynamically G unfolding Conformational stability • Biochemically Enzyme activity Storage stability Operational stability
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)
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)
Monitoring protein stability Thermal inactivation • G= H - TS 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
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
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)
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
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)
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
