Protein Stability

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Protein Stability

• Willem J.H. van Berkel
• Laboratory of Biochemistry
• Wageningen University
• The Netherlands

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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

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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

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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

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Enzyme Stability

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

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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

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Enzyme Stability

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

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Enzyme Stability

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

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Enzyme Stability

• Factors affecting protein folding and activity
• Hydrogen bonds
• Ionic bonds
• Van der Waals forces

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Folding of a polypeptide chain

• Non-covalent amino acid interactions
• Hydrogen bonds CO . HN
• CO Glu, Asp, Gln, Asn
• NH Lys, Arg, Gln, Asn, His
• OH Ser, Thr, Glu, Asp, Tyr

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Folding of a polypeptide chain

• Non-covalent amino acid interactions
• Ionic bonds COO- . H3N
• COO- Glu, Asp pKa lt 5
• NH3 Lys, Arg pKa gt 10

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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

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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)

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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

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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

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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

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Protein Folding

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

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Enzyme catalysis

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

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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)

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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

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Protein Inactivation

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

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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

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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

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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

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Protein Inactivation

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

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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

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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

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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

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Protein Inactivation

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

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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

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Protein Inactivation

• General mechanisms
• Disturbance of balance of stabilising and
  destabilising interactions by weakening or
  strengthening charge or hydrophobic interactions
• Covalent modifications
• Breaking disulfide bonds

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Monitoring protein stability

• Stages of enzyme inactivation
• Reversible inactivation
• Partial unfolding
• Chemical alteration
• Irreversible inactivation
• Complete unfolding, aggregation
• Chemical modification, proteolysis

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Monitoring protein stability

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

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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)

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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)

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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

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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

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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)

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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

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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)

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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