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Protein Stability

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Title: Protein Stability


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 CO . HN
  • CO 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 lt 5
  • NH3 Lys, Arg pKa gt 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
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16
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

17
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

18
Protein Folding
  • Fold classification
  • Three-dimensional structures (X-ray, NMR)
  • Sequence comparisons
  • Protein homology modeling
  • Structure prediction
  • No simple relation with protein stability

19
Enzyme catalysis
  • Active site topology
  • Spatial arrangement of catalytically active
    groups
  • Recognition of substrates and cofactors
  • Conserved mechanisms
  • Substrate specificity
  • Enzyme families

20
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)

21
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

22
Protein Inactivation
  • Irreversible inactivation
  • Proteolysis
  • Partial unfolding may increase proteolytic
    susceptibility (surface loops)
  • Integrity protein can be studied by (limited)
    proteolysis

23
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

24
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

25
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

26
Protein Inactivation
  • Low temperature
  • Freezing
  • Concentration of solutes
  • Changes in pH and ionic strength
  • Increase in oxygen sensitivity
  • Storage in liquid nitrogen

27
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

28
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

29
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

30
Protein Inactivation
  • Oxidation
  • Oxygen, hydrogen peroxide, oxygen radicals
  • Tyr, Phe, Trp, Cys, Met
  • UV-radiation
  • Cys, Trp, His

31
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

32
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

33
Monitoring protein stability
  • Stages of enzyme inactivation
  • Reversible inactivation
  • Partial unfolding
  • Chemical alteration
  • Irreversible inactivation
  • Complete unfolding, aggregation
  • Chemical modification, proteolysis

34
Monitoring protein stability
  • How do we measure protein stability?
  • Thermodynamically ?G unfolding
  • Conformational stability
  • Biochemically Enzyme activity
  • Storage stability
  • Operational stability

35
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)

36
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)

37
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

38
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

39
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)

40
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

41
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)

42
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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