This action might not be possible to undo. Are you sure you want to continue?
Protein stability and folding
Upon biosynthesis, a polypeptide folds into its native conformation which is structurally stable and functionally active. The conformation adopted ultimately depends upon the amino acid sequence of polypeptides, which explains why different polypeptide types have different characteristic conformations. The major stabilizing forces of a polypeptide’s overall conformation are: 1. Hydrophobic interactions 2. Electrostatic interactions 3. Covalent linkages Hydrophobic interactions Hydrophobic interactions are the single most important stabilizing influence of protein native structure. The “hydrophobic effect” refers to the tendency of non-polar substances to minimize contact with a polar solvent such as water. Non-polar amino acid residues constitute a significant proportion of the primary sequence of virtually all polypeptides. These polypeptides will fold in such a way as to maximize the number of such non-polar residue side chains buried in the polypeptide’s interior away from the surrounding aqueous environment. situation is most energetically favorable. Electrostatic attraction Stabilizing electrostatic interactions include: i. ii. iii. Van der Waals forces − which are relatively weak Hydrogen bonds Ionic interactions This
Although nowhere near as strong as covalent linkages, the larger number of such interactions existing within a polypeptide renders them collectively quite strong. Table: - Approximate bond energies associated with various (non-covalent) electrostatic interactions, as compared to a carbon-carbon single bond Bond type Van der Waals forces Hydrogen bond Ionic interactions Carbon-carbon bond Covalent linkages 1 Bond strength (kj/mol) 10 20 86 350
Disulfide bonds represent the major covalent bond type which can help stabilize polypeptides native 3-D structure. Intracellular proteins Although generally harboring multiple cystein residues, these rarely form disulfide linkages, due to the reducing environment which prevails inside the cell. Extracellular proteins In contrast, these are usually exposed to a more oxidizing environment, conductive to disulfide bond formation. In many cases the reduction (i.e. polypeptide’s native conformation. However, in other cases (particularly in disulfide rich proteins) disruption of this covalent linkage does render the protein less stable conformationally. In these cases the disulfide linkages likely serve to ‘lock’ functional and/or structurally important elements of domains- tertiary structure in place. breaking) of disulfide linkages has little effect upon the
Proteins are static, rigid structures; but this is not the case. A protein’s constituent atoms are constantly in motion and groups ranging from individual amino acid side chains to entire domains can be displaced via random motion by anything up to approximately 0.2 nm. A protein’s conformation therefore displays a limited degree of flexibility and such movement is termed “breathing” Breathing can sometimes be functionally significant by, for example, allowing small molecules to diffuse in/out of the protein’s interior. In addition to breathing, some proteins may undergo more marked (usually reversible) conformational changes. binding to an antibody) Such changes are usually functionally significant. Mot often they are induced by biospecific ligand interactions (e.g. binding of a substrate to an enzyme or antigen
Factors influencing intrinsic stability
The factors influencing the intrinsic stability of native polypeptide conformation have largely been elucidated via the study of proteins which function under relatively mild environmental conditions. 3-D structure of a number of homologous proteins derived from various psycrophiles, mesophiles, thermophiles and hyperthermophiles have now been determined. 2
This facilitates the identification of changes in structural features that helps render the protein stable under its particular native physiological conditions. Thermodynamics analysis reveals that the of marginal stability between the native versus
denatured state extends to proteins isolated from such extreme environments. It might be expected that proteins isolated from thermophiles and hyperthermophiles would exhibit an increased level of intramolecular stabilizing interactions, in order to compensate for the destabilizing influence of elevated temperature. Conversely, it could be predicted that, in order to remain at appropriate degree of conformational flexibility, proteins from psychrophiles would display decreased levels of such stabilizing interactions. Thermal stability Increased thermal stability is generally related to one or more of the following structural adaptations: 1. 2. 3. 4. An increase in the number of intramolecular polypeptide hydrogen bonds. An increase in the number of salt bridges. Increased polypeptide compactness (improved packing of hydrophobic regions) Extended helical regions.
Stability of proteins derived from psychrophiles Enhanced stability/functional flexibility of proteins derived from psychrophiles appear to be achieved by one or more of the following adaptations: 1. 2. 3. 4. Fewer salt links Reduced aromatic interactions within the hydrophobic core (reduction in hydrophobicity) Increased hydrogen bonding between the protein surfaces of the surrounding solvent. Occurrence of extended surface loops.
Conformational stability of proteins
Stability The stability of a protein, i.e. its usefulness as a biologically active molecule, depends on the particular environment and the exposure to conditions that can promote chemical deterioration or conformational changes.
1. in vivo stability
⇒ Under in vivo condition, protein stability (turnover) is governed by the action of proteolytic enzymes. 3
⇒ Protein life-times ranges from milliseconds (hormones, protein kinases) to years (collagen) ⇒ Of all known proteins, crystallin (eye lens) has the longest half life; it exhibits zero turnover and remains chemically unchanged throughout the life of the organism.
2. in vitro stability
⇒ The stability of an isolated protein in solution, i.e. removed from native environment, is limited by deleterious chemical reactions and/or conformational changes (denaturation). ⇒ Destabilization can be Irreversible ⇒ Destabilization caused by chemical reactions is permanent or irreversible. ⇒ Permanent destabilization is invariable deleterious, because it is accompanied by inactivation. Reversible ⇒ Structural change induced by changes in the solvent environment is reversible or temporary. In vitro stability include i. ii. Chemical (covalent) stability Conformational (non-covalent) stability in solution - Irreversible or Permanent - Reversible or Temporary
iii. Operational stability iv. Storage stability (i) Chemical stability ⇒ The peptide group is by nature reactive, as are several of the amino acid side chains. ⇒ They are thus subjected to attack by many reagents and can undergo the following reactions: i. ii. Hydrolysis (enzymatic or chemical) Oxidation, particularly serine
iii. Deamination, particularly asparagines iv. Phosphorylation and glycolation v. Β-elimination vi. Isopeptide formation vii. Racemization viii. S-S interaction and/or thiol/S-S-exchange (disulfide scrambling) ix. Maillard reaction (NH2+ reducing sugar) x. Chemical modification (immobilization, crosslinking)
(ii) Conformational (non-covalent) stability in solution ⇒ In addition to chemical reactions, which reduce the biological activity, isolated proteins in solution can also be inactivated by changes in their tertiary and higher order structures. 4
⇒ Such changes can, under certain circumstances can be reversed and the full activity restored to protein. ⇒ Conformational destabilization is produced by the following environmental changes: i. ii. iii. iv. v. vi. vii. Extremes of pH Hydrophobic aggregation (e.g. by detergents) Pressure Shear forces Temperature Sorption at interfaces (e.g. foaming) Metal binding
viii. Solvent effect, lyotropism (salting in) ⇒ Some of the above effect, used under right conditions, can be employed so as to stabilize or reactivate proteins. Such stabilization treatments include: i. ii. Immobilization, e.g. in gel, or column, or within cells Binding of cofactors/substrates/metals
iii. Low temperatures (unfrozen) iv. Crosslinking reactive side chains v. Hydrophilization, i.e. derivatization with hydrophilic groups vi. Lyotropism (salting out) (iii) Operational stability ⇒ Proteins used as processing aids, e.g. biocatalysts, need to be able to function over considerable periods of time (the longer the better) ⇒ The methods that may be used to stabilize an industrial enzyme against chemical attack or high temperatures differ dramatically from those that can be applied to stabilize therapeutic products destined for injection or ingestion. ⇒ In latter case, operational stability has no meaning, because each preparation in used just once and its efficacy is then determined by its in vivo performance. (iv) Storage stability ⇒ No protein can be purified to the extent that all traces of proteolytic enzymes are removed. ⇒ In order to obtain a commercially viable product, it is necessary to subject the protein to some form of treatment that renders it stable during processing, distribution and storage, with an acceptable shelf life, preferably at ambient temperatures. Stabilizing treatment of liquid products includes: i. ii. Salt suspensions, typically in 3M ammonium sulfate. Concentrated (50%) glycerol solutions, coupled with shipping and storage at -20oC. 5
Solid-state stabilization methods include:
Immobilization Deep freezing
iii. Freeze drying iv. Vacuum/spray drying
Over the past two decades, freeze-drying has established itself as the favored stabilization method, especially in the manufacture of therapeutic products and biochemical reagents.
Conformational stability in solution
⇒ The simplest model treats protein stability in terms of two state in dynamic equilibrium: N native state ↔ Denatured state D………..(i) The native (N) state is equally defined in terms of specificity and biological activity. The denatured (D) state refers to a macromolecule with perfectly flexible links between residues. ⇒ Denaturation can be associated with changes in one or more levels of structure, and can be proved by various spectroscopic and other physical techniques. ⇒ According to the two-state N/D model, denaturation and renaturation are cooperative, all-or-none process; no intermediate species exists with appreciable life times or concentrations. ⇒ Denaturation can therefore often be treated by simple equilibrium thermodynamics and the process in dilute solution can then be quantitatively described by an equilibrium constant, k, of the form – k = [D]/[N]……………(ii) Where, the second bracket denotes concentrations.
⇒ Thermal denaturation is sensitive to the amino acid composition. ⇒ In protein engineering, the aim is frequently increase the thermal stability by single point mutation. ⇒ It should be remembered that an increase in stability is not always accompanied by corresponding enhancement activity. ⇒ Studies on several enzymes have led to the general results that four reactions contribute to thermal inactivation: i. ii. Hydrolysis Deamination (of greatest importance)
iii. –SH oxidation iv. –S-S- rearrangement 1.
Denaturation by change in temperature
a. Heat denaturation b. Cold denaturation 6
This is done by three ways:
c. Freeze denaturation (a) Heat denaturation ⇒ One aim of protein engineering is the production of mutants with superior thermal stability, e.g. enzyme destined for the catalysis of biotransformations on an industrial scale, or for the use in products, such as detergents. ⇒ It is not immediately apparent how amino acid substitutions can enhance resistance against the chemical reactions shown above, unless the sensitive residues, e.g. as glutamine, can be considerable reduced in number or eliminated from the peptide chain altogether. (b) Cold denaturation ⇒ More recently, cold denaturation has been established as a real and probably universal phenomenon, of great ecological significance. ⇒ Cold-induced protein transition plays an important role in natural cold resistance and cold acclimation of many organisms. Features associated with cold denaturation: 1. 2. 3. 4. 5. The process is completely reversible, even at very high protein concentration. The denaturation/renaturation cycle appears to exhibit hysteresis, the extent of which is unaffected by the number of cycling process. The destabilizing effect of cryosolvents (e.g. aqueous methanol) on TH and TL are not identical. Glycerol and other polyhydroxy compounds (PHC) are well known to stabilize proteins against heat inactivation. The thermodynamics of cold inactivation are mirror image of those associated with heat denaturation. (c) Freeze denaturation ⇒ The major injurious effect of freezing is not low temperature, but the combination of all soluble species while ice separates from the mixture as a pure water phase. ⇒ Complex relationships exists between the initial protein concentration and the degree of freeze denaturation observed at different subzero temperatures. ⇒ Freezing also increases the concentration of buffers and other additives present in the solution by order of magnitude. ⇒ This can lead to the precipitation of acid and/or salts and large changes in the perceived pH, which in themselves can cause a protein to become inactivated.
2. Denaturation by change in pressure
In vitro protein denaturation by pressure is not of great practical importance, because very high pressure (>2 K bar) is required. The relationship between stability and pressure are therefore extremely complex.
3. Shear denaturation
Closely related pressure denaturations are the effects of shear on protein stability. Since protein purification procedures involve mixing, flow in tubes, ultrafiltration, passage through pumps and so on, the effects of shear are of most importance. Similarly immobilized proteins with fluid flowing past are also subject to shear degradation, e.g. a coating of anticoagulant on immobilized enzyme on a tube surface. The kinetics of enzyme reactions is also altered by shear.
4. Chemical denaturation
Chemical denaturations have effects on N-D equilibrium activities. Such effects may rely on electrostatic interactions or they may be caused by salting in/out phenomena. They may also be caused by specific binding phenomena or may be classified as general solvent effects. The stabilization for a given additive concentration observed at low temperatures far exceeds that at high temperatures.
5. Surface denaturation
Since proteins are amphiphilic polyelectrolytes, they exhibit some degree of surface activity, e.g. they absorb to interfaces. Hence, protein act as emulsifying/dispersing agent, as in the stabilization of fat in blood or milk, or the stabilization of air babbles in ice cream. ⇒ When surface forces are strong and coupled with a low N-state stability, sorption induces surface denaturation, as, for instances, in the precipitation of blood proteins on contact with some plastic materials.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue reading from where you left off, or restart the preview.