conformational stability of proteins

8/8/2019 Conformational Stability of Proteins

1/8


2/8

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. breaking) of disulfide linkages has little effect upon the

polypeptides 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.

Breathing

Proteins are static, rigid structures; but this is not the case.

A proteins 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 proteins 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 proteins interior.

In addition to breathing, some proteins may undergo more marked (usually reversible)

conformational changes. 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

binding to an antibody)

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


3/8

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. An increase in the number of intramolecular polypeptide hydrogen bonds.

2. An increase in the number of salt bridges.

3. Increased polypeptide compactness (improved packing of hydrophobic regions)

4. 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. Fewer salt links

2. Reduced aromatic interactions within the hydrophobic core (reduction in hydrophobicity)

3. Increased hydrogen bonding between the protein surfaces of the surrounding solvent.

4. 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


4/8

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 or Permanent

- Reversible or Temporary

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. Chemical (covalent) stability

ii. Conformational (non-covalent) stability in solution

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. Hydrolysis (enzymatic or chemical)

ii. Oxidation, particularly serine

iii. Deamination, particularly asparagines

iv. Phosphorylation and glycolation

v. -elimination

vi. Isopeptide formation

vii. Racemization

viii. S-S interaction and/or thiol/S-S-exchange (disulfide scrambling)

ix. Maillard reaction (NH 2+ 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


5/8

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. Extremes of pH

ii. Hydrophobic aggregation (e.g. by detergents)

iii. Pressure

iv. Shear forces

v. Temperature

vi. Sorption at interfaces (e.g. foaming)

vii. 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. Immobilization, e.g. in gel, or column, or within cells

ii. 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. Salt suspensions, typically in 3M ammonium sulfate.ii. Concentrated (50%) glycerol solutions, coupled with shipping and storage at -20 oC.

Solid-state stabilization methods include:5


6/8

i. Immobilization

ii. 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.

Denaturation mechanisms

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. Hydrolysis

ii. Deamination (of greatest importance)

iii. SH oxidation

iv. S-S- rearrangement

1. Denaturation by change in temperature

This is done by three ways:a. Heat denaturation

b. Cold denaturation

6


7/8

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. The process is completely reversible, even at very high protein concentration.

2. The denaturation/renaturation cycle appears to exhibit hysteresis, the extent of which is unaffected

by the number of cycling process.

3. The destabilizing effect of cryosolvents (e.g. aqueous methanol) on T H and T L are not identical.

4. Glycerol and other polyhydroxy compounds (PHC) are well known to stabilize proteins against

heat inactivation.

5. 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

7


8/8

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.

8

conformational stability of proteins

Documents