Protein stability(molecular biology)

description of protein stability and the interactions between them and measuring techniques of protein stability

1. Protein stability D.INDRAJA

2. Protein stability Protein stability is the net balance of forces, which determine whether a protein will be in its native folded conformation or a denatured state . Protein stability normally refers to the physical (thermodynamic) stability, not the chemical stability . Structural stability of protein is largely understood by studying various structures and conformations of protein Chemical Stability Chemical stability involves loss of integrity due to bond cleavage . deamination of asparagine and/or glutamine residues , hydrolysis of the peptide bond of Asp residues at low pH , oxidation of Met at high temperature, elimination of disulfide bonds disulfide interchange at neutral pH Other processes include thiol -catalyzed disulfide interchange and oxidation of cysteine residues.

3. Measuring protein stability The net stability of a protein is defined as the difference in free energy between the native and denatured state: Both GN and GU contribute to G It is measuring the energy difference between the U (unfolded) and F (folded) states . The average stability of a monomeric small protein is about kcal/mol, which is very small ! Δ DG = G N - G U = - RTlnK K=e -ΔG/RT = e -10x1000/(2x298) =2x 10 7 i.e . in aqueous solution, at room temperature, the ratio of folded : unfolded protein is 2x 10 7 : 1 Decreasing the energy of the folded state or increasing the energy of the unfolded state have the same effect on ΔG.

4. Protein Stability - Importance • Protein stability is important for many reasons: • Providing an understanding of the basic thermodynamics of the process of folding . • Increased protein stability may be a value in food and drug processing, and in biotechnology and protein drugs. Treatments and drugs that can specifically induce and sustain a strong chaperone and protease activity in cells Factors Affecting Protein Stability pH : proteins are most stable in the vicinity of their isoelectric point, pI . In general, electrostatic interactions are believed to contribute to a small amount of the stability of the native state; however, there may be exceptions. 2 ) Ligand binding : It has been known for a long time that binding ligands , e.g. inhibitors to enzymes, increases the stability of the protein. This also applies to ion binding --- many proteins bind anions in their functional sites. 3 ) Disulfide bonds : It was observed that many extracellular proteins contained disulfide bonds; whereas intracellular proteins usually did not exhibit disulfide bonds.

5. For many proteins, if their disulfides are broken (i.e. reduced) and then carboxymethylated with iodoacetate , the resulting protein is denatured, i.e. unfolded, or mostly unfolded Disulfide bonds are believed to increase the stability of the native state by decreasing the conformational entropy of the unfolded state due to the conformational constraints imposed by cross-linking ( i . e. decreasing the free energy of the unfolded state ). Not all residues make equal contributions to protein stability. In fact, it makes sense that interior ones, inaccessible to the solvent in the native state, should make a much greater contribution than those on the surface, which will also be solvent accessible in the unfolded state

6. Protein stability associated functions Controlling the stability of cellular proteins is a fundamental way by which cells • regulate growth, • differentiation, • survival, and • development. Measuring the turnover rate of a protein is often the first step in assessing whether or not the function of a protein is regulated by proteolysis under specific physiological conditions.

7.   Factors affecting protein stability in cell A network of highly conserved molecular chaperones and chaperone-related proteases controls the fold-quality of proteins in the cell. Most molecular chaperones can prevent protein aggregation by binding misfolding intermediates (for example, the GroEL / GroES or the DnaK / DnaJ / GrpE system).

8. Some molecular chaperones and chaperone-related proteases, such as the proteosome , can also hydrolyse ATP to forcefully convert stable harmful protein aggregates into harmless natively refoldable , or protease-degradable, polypeptides. Molecular chaperones and chaperone-related proteases thus control the delicate balance between natively folded functional proteins and aggregation-prone misfolded proteins eventually affecting the stability of protein, which may form during the lifetime and lead to cell death. Therapeutic approaches include treatments and drugs that can specifically induce and sustain a strong chaperone and protease activity in cells and tissues prone to toxic protein aggregations. Molecular chaperones and the proteases are major clearance mechanisms to remove toxic protein aggregates from cells, delaying the onset and the outcome of protein- misfolding diseases

9. Techniques Protein stability is measured with several methods such as Differential Scanning Calorimetry (DSC) Pulse-Chase Method Circular Dichroism (CD) Spectroscopy Fluorescence-based Activity Assays  

10. Differential Scanning Calorimetry (DSC) This technique has been widely used in characterizing the stability of proteins in their native form by measuring the amount of heat required to denature a particular biomolecule (e.g., protein). Generally, molecules with higher thermal transition midpoint (T m ) are considered more stable than those with lower transition midpoints. Due to its accuracy and high reproducibility, DSC is recognized as the “gold standard” for thermal stability analysis and can be used in characterizing and selecting the most suitable proteins in biotherapeutic development as well as for ligand interaction studies. Additionally, DSC offers the following advantages over other protein stability determination methods: Simple sample preparation Thermodynamic parameter determination Can use solid and liquid samples Can be used to detect denaturation temperatures and irreversible-reversible phase changes

11. Pulse-Chase Method Despite the fact that it involves labelling cells with radioactive precursors or pulse, the Pulse-Chase method has traditionally been the method of choice for determining protein stability. Many researchers favour this method since it allows for the accurate measurement of protein half-life and the determination of its subcellular localization. There are at least two popular non-radioactive versions of this method – the bleach-chase method and the cycloheximide chase method. Bleach-chase method .  In this method, the protein of interest is fused with a fluorophore and bleached with a brief pulse of light to produce a fluorescently and non-fluorescently protein population. The rate of degradation is correlated with the rate of fluorescence recovery after the bleaching process. Cycloheximide -chase method .  Instead of adding radioactive precursors, cycloheximide is added to inhibit protein synthesis and allow researchers to observe and assess the degradation of proteins as a function of time

12. Circular Dichroism (CD) Spectroscopy CD spectroscopy provides information on the conformation and stability of proteins by measuring the differences in absorption between left- and right-handed polarized light resulting from the structural asymmetry of the protein of interest. While it provides low-resolution information, CD spectroscopy is commonly used in most laboratories because it only requires a small amount of sample, is non-destructive and relatively easy to operate. Fluorescence-based Activity Assays Basically, fluorescence-based activity assays can be used to indirectly measure protein stability and provide information on protein activity and functionality. However, since it is highly susceptible to artifacts , coming up with reproducible results can be a challenge. In addition, the dyes used to track the stability of the proteins can affect the stability of the protein of interest .