Protein Stability and folding.pptx/pdf..

Protein Stability and Folding

1. Protein Stability and Folding Dr.J . Vardhana Assistant Professor, Dept of Biotechnology VISTAS

2. Protein Stability Native proteins are only marginally stable under physiological conditions -> high turnover Free energy required for denaturation is ~0.4 kJ/mol/ residue -> fully folded 100 residue protein is ~40 kJ/mol more stable than its unfolded form = energy of 2 hydrogen bonds But energy of all noncovalent interactions within a protein is in the order of thousands of kJ/mol => native structure results from a delicate balance of powerful counteracting forces

3. Proteins are stabilized by several forces Protein structures are governed mainly by hydrophobic effects and to lesser extend by interactions between polar residues The hydrophobic effect causes nonpolar substances to minimize their contact with water (degree of order, entropy, of water is decreased because water has not to form “cages” around the hydrophobic groups) Relative hydropathy of residues: energy required to solubilize a given amino acid in water

4. Electrostatic interactions contribute to protein stability Relatively week van der Waals forces are nevertheless important to stabilize the protein in its native state But hydrogen bonds, which are a central feature of protein structures, particularly secondary structures, make only a minor contribution to the overall stability of a protein Because of extensive hydrogen bonding of surface residues to water, difference between native and unfolded energy of hydrogen bonding is ~-2 to 8 kJ/mol Ion pairing / salt bridges , i.e. between Lys+ and Asp- 75% of ionized residues are paired, mostly on surface, but they have only a small stabilizing effect (paid by loss of entropy and loss of solvation free energy) => poorly conserved

5. Examples of ion pairs in myoglobin - Oppositely charged side chains from groups that are far apart in sequence closely approach each other through the formation of ion pairs

6. Disulfide bonds cross-link extracellular proteins Intrachain or inter-chain disulfide bonds form during folding of the protein While they are not necessary for the structure/function of most proteins, they “lock in” a particular backbone fold - Disulfide bonds are rarely in cytoplasmatic protein because the cytosol is a reducing environment that would cleave the disulfide bridges Most disulfide bridges occur in secreted proteins, i.e. they are stable in the more oxidizing extracellular environment

7. Metal ions stabilize some small domains Metal ions, bound within a protein, can also serve to stabilize the protein structure, through internal cross-linking For example zing fingers are motifs that frequently occur in DNA binding proteins Zn 2+ is tetrahedrally coordinated by side chain Cys, His and occasionally Aps and Glu Zn 2+ allows short stretches of polypeptides, 25-60 residues, to fold into stable units Zinc fingers are not stable in the absence of Zn 2+ Zn 2+ is stable and not oxidized/reduced (unlike Cu, or Fe)

8. A zing fin g er mo tif Coo rdin a t ed by Cys and His

9. Protein Folding Protein folding is directed largely by residues that occupy the interior of the folded protein How does a protein fold into its three dimensional structure Does not occur through sampling of all possible conformations ! This would take longer than the universe exists (n residues -> 2n torsion angles, each has 3 stable conformations ->3 2n = ~10 n possible conformations, 10 -13 sec for each conformation -> t = 10 n /10 13 => for n=100 residues t = 10 87 sec, 20 Mia years = 6 10 17 sec)

10. A ) Pro teins f ollo w f ol ding pathways Many proteins fold into their native conformation in less than a few seconds They follow directed pathways, not random Folding occurs locally by the formation of secondary structures Followed by a hydrophobic collapse of the structure to adopt a molten globule = has most of the secondary structures formed but not yet the proper tertiary structure Proteins fold in a hierarchical manner Cooperative process

11. P rotein di sul fide i s omera s e acts d u ri n g protein folding Proteins fold more slowly in vitro than they fold in vivo This is frequently due to the formation of non- native disulfide bridges which are then slowly exchanged to the native ones In vivo, disulfide bond formation is catalyzed by and enzyme: protein disulfide isomerase (PDI) PDI binds a variety of unfolded proteins via a hydrophobic patch to form a mixed disulfide

12. Mechanism of protein disulfide isomerase

13. Mechanism of protein disulfide isomerase

14. B) Mo l ec ul ar c h apero n es a ss i s t protein folding Proteins begin to fold as they are synthesized and grow on the ribosome In vivo, a peptide chain folds in the presence of a very high concentration of other proteins Molecular chaperones are essential proteins that help to fold newly synthesized or partially unfolded proteins to re-fold correctly Many molecular chaperones were first described as heat shock proteins (Hsp) , their expression is strongly induced upon heat treatment of cells

15. C h apero n e acti v ity re qu ires A TP Classes of molecular chaperones in prokaryotes and eukaryotes Hsp70 family, function as monomers with the cochaperone Hsp40 , folds newly made proteins Chaperonins , large multisubunit proteins (see below) Hsp90 proteins, folding of proteins in signal transduction such as steroid receptors Trigger factor , associate with ribosome and prevent improper folding of newly made All of them operate by binding to solvent-exposed hydrophobic surfaces and subsequent release All are ATPases

16. The GroEL/ES chaperonin forms closed chambers in which proteins fold The chaperonins in E. coli consist of two types of subunits, GroEL and GroES Structure: 14 identical 549-residue GroEL subunits arranged in two stacked rings of seven subunits each Complex is capped at one end by domelike heptameric ring of 97 Aa GroES subunits Bullet-shaped complex with C7 symmetry Central chamber of ~45 Å in which peptides fold

17. X-ray structure of the GroEL-GroES-(ADP) 7 complex GroES (cap) Gr oEL (cis) GroEL (tr ans)

18. Some diseases are caused by protein misfolding At least 20 – usually fatal – human diseases are associated with extracellular deposition of normally soluble proteins as insoluble fibrous aggregates = amyloids (starch-like) Amyloidoses: set of rare inherited diseases in which mutant forms of normally soluble proteins, such as lysozyme or fibrinogen, accumulate as amyloids Symptoms usually become apparent only later in life (30-70 years) progress over 5-15 years till death

19. Applications of hydrophobicity Using a hydrophobicity scale that assigns a value to each amino acid we can plot the variation of hydrophobicity along the sequence of a protein. This is called a hydrophobicity profile . Hydrophobicity profiles have been used to predict the positions of turns between elements of secondary structure, exposed and buried residues, membrane-spanning segments, and antigenic sites. Use of hydrophobicity profiles to predict the positions turns between helices and strands of sheet Figure shows the hydrophobicity profile of hen egg white lysozyme. It has pronounced minima at the following residues: 17, 44, 70, 93, and 117.

20. Figure shows the structure of hen egg white lysozyme, from which it is possible to check the correlation between turns in the structure and the positions of the minima in the hydrophobicity profile. Four of the major minima in the hydrophobicity profile appear at or near the positions of turns. Another minimum occurs in a surface-exposed region, but in the structure this one corresponds to a strand of a β sheet rather than to a turn. One of the minima is within a helix. Conversely, many of the turns do not correspond to pronounced minima in the hydrophobicity plot. Hydrophobicity profiles provide useful information, but do not unambiguously predict all turns in a protein structure.

21. The helical wheel O.B. Ptitsyn observed that α helices in globular proteins often have a ‘hydrophobic face’ turns inwards towards the protein interior, and a ‘hydrophilic face’ turned outwards towards the solvent. Each residue in an α helix appears at a position 100° around the circumference of the helix from its predecessor. Therefore, to achieve Ptitsyn's effect, the sequence of residues should alternate between hydrophobic and hydrophilic with a periodicity of approximately four. To check this relationship, the residues can be projected onto a plane perpendicular to a helix axis, a diagram called a helical wheel . This example shows the sequence of an α helix of sperm whale myoglobin. Charged and polar residues appear in green; others in black.

22. The helix has a hydrophobic face—which points to the inside of the structure, and a hydrophilic face—which points outside. In this picture the hydrophilic face is at the bottom of the diagram. From such a pattern of hydrophobicity we can predict whether a region of an amino acid sequence is likely to form an α helix in the native protein structure.

23. Superposition of structures Some aspects of sequence analysis carry over fairly directly into structural analysis, some must be generalized, and others have no analogues at all. As in the case of sequences, a fundamental question in analysing structures is to devise and compute a measure of similarity. If two molecules have very similar structures, we can imagine superposing them so that corresponding points are as close together as possible. Then the average distance between corresponding points is a measure of the structural similarity. In practice it is conventional to report the root-mean-square ( r.m.s. ) deviation of the corresponding atoms: where d i is the distance between the i th pair of atoms after optimal fitting, and n is the number of points. This assumes that we have prespecified the correspondence between the points. If the correspondence is not known, we must first determine it and only then calculate the r.m.s. deviation of the alignable substructures.

24. Structural alignment of bovine γ- chymotrypsin and S. aureus epidermolytic toxin A Bovine chymotrypsin and S. aureus epidermolytic toxin A are both members of the chymotrypsin family of proteinases. Figure shows a structural superposition of PDB entries 8GCH (γ-chymotrypsin) (black) and 1AGJ ( S. aureus epidermolytic toxin A) (green). The molecules share the common chymotrypsin-family serine proteinase folding pattern, and the Ser-His-Asp catalytic triad (ball-and-stick). Structural superposition of γ-chymotrypsin [8GCH] (black) and S. aureus epidermolytic toxin A [1AGJ] (green). The sidechains of the catalytic triads are shown. Observe that the region around the active site is the best-conserved part of the protein.