Biochemistry 601

September 8, 1999 The Process of Protein Folding

Last modified 8/23/99
Dr. Landry

Rm. 6055



Native State Stabilization

The net stabilization of the native state conformation of a protein results from the balance of large forces that favor both folding and unfolding. For a theoretical protein, the energetic contributions to native state stabilization may be distributed as follows:
Folding                                   Unfolding

hydrophobic collapse                conformational entropy
intramolecular H-bonding         H-bonding to solvent (water)
van der Waals interactions

Contributions to the Free Energy of the reaction,  U-->N:

-200 kCal/mole                         +190 kCal/mole

Thus the net free energy of folding is 10 kCal/mole.
hydrophobic collapse
hydrophobic sidechains coalesce in the interior of the protein structure [Much of the free energy in this term is entropic in nature. The molecular explanation goes as follows: solvent-exposed surface area is reduced and thus fewer water molecules must be ordered]

The folding behavior of proteins is well approximated by a heteropolymer model composed of two residue types, hydrophobic and hydrophilic, in a "poor" solvent.
van der Waals interactions
weak dipole-dipole interactions between closely packed molecules

conformational entropy
entropy associated with the multiplicity of conformational states of the disordered polypeptide chain

Interactions stabilizing the native state are cooperative and redundant

The thermal melting behavior of protein structure indicates a "catastrophic" transition to the unfolded state, a characteristic of highly cooperative systems. A typical point mutant causes little or no change in the structure of the protein, but reduces protein stability as indicated by the lower melting temperature. Note that the mutant still undergoes cooperative unfolding. [Many amino acid substitutions in protein sequences have no detectable effect on protein function. Mutations causing functional defects usually occur in an enzyme active site or interfere with intermolecular assembly (e.g., collagenopathies).]

Hemoglobin S

Note in the kinemage that the structure of hemoglobin S is the same as normal hemoglobin, except for the substitution of Val for Glu at position 6. Aggregation of hemoglobin S into rod-like structures distorts the red blood cells into their characteristic "sickle" shape.

Sequence Specifies Structure

Protein folding can be studied in vitro by unfolding the protein with high concentrations (8 M) of a chaotropic agent such as guanidine-HCl or urea, and then permitting the protein to refold by removal of the chaotrope by dialysis or dilution. [Note the structural similarity of chaotropic agents and the peptide unit. Remember the old saying, "Like dissolves like"? The polypeptide tends to maximize its interaction with a "good" solvent by unfolding.]

In the classic refolding experiments by Anfinsen, it was shown that the information specifying the active conformation of ribonuclease A is contained in the amino acid sequence. Since the native state was achieved by either of two refolding paths, the native state lies at a global free energy minimum.

Folding Follows Pathways

The Levinthal Paradox

Consider how long it would take a 100-residue polypeptide to complete a random search for the native state.

Assume there are three (3) possible conformational states for each residue and that it takes 10**(-13) sec to interconvert between each state. For the 100-residue polypeptide, there are 3**100 [or 5x10**47] possible conformational states. Assuming a single unique native state conformation, it would take (5x10**47)(10**(-13)) sec = 5x10**34 sec or 1.6x10**27 yr. This absurd result clearly shows that protein folding does not occur by random search.

Recent in vitro studies demonstrate that protein folding follows a path(s) characterized by retention of partially correct intermediates.

Molecular Chaperones Block Aggregation of Folding Intermediates

The efficiency of protein folding can be compromised by aggregation of folding intermediates that have exposed hydrophobic surfaces. Such aggregates are essentially irreversible. Molecular chaperone proteins bind reversibly to folding intermediates, prevent aggregation, and promote their passage down the productive folding path. Molecular chaperones also are known as heat shock proteins because they are synthesized in much greater amounts by cells subjected to a wide variety of stresses, including elevated temperature and oxidative stress.

Specialized Enzymes Catalyze Key Steps in Protein Folding

Peptidylprolyl cis/trans isomerase (PPIase)

PPIase catalyzes the cis/trans isomerization of X-pro peptide bonds, where X is any amino acid. As previously discussed, the partial double bond character of the peptide bond restricts its rotation to values that keep the atoms bonded to the CO and N in a plane. The bond may adopt either the cis or trans conformation while satisfying this requirement. For all but X-pro peptide bonds, the trans conformation is strongly favored by steric interference in the cis configuration. For X-pro peptide bonds, a similar level of steric interference occurs in both cis and trans conformations; thus, the cis conformer is more favored for X-pro bonds than for bonds between other pairs of amino acids.

Steric interference between neighboring residues in the amino acid sequence is an example of a local interaction. The conformational behavior of an unfolded polypeptide is dominated by local interactions; whereas, long-range interactions stabilize secondary and tertiary structure in native proteins. In an unfolded polypeptide, a given peptide bond is much more likely to be trans (96%) than cis (4%); except in the case of an X-pro peptide bond where the likelihood of the cis conformer is significantly increased (%20). This relatively high probability of cis presents a significant barrier to the folding of a protein for which the native secondary and tertiary structure demands the trans conformation. Futhermore, some proteins contain a cis X-pro bond in the native structure, so molecules with a trans X-pro bond must interconvert to cis in order for the protein to complete folding. PPIase catalyzes cis/trans interconversion, achieving as much as a 300-fold rate enhancement.

Cyclophilin and FK506-binding Protein are PPIases

Immunosuppression by cyclosporin results from the formation of a ternary complex of cyclosporin, cyclophilin, and calcineurin. Cyclophilin was named in recognition of its affinity for cyclosporin, and it was later identified as a PPIase. Indeed, the PPIase activity of cyclophilin is inhibited by cyclosporin. However, immunosuppression results from inhibition of the protein phosphatase calcineurin which is tightly bound by the cyclophilin/cyclosporin complex. FK506 is another immunosuppressant which is chemically unrelated to cyclosporin. FK506 is bound by FK506-binding protein (another PPIase) resulting in formation of a calcineurin inhibitor.

Protein Disulfide Isomerase (PDI)

Disulfide bonds form and break by an exchange mechanism. Free thiol groups of cysteine residues in proteins are oxidized to form disulfide bonds by the reduction of preexisting disulfide bonds. Intracellularly, protein thiols may be oxidized initially by the specialized redox molecule, glutathione (a cysteine-containing tripeptide). The first exchange reaction between glutathione dimer (G-S-S-G) and the protein yields a mixed disulfide. The mixed disulfide then undergoes an intramolecular exchange reaction resulting in the discharge of reduced glutathione and formation of the intrachain disulfide bond. The polypeptide may then isomerize through the exchange of disulfide bonding partners. [In Anfinsen's experiment, "scrambled" ribonuclease isomerized to the native disulfide pattern after addition of a small amount of reduced beta-mecaptoethanol to break one of the disulfide bonds.] In cells, these isomerization reactions are catalyzed by PDI. The mechanism of PDI catalysis involves the transient formation of a disulfide bond between PDI and the polypeptide.

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