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Protein Folding
Protein Folding. Source: QS Study

Protein Folding & Its Pathways

Protein Folding

Studies of protein stability and renaturation suggest that protein folding is directed largely by the residues that occupy the interior of the folded protein. But how does a protein fold to its native conformation? One might guess that this process occurs through the protein’s random exploration of all the conformations available to it until it eventually stumbles onto the correct one. A simple calculation first made by Cyrus Levinthal, however, convincingly demonstrates that this cannot possibly be the case: Assume that an n-residue protein’s 2n torsion angles, ϕ and ψ, each have three stable conformations. This yields 32n ≈ 10n possible conformations for the protein (a gross underestimate because we have completely neglected its side chains). Then, if the protein could explore a new conformation every 10−13 s (the rate at which single bonds reorient), the time t, in seconds, required for the protein to explore all the conformations available to it is

[latexpage] $t =\frac{10^n}{10^13}$

For a small protein of 100 residues, t = 1087 s, which is immensely greater than the apparent age of the universe (∼13.7 billion years = 4.3 × 1017 s). Clearly, proteins must fold more rapidly than this.

Proteins Follow Folding Pathways

Experiments have shown that many proteins fold to their native conformations in less than a few seconds (and even microseconds for some small proteins). This is because proteins fold to their native conformations via directed pathways rather than stumbling on them through random conformational searches. Thus, as a protein folds, its conformational stability increases sharply (i.e., its free energy decreases sharply), which makes folding a one-way process.

Hypothetical protein folding pathway
Hypothetical protein folding pathway. Source: Fundamentals of Biochemistry,5th edition,

Experimental observations indicate that protein folding begins with the formation of local segments of secondary structure (α helices and β sheets). This early stage of protein folding is extremely rapid, with much of the native secondary structure in small proteins appearing within 5 ms of the initiation of folding. Because native proteins contain compact hydrophobic cores, it is likely that the driving force in protein folding is what has been termed a hydrophobic collapse. The collapsed state is known as a molten globule, a species that has much of the secondary structure of the native protein but little of its tertiary structure. Theoretical studies suggest that helices and sheets form in part because they are particularly compact ways of folding a polypeptide chain.

Over the next 5 to 1000 ms, the secondary structure becomes stabilized and tertiary structure begins to form. During this intermediate stage, the nativelike elements are thought to take the form of subdomains that are not yet properly docked to form domains. In the final stage of folding, which for small, singledomain proteins occurs over the next few seconds, the protein undergoes a series of complex rearrangements in which it attains its relatively stable internal side chain packing and hydrogen bonding while it expels the remaining water molecules from its hydrophobic core.

In multidomain and multisubunit proteins, the respective units then assemble in a similar manner, with a few slight conformational adjustments required to produce the protein’s native tertiary or quaternary structure. Thus, proteins appear to fold in a hierarchical manner, with small local elements of structure forming and then coalescing to yield larger elements, which coalesce with other such elements to form yet larger elements, etc.

Folding, like denaturation, is a cooperative process, with small elements of structure accelerating the formation of additional structures. A folding protein must proceed from a high-energy, high-entropy state to a low-energy, low-entropy state. An unfolded polypeptide has many possible conformations (high entropy). As it folds into an ever- Section 5 Protein Folding decreasing number of possible conformations, its entropy and free energy decrease. The energy–entropy diagram is not a smooth valley, but a jagged landscape. The minor clefts and gullies on the sides of the funnel (false energy minima) represent partially folded conformations that are temporarily trapped until, through random thermal activation or the action of molecular chaperones, they overcome an “uphill” free energy barrier and can then proceed to a lower energy conformation. Evidently, proteins have evolved to have efficient folding pathways as well as stable native conformations.

Understanding the process of protein folding as well as the forces that stabilize folded proteins is essential for elucidating the rules that govern the relationship between a protein’s amino acid sequence and its three dimensional structure. Such information will prove useful in predicting the structures of the millions of proteins that are known only from their sequences.

Protein Disulfide Isomerase Acts during Protein Folding

Even under optimal experimental conditions, proteins often fold more slowly in vitro than they fold in vivo. One reason is that folding proteins often form disulfide bonds not present in the native proteins, and then slowly form native disulfide bonds through the process of disulfide interchange.

Energy–entropy diagram for protein folding.
Energy–entropy diagram for protein folding. Source: Fundamentals of Biochemistry,5th edition,

Protein disulfide isomerase (PDI) catalyzes this process. Indeed, the observation that RNase A folds so much faster in vivo than in vitro led Anfinsen to discover this enzyme.

PDI binds to a wide variety of unfolded polypeptides via a hydrophobic patch on its surface. A CysSH group on reduced (SH-containing) PDI reacts with a disulfide group on the polypeptide to form a mixed disulfide and a CysSH group on the polypeptide. Another disulfide group on the polypeptide, brought into proximity by the spontaneous folding of the polypeptide, is attacked by this CysSH group. The newly liberated CysSH group then repeats this process with another disulfide bond, and so on, ultimately yielding the polypeptide containing only native disulfide bonds, along with regenerated PDI. Oxidized (disulfide-containing) PDI also catalyzes the initial formation of a polypeptide’s disulfide bonds by a similar mechanism. In this case, the reduced PDI reaction product must be reoxidized by cellular oxidizing agents in order to repeat the process.


About Fahmida Akter Bristi

I am currently doing my Bachelor degree. I love to write by exploring knowledge that is new to me. Hope this effort of mine benefits you all. Right now, I am the head of Project R. Franklin & Project Waksman in Society & Science Foundation. Knock me anytime. Email:

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