Protein Folding Explained

Protein folding is a crucial process that gives rise to 3D structures essential for biological function. It is a delicate process that can be disrupted by extreme temperatures, pH, chemicals and space limitations. Miss folding can lead to aggregation and denatured proteins that are no longer functional.


It is assisted by heat shock proteins called chaperones.


The shape of a protein determines how it functions. From insulin that controls sugar levels in the blood to antibodies that fight coronavirus, most biological processes involve proteins. But protein folding is a difficult task. It has been estimated that an unfolded polypeptide chain has an astronomical number of possible shapes, or conformations. [1]

The native structure of a protein is determined by its amino acid sequence and the energy available to fold it, which includes hydrogen bonds between polar amino acids, Van der Waals interactions with water molecules, and the energy of entropy. Whether a protein can adopt its native conformation also depends on the environment, such as its solvent (water or lipid bilayer), pH, temperature, salt concentrations, the presence of cofactors and molecular chaperones, and the kinetic energy of surrounding protein molecules.

It is therefore not surprising that a large number of different proteins have been observed in various states, including aggregated or misfolded forms. In these states, they may not bind to other proteins and thus fail to carry out their cellular function. In order to understand why this happens, biophysical techniques such as stopped flow and fluorescence spectroscopy can be used to study the dynamics of a small sample of a protein.

Hydrophobic Interactions

Many of the steps in protein folding involve short-range interactions between a limited set of amino acid residues that form clusters. Then these regions coalesce to form ordered backbone structures (a-helixes or b-sheets). The final stage is the formation of tertiary structure as long-range contacts between amino acids are formed.

The hydrophobic effect is a strong driving force in the early stages of protein folding. This is a result of the patterns of non-polar side chains that are formed in the protein sequence. The dimensions of these patterns are critical, and a protein with a larger number of fully buried polar residues can be significantly more destabilized than one with a similar number of exposed polar residues.

The interaction of these residues with water molecules is crucial to the protein’s stability. Recent studies have shown that the number of hydrogen bonds formed by a water molecule with a protein is relatively constant, both in bulk water and at the protein-water interface10,11.

Disulfide Bonds

Protein folding often involves covalent bonding through disulfide bonds formed between the thiol groups of two cysteine residues. These links (also known as disulphide bridges or S-S bonds) are essential for many functions, such as stability and tertiary structure. The formation of these bonds is a key step in the transition between the amorphous “molten globule” state and the native folded structure.

The cytosol in eukaryotic cells contains reducing agents that keep cysteine residues in their reduced form, preventing the formation of disulfide bonds. To overcome this, proteins must fold in a redox-active environment such as the endoplasmic reticulum, where a special enzyme called PDI is required to oxidatively form and break these bonds as needed.

Despite great progress over the past 30-plus years, the exact mechanism by which PDI recognizes incorrectly oxidized proteins and promotes their proper rearrangement remains unclear. It is likely that different PDI paralogues have cysteines with a range of electron affinities and that each binds to only a fraction of the possible disulfide pairs found in the target protein.

Secondary Structure

Protein folding involves the formation of secondary structures such as a-helixes and beta pleated sheets. These structures form from the peptide backbone of the polypeptide chain. The secondary structure forms in a manner that gives the protein its geometric shape. These structures then interact with each other to give the protein its tertiary structure. The tertiary structure of a protein is determined by interactions and bonding between amino acid side chains. It is generally accepted that evolution has designed protein sequences so that the overall energy landscape of the molecule largely favors folding. This is known as the “folding funnel” theory.

Many methods have been developed to predict the three-dimensional structure of proteins. These methods use information such as amino acid type and polarity, phi and psi angle preferences and hydrogen bond donor and acceptor positions in the protein’s peptide backbone to construct models of a protein’s secondary structure.

X-ray crystallography is another method used to determine protein structure. This technique uses a pattern of deflected X-rays to determine the positions of the thousands of atoms that make up a protein.

Tertiary Structure

The final stage of protein folding is when the secondary structures fold into each other to give a three-dimensional shape to proteins. This is called the tertiary structure of a protein and it is mainly stabilized by interactions between amino acid side chains, which are the unique chemical groups that distinguish each amino acid from other proteins. These are either positively or negatively charged, polar uncharged, or non-polar and can interact with each other through hydrogen bonds, salt bridges, and disulfide bonds.

The primary structure of a protein is encoded by its amino acid sequence and this dictates the type of secondary structures that will form, for example alpha helices or beta pleated sheets. The sequence also determines the location of these secondary structures within a protein.

The interactions that occur between these different layers of a protein are what make it functional. If these interactions are disrupted by temperature, pH changes, chemicals, or molecular crowding then a protein will lose its higher-order structures and return to its unstructured string of amino acids state, this is called denaturing a protein.