Protein Folding: 5 Things to Know About

Read this article to learn about five things to know about protein folding.

The five things are: (1) Creating a Functional Protein (2) The Quest to Understand Protein Folding (3) Finding the Energy to Fold (4) The Process of Folding and (5) Fold Recognition (Threading).

Proteins are the biological workhorses that carry out vital functions in every cell. To carry out their task, proteins must fold into a complex three-dimensional structure, but how and which shape a protein should attain is determined protein itself.

Of all the molecules found in living organisms, proteins are the most important. They are used to support the skeleton, control senses, move muscles, digest food, and defend against infections and process emotions. Proteins come in all shapes and sizes. They can be round (like haemoglobin), long (like collagen), strong (like spectrin which protects erythrocytes (the cells that carry oxygen from the lungs to our tissues) from the powerful shearing forces they are exposed to), or elastic (like titin, which controls muscle stretching and contraction).

The name protein is derived from the Greek word protos, meaning ‘primary’ or ‘first rank of importance? ? And with good reason. These are the most abundant component within a cell’. More than half the dry weight of a cell is made up of proteins and they have a range of indispensable roles; for example, enzymes, the biocatalysts that carry out crucial biochemical reactions in every cell that would otherwise be too slow to sustain life.

What is remarkable is that the more than 100,000 proteins in our bodies are produced from a set of only 20 building blocks, known as amino acids. All amino acids have the same basic structure an amino group, a carboxyl group and a hydrogen atom, but differ due to the presence of a side-chain. This side-chain varies dramatically between amino acids, from a simple hydrogen atom in the amino acid glycine to a complex structure found in tryptophan.

Depending on the nature of the side-chain, an amino acid can be hydrophilic (water-attracting) or hydrophobic (water-repelling), acidic or basic; and it is this diversity in side-chain properties that gives each protein its specific character.

Thing # 1. Creating a Functional Protein:

The sequence of amino acids in a protein defines its primary structure. The blueprint for each amino acid is laid down by sets of three letters known as base triplets that are found in the coding regions of genes. These base triplets are recognized by ribosomes, the protein building sites of the cell, which create and successively join the amino acids together. This is a remarkably quick process: a protein of 300 amino acids will be made in little more than a minute.

The result is a linear chain of amino acids, but this only becomes a functional protein when it folds into its three- dimensional (tertiary structure) form. This occurs through an intermediate form, known as secondary structure, the most common of which are the rod-like a-helix and the plate-like 3-plated sheet. These secondary structures are formed by a small number of amino acids that are close together, which then, in turn, interact, fold and coil to produce the tertiary structure that contains its functional regions (called domains).

Although it is possible to deduce the primary structure of a protein from a gene/s sequence, its tertiary structure cannot be determined (although it should become possible to make predictions when more tertiary sequences are submitted to databases).

It can only be determined by complex experimental analyses and, at present, this information is only known for about 10% of proteins. It is therefore not yet known how an amino-acid chain folds into its tertiary structure in the short time scale (fractions of a second) that occurs in the cell.

So, there is a huge gap in our knowledge of how we move from protein sequence to function in living organisms: the line of sight from the genetic blueprint for a protein to its biological function is blocked by the impenetrable jungle of protein folding.

Thing # 2. The Quest to Understand Protein Folding:

One of the most important results in understanding the process of protein folding was a thought provoking experiment that was carried out by Christian Anfinsen and colleagues in the early 1960s. They investigated a protein called ribo-nuclease, which they isolated from the pancreatic tissue of cattle. This enzyme, made up of 124 amino acids, cleaves any ribonucleic acid (RNA) that could be harmful to the cell, such as truncated RNA that would not make a fully operational protein.

It briefly binds RNA in a binding site and requires several sulphur-containing amino-acid cysteine residues in the protein, which form bonds with each other (called disulphide bridges) and hold the protein structure together. Ribo-nuclease can be denatured by adding certain chemicals or by heat. The disulphide bridges break and other forces of attraction between amino acids disappear, which makes the enzyme collapse into a tangled, useless ball.

The ribo-nuclease then folds back to its natural functional state on its own. So, Anfinsen concluded that the amino- acid sequence determines the shape of a protein, a finding for which Anfinsen received the Nobel Prize in Chemistry in 1972.

Thing # 3. Finding the Energy to Fold:

As with all processes in nature, protein folding also needs energy, the process has to obey the laws of thermodynamics. A protein always folds so that it achieves the lowest possible energy just as we always try to adopt the most comfortable position, in which we need to move about least, when going to sleep. It is thought that this is achieved by using an energy gradient or funnel along the path from the random tangle to the folded protein.

Alan Fersht of Cambridge University used the following analogy to illustrate this model: if you blindfold a golfer and let him hit the ball in any direction he likes, the probability that he will hold the ball is almost infinitesimal.

The same is true of a protein, finding the right form by chance. However, if all parts of the golf course slope toward the hole, which is at the lowest point in the area, even a blindfolded golfer has a good chance of finding the hole. So, fixed reaction pathways are not necessary, as each protein seeks out its natural shape through a funnel of declining energy; it can take many folding routes and still reach its target of the completed tertiary structure (Fig. 8.8).

Thing # 4. The Process of Folding:

The folding pathway of a large polypeptide chain is very complicated, and not all the principles that guide the process have been worked out. However, many plausible models have attempted to describe protein folding. One model views folding as a hierarchical process, where local secondary structures form first. Under this model, a helices and β sheets form first, with longer range interactions between helices and sheets forming super-secondary structures later. This process continues until the entire polypeptide folds. An alternative model describes folding as a spontaneous collapse of the polypeptide into a compact state. This collapsed state is known as a molten globule.

It may be that the actual folding process of proteins incorporates features of both models. Instead of following a single pathway, a population of peptide molecules may take a variety of routes. Thermodynamically, the folding process can be viewed as a kind of free-energy funnel, where the unfolded states are characterized by a high degree of conformational entropy and relatively high free energy.

In a trivialized definition, entropy is a measure of chaos, a measure of all different conformational states that the protein can be in. Obviously, there is more chaos in the protein in its unfolded state. On the other hand, high free energy is a measure of unstableness, which is higher in a protein’s unfolded state. Therefore, as folding proceeds, the narrowing of the funnel represents a decrease in the number of conformational states present. At the bottom of the funnel, also known as the global minimum, assemblies of folding intermediates are reduced to a single conformation.

It is important to realize that although we often describe the free energy funnel as having one global minimum that is, one native conformation a protein can have a small set of native conformations, each one important for its biological function(s).

It has been experimentally confirmed that not all proteins fold spontaneously in the cell. For many proteins the folding process is facilitated by the action of specialized proteins known as chaperones. Molecular chaperones are proteins that interact with partially folded or improperly folded polypeptides to facilitate correct folding pathways of provide microenvironments so that folding can occur.

Chaperones are not the only proteins to facilitate protein folding. Two enzymes, protein disulfide isomerase (PDI) and peptide prolylcis-trans isomerase (PPI), catalyze isomerization reactions and are required for the folding pathways of a number of proteins.

Thing # 5. Fold Recognition (Threading):

Threading uses a database of known three-dimensional structures to match sequences without known structure with protein folds. This is accomplished through a scoring function that assesses the fit of a sequence to a given fold. These scoring functions are usually derived from a database of known structures and generally include a pair-wise atom contact and solvation terms. Threading methods are very similar to comparative modeling in that threading compares a target sequence against a library of structural templates, producing a list of scores.

The scores are then ranked and the fold with the best score is assumed to be the one adopted by the sequence. The methods to fit a sequence against a library of folds can be extremely elaborate computationally, such as those involving double dynamic programming.