Question

_Describe what was done in the famous experiment by Christian Anfinsen on RNase A, and which two conclusions could be drawn from it.

_Protein folding would require billions of years if proteins would explore all possible conformations before settling into the native fold. Describe how most proteins are able to fold within seconds to minutes.

Answer 1

1. The Anfinsen Experiment in Protein Folding:

Disulfide bridges can be disrupted by treating a protein with 2-mercaptoethanol (HS-CH2-CHOH). The bond between the two sulfurs in the protein is broken and a new bond is created between two sulfurs at the end of two molecules of 2-mercaptoethanol. (2-mercaptoethanol used to be called β-mercaptoethanol or βME.) Treatment with 2-mercaptoethanol is now standard procedure for denaturing proteins. For example, 2ME is always included when proteins are prepared for SDS polyacrylamide gel electrophoresis.

Anfinsen wanted to show that the information for protein folding resided entirely within the amino acid sequence of the protein. He choose ribonuclease A as his model for folding but he couldn't completely denature the protein unless he treated it with the denaturant urea plus 2ME to break the disulfide bridges.

Under those conditions, the protein unfolded. It would refold spontaneously once he removed urea and 2ME from the folding solution. Ribonuclease A regained biological activity under those conditions. This demonstrated that refolding could take place in vitro.

Anfinsen discovered that removing 2ME but not urea led to recovery of 1% of the activity. This is attributed to the formation of random disulfide bridges between the 8 cysteines present in the protein. There are 105 different possibilities (7x5x3x1) so the 1% recovery makes sense. It also shows that the correct three-dimensional conformation must be achieved fairly rapidly when urea is removed since most of the protein under those conditions becomes active.

However, recovery is not 100%. Mistakes are made in vitro and presumably in vivo as well. This led to the discovery of an enzyme called protein disulfide isomerase (PDI)—an enzyme that catalyzes reduction of incorrect disulfide bonds and allows a protein trapped in an incorrect conformation to unfold and try again.

PDI is a ubiquitous enzyme as expected from its important role in proper folding. The active site of the enzyme contains a disulfide (shown as two yellow sulfur atoms in the figure). Thus, the enzyme acts very much like 2-mercaptoethanol, catalyzing a disulfide exchange reaction where the incorrect disulfide bridge in the misfolded protein is reduced and PDI is oxidized. (The correct name of the protein is "thiol-disulfide oxidoreductase. Oxidoreductases form a large class of very important enzymes.)The enzyme preferentially recognizes incorrect disulfide bridges since these tend to be exposed on the surface of the misfolded protein, whereas correct disulfide bridges are usually buried in the hydrophobic interior of the correctly folded protein.

Figure is attached below.(FIG1, FIG2, FIG3)

2. Protein Folding and Processing
Translation completes the flow of genetic information within the cell. The sequence of nucleotides in DNA has now been converted to the sequence of amino acids in a polypeptide chain. The synthesis of a polypeptide, however, is not equivalent to the production of a functional protein. To be useful, polypeptides must fold into distinct three-dimensional conformations, and in many cases multiple polypeptide chains must assemble into a functional complex. In addition, many proteins undergo further modifications, including cleavage and the covalent attachment of carbohydrates and lipids, that are critical for the function and correct localization of proteins within the cell.

Because native proteins are only 5–10 kcal/mol more stable than their denatured states, it is clear that no type of intermolecular force can be neglected in folding and structure prediction. Although it remains challenging to separate in a clean and rigorous way some types of interactions from others, here are some of the main observations. Folding is not likely to be dominated by electrostatic interactions among charged side chains because most proteins have relatively few charged residues; they are concentrated in high-dielectric regions on the protein surface. Protein stabilities tend to be independent of pH (near neutral) and salt concentration, and charge mutations typically lead to small effects on structure and stability. Hydrogen-bonding interactions are important, because essentially all possible hydrogen-bonding interactions are generally satisfied in native structures. Hydrogen bonds among backbone amide and carbonyl groups are key components of all secondary structures, and studies of mutations in different solvents estimate their strengths to be around 1–4 kcal/mol or stronger. Similarly, tight packing in proteins implies that van der Waals interactions are important.

Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research.

Prior to the mid-1980s, the protein folding code was seen a sum of many different small interactions—such as hydrogen bonds, ion pairs, van der Waals attractions, and water-mediated hydrophobic interactions. A key idea was that the primary sequence encoded secondary structures, which then encoded tertiary structures. However, through statistical mechanical modeling, a different view emerged in the 1980s, namely, that there is a dominant component to the folding code, that it is the hydrophobic interaction, that the folding code is distributed both locally and nonlocally in the sequence, and that a protein’s secondary structure is as much a consequence of the tertiary structure.

Hence ,this question's answer is given.