Proteins- Folding

Yup, yet another post about proteins...

You should understand Anfinsen's experiment and know the four major conclusions from it.

In Anfinsen's experiment, a protein was denatured by being placed in a reducing environment, breaking disulfide bonds. The protein was then placed back in an oxidising environment to see if the same disulfide bonds would form. Out of the 104 possible combinations of disulfide bonds that could have formed, only one formed- the one that allowed for correct functioning of the protein. From this experiment four major conclusions could be drawn:

1. Correct protein structure is necessary for correct function.
2. Disulfide bonds stabilise proteins- when broken, the protein is unable to carry out its proper function.
3. Correct disulfide bonds are formed as proteins fold.
4. All of the information for a protein to fold correctly is contained in the protein sequence, so even if a protein is denatured it may be able to renature again if placed in a favourable environment.

You should be able to describe the different types of van der Waals interactions.

Van der Waals interactions involve the formation of weak dipoles in molecules. A molecule with, say, a C=O bond may have a dipole, with the O being slightly negatively charged and the C being slightly positively charged. This slight dipole can allow the molecule to interact with other molecules with slight dipoles. Additionally, the dipole can induce a dipole in other molecules- the electronegative O may repel electrons at the end of a neighbouring molecule, for instance.

Another type of van der Waals interaction is London dispersion forces. These occur between nonpolar molecules. Electrons in molecules do not remain in the same place all the time. At times, there will be more electrons in some parts of the molecule and fewer in other parts. This can create temporary dipoles which can then interact with each other. Larger molecules tend to display stronger London dispersion forces, probably due to their larger number of electrons and greater mass.

You should know how the placement of hydrophobic (nonpolar) and hydrophilic (polar) amino acid side chains in an alpha-helix determines the characteristics of the helix and hence how it packs into a protein structure. You should know the definition of an amphipathic alpha-helix.

As mentioned previously, all amino acid side chains face outwards from the helix. If these chains are hydrophilic they will interact better with water, whereas if the chains are hydrophobic they will interact better with lipids. If the protein is to exist in an aqueous environment such as in the cytosol, it is more energetically favourable if hydrophilic amino acids are facing towards the outside of the protein and hydrophobic amino acids are facing towards the inside. Alternatively, if the protein is to insert in the membrane, then it is more energetically favourable to have hydrophobic amino acids in the part of the chain that will be inserted into the membrane, and hydrophilic amino acids at the ends. Alpha-helices with hydrophobic parts and hydrophilic parts are known as amphipathic.

You should know how the placement of hydrophobic (nonpolar) and hydrophilic (polar) amino acid side chains in a beta-sheet determines the characteristics of the sheet and hence how it packs into a protein structure. You should know the definition of an amphipathic beta-sheet.

As also mentioned previously, every second amino acid side chain is above the beta sheet, while the others are below the sheet. Like alpha-helices, hydrophobic side chains tend to face into the protein while hydrophilic side chains tend to face outwards. Amphipathic beta-sheets are beta-sheets with hydrophobic parts and hydrophilic parts.

You should understand how hydrophobic regions of motifs and secondary structure determine how they pack in the tertiary structure of a protein.

As I just mentioned, hydrophobic regions tend to be hidden within proteins. Sometimes they are sandwiched between hydrophilic regions.

You should know the thermodynamic descriptions of the effects of protein folding on polar and nonpolar amino acid and water molecules. In particular you should know what the changes in enthalpy (Delta H) and entropy (-T Delta S) occur due to water molecules and hydrophobic (nonpolar) amino acids when a protein folds and why the entropic effect on water drives the folding.

A simplified explanation of enthalpy is that it is the change in heat. Generally heat is absorbed to break bonds and released when bonds are formed. A simplified explanation of entropy is that it is the change in "randomness" of the molecules. If I remember correctly, the second law of thermodynamics is that everything naturally wants to descend towards chaos, sorta like a class of schoolchildren who naturally get rowdier and rowdier the longer that they are left without a teacher.

One important concept is that the total free energy change, Delta G, is equal to the sum of Delta H for the chain and Delta H for the solvent minus T Delta S for the chain and T Delta S for the solvent. If Delta G is negative, then protein folding is favourable; if it is positive, then the folded conformation is not energetically favourable.

Let's have a look at what happens if you leave a protein to fold in a vacuum, where there is no solvent around. Delta H is favourable as there are plenty of hydrogen bonds and van der Waals interactions going on between different parts of the molecule, hence releasing energy as heat. Delta S, on the other hand, is unfavourable as this folding means that the protein is becoming more ordered and thus there is less entropy. Overall, however, Delta H is larger than Delta S, so the overall result is a negative Delta G and a shiny new folded protein.

Now let's have a look at what happens to hydrophobic groups in an aqueous solvent. Delta H for the chain this time is unfavourable. I think this is because the water gets in the way of some of the electrostatic reactions between parts of the molecule. Delta S for the chain is also unfavourable, again as the folding of an ordered chain means that there is a decrease of entropy. Delta H for the solvent is favourable, possibly because the "pushing aside" of water molecules by the protein as it folds means that the water molecules have more interactions with each other. Also, after folding, fewer water molecules are required to form a "cage" around the hydrophobic residues, resulting in greater "freedom" of the water molecules after folding than before folding. This means that entropy has increased. In fact, the entropy of water is one of the major driving forces behind the folding of proteins.

Hydrophilic groups in an aqueous solvent, however, do not have quite the same effect. Hydrophilic residues naturally want to interact with water, and so folding, which reduces these interactions, results in an unfavourable Delta H for the chain. Delta S for the chain remains unfavourable due to the formation of an ordered chain. The entropy of water is not decreased as much when hydrophilic groups are present as compared to hydrophobic groups. Overall, Delta G for this situation is relatively neutral.

Sorry if that was a terrible explanation- it's a topic I don't understand that well, partly because I don't have any kind of physics background.

You should understand the Leventhal paradox and the concept of an energy landscape funnel which enables a polypeptide chain to start from a large range of conformations but end up in a single native folded state. You should know that protein folding is an energetically favourable process and be aware of the possibility of relatively stable misfolded states.

One of the hypotheses around protein folding was that every combination is tested to find the lowest energy conformation. However, the Levanthal paradox states that there are so many folding possibilities for your average protein that it would take far too much time to test them all. Another model of protein folding involves an "energy landscape funnel" showing multiple different routes that a protein might undertake to reach its native "folded state." Along these routes, intermediates may be formed of progressively lower energy levels. There may, however, be some stable misfolded states mixed in among the intermediates.

You should know that some proteins require no assistance in folding (generally small simple proteins) whilst some require the assistance of heat shock proteins such as Hsp70 which bind to exposed hydrophobic regions in unfolded or partially folded proteins, protecting them from aggregating with other unfolded or partially folded proteins. You should know that the folding process can occur by association with Hsp70 but in larger more complex proteins the protein is delivered to the Hsp60/Hsp10 chaperones (GroEL/GroES in bacteria) which provide a hollow barrel shaped environment to allow folding to occur secluded from the rest of the cell. You should know why heat shock proteins are so called.

Whoa... that dot point was somewhat lengthy, and it covered quite a bit. The only thing that really needs additional explanation here is why heat shock proteins are called that. Heat shock proteins are called heat shock proteins as their synthesis tends to increase in situations of high heat. This may be because there is an additional risk of protein denaturation at higher temperatures, and thus more heat shock proteins are needed to counterbalance this.