Getting to Know Your Protein Trivia Quiz

Answer: X-ray crystallography

X-ray crystallography is pretty much unmatched in the detail with which it can analyse protein structures. Solving protein structures to atomic detail often allows the protein's mechanism of action to be deduced (as was the case for ATP synthase). There are several limitations to x-ray crystallography, however. Proteins must be crystallised prior to analysis and this is difficult for proteins which are large and/or dynamic.

It is for this reason that the 26S proteasome, which is essential to our well-being, has proven difficult to analyse using this technique. Additionally, some argue that the crystallisation conditions are too far removed from physiological conditions and so the structures observed cannot be seen as wholly representative.

Answer: Alpha helices and beta sheets

Far UV CD gives a less detailed account of the protein structure than techniques such as X-ray crystallography and electron microscopy, but it can reliably inform on the proportion of the protein which is alpha helical and how much is made up by beta sheets. These structures are formed due to the interactions of residues via hydrogen bonds.

Near UV CD gives information about the tertiary structure of a protein. Specifically, they inform us about the chemical environments of aromatic amino acids that are found in protein interiors.

Answer: Infrared spectroscopy

While visible light has a very narrow range of wavelengths (380 nm-700 nm), infrared radiation has longer wavelengths and is defined by a broader range (700 nm-1 mm). By starting at, say 700 nm infrared radiation and firing it at a sample containing your compound, you can increase the wavelength and measure which wavelengths are absorbed by the compound. Different functional groups/chemical bonds absorb different wavelengths.

The wavelengths that different chemical groups absorb are well documented.

For example, a primary alcohol absorbs infrared radiation at 9434-9615 nm, whereas a tertiary alcohol absorbs at 8333-8696 nm. This may also be used to characterise R groups of amino acids.

Answer: The peptide fingerprint

Proteins are made unique by the sequence of amino acids in their chains. If trypsin is used to digest a protein, it will cleave after basic residues (arginine and lysine). The number of amino acids in between these arginines and lysines will vary from protein to protein and so a protein can be identified by observing its peptide fingerprint.

However, these fragments are tiny and need a very sensitive method of detection, such as mass spectrometry. One technique known as MALDI-TOF involves the ionisation of these peptide fragments, before they are accelerated through a column towards a detector which determines their mass.

Answer: Affinity chromatography

There are several types of chromatography. Some discriminate on the basis of size, some on charge, and others on hydrophobicity. Affinity chromatography is much more specific and requires a greater prior knowledge of the protein in question. For example, if a group wish to isolate an enzyme from a cell lysate, they may ligate known substrate molecules of that enzyme to a column before passing the lysate over said column.

Other groups use more advanced techniques to introduce novel groups into the protein so that it can be bound to a column.

For example, by introducing a poly-histidine tail onto a protein, it will then bind to a column to which zinc is attached.

Answer: Exons

As is dictated in the Central Dogma of Biology, DNA makes RNA, which makes protein. The DNA sequence is first transcribed to a molecule of mRNA, which leaves the nucleus and can be translated by the ribosome. However, before leaving the nucleus, mRNA must undergo processing.

This includes cutting out the non-coding regions of RNA (called introns) between the regions of RNA which code for the protein (called exons). This cutting is performed by a ribonucleoprotein called a splicesome (it splices out the introns!).

The splicesome also stitches the exons back together to form a continuous coding sequence. Specific nucleotide sequences may betray the points where an intron ends and an exon starts, allowing us to have some idea of which regions of a gene actually possess the protein-coding material by observing the DNA sequence alone.

Answer: Bradford reagent

The Bradford assay is a colorimetric technique. The red Bradford reagent is added to the sample and becomes blue upon binding protein. The extent of this red-to-blue transition is proportional to the amount of light at wavelength 595 nm absorbed by the sample, which is in turn proportional to the amount of protein present.

The Bradford assay is therefore the assay of choice for a quick and easy protein quantification.

Answer: A stable pH gradient across the gel

It is important that the pH gradient becomes more alkaline (i.e. that the pH increases) towards the negative electrode. Small molecules are added to solution. If they have a charge, they will move towards the electrode of opposite charge (i.e. negatively charged species will move towards the positive electrode and vice versa). Because of the pH gradient, the charged species will only move as far as the point where the pH of the gel is equal to the pI of the molecule.

The pI is the pH at which the net charge of the molecule is zero.

Therefore, arginine (an amino acid with pI of 10.76) will exist as a positively charged entity anywhere in the gel where the pH is lower than 10.76. This positivity means that arginine moves towards the negative electrode, and will do so until it reaches a point where the pH is 10.76 and its net charge is zero.

Answer: Reconstitution

When investigating which protein components of a cell are needed for a particular process, you may remove specific proteins from the cell, for example by knocking out the gene which codes for that protein. If the process under investigation stops after this protein is removed, it tells us that this protein is necessary for this cellular process.

But is it sufficient? This question is answered using reconstitution experiments. For example, if an in vitro system was set up and found that this cellular process could occur with only this protein present, then this protein is sufficient to carry out this process.

This is rarely the case, however, as several proteins are often necessary to perform cellular processes. The set of proteins are established through repeating these reconstitution experiments (trial-and-error).

Answer: Molten globules

Proteins fold from a denatured, unstructured state, into a natively folded state. Often this happens so rapidly that the stages in between cannot be observed. Sometimes, semi-stable intermediates exist long enough to be observed (these are called kinetic intermediates).

The molten globule is also seen as an intermediate (it is called a thermodynamic intermediate). It is not observed as part of the folding pathway, but is generated by exposing the protein to mildly denaturing conditions (e.g. pH4).

The molten globule is, however, believed to share many properties with the intermediates which transiently exist during the folding process. Study of these proteins may therefore improve our understanding of how proteins fold.