Highlights Lecture #7 Spring 2017

1. It is extraordinarily difficult to impossible to predict the precise 3D strucutre of a protein simply from the primary sequence. The dilemma is described in Levinthal’s paradox.

2. The following forces stabilize protein structure

  • Primary = peptide bonds (covalent)
  • Secondary = hydrogen bonds
  • Tertiary = hydrogen bonds, ionic interactions, hydrophobic interactions, disulfide (covalent) bonds, ionic interactions

3. Misfolding of proteins is a factor in diseases such as mad cow disease or a human disesase called Creutzfeld-Jacob disease. These misfolded proteins are normal brain proteins and are called prions when they fold improperly. Cells have protections against misfolding in the form of complexes called chaperones.

4. Globular proteins that are soluble in water tend to have polar amino acids on the outside in contact with water and non-polar amino acids on the inside associating with each other (thus avoiding water).

Protein Purification

1. To study a protein, one must purify it away from all the other proteins in a cell. Steps in the purification typically include 1) busting cells open; 2) centrifuging cellular components apart from the cytoplasm; and 3) using techniques that separate molecules by several different processes.

2. Most proteins require using more than one method to be purified.

3. Techniques for protein purification often rely on the structural components of proteins. Isolation of proteins from cells requires breaking open the cells, centrifugation to remove insoluble debris, and the application of protein isolation techniques to purify desired proteins from the soluble fraction of the cells.

4. Gel exclusion chromatography is a technique for isolating proteins on the basis of their different sizes. The method uses ‘beads’ with uniformly sized holes in them. The holes are openings to tunnels through the bead. Small molecules that fit into the holes travel throught the tunnels and take longer to pass through the column than large molecules that do not fit into the holes.

5. Affinity chromatography uses the structure of a molecule that a protein binds (such as ATP) as a means of purifying the protein. For example, proteins that bind ATP would be retained by a column full of beads with ATP on their surface. The non-ATP-binding proteins will pass through first. ATP-binding-proteins can be removed from the column by adding ATP.

6. Ion exchange chromatography uses beads that don’t have holes, but instead have charged molecules linked to them. Anion exchange chromatography has positive ions linked to the beads and a chloride counterion. When a solution of charged molecules passes through the column, the negative chloride ions are replaced by the negatively charged molecules, which ‘stick’ to the positive ions linked to the column. Cation chromatography uses negative ions linked to the beads with sodium or potassium counterions.

7. Gel electrophoresis involves gelatinous support materials (agarose – for DNA or polyacrylamide – usually for proteins) and an electric current that drives molecules through the gel. Electrodes are arranged such that the “top” or beginning of the gel is where the negative electrode is placed and the positive electrode is placed at the bottom or opposite end of the gel. DNA is negatively charged, so it is repelled away from the top and towards the bottom of the gel. Separation is on the basis of size. Large molecules travel slowest in the gel, whereas the small molecules travel fastest. DNA fragments appear as bands on a gel and bands can be excised separately from the other bands for further manipulation.

8. Proteins can have negative, positive, or no charge. To be separated in electrophoresis, they must be converted from folded entities with their native charges into rod-like structures with uniform negative charge. This is done using the detergent called SDS. When mixed with a protein, it unfolds the protein and coats it with negative charges. This coated protein is loaded onto a gel of polyacrylamide and the complexes are sorted on the basis of size, just like the DNA fragments were – smallest moves fastest and largest moves slowest. The technique is called SDS-PAGE.

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