Topic 7: Tertiary Structure of Proteins Flashcards
What are the 4 types of bonds found in tertiary and quaternary structures?
- Disulfide bridges
- Salt bridges
- Hydrogen bonding
- Hydrophobic interactions
Give examples of agents that cause denaturation and how they do so?
Heat: The weak side-chain attractions in globular proteins are easily broken by heating. One example of this is cooking meat converts some of the insoluble collagens into soluble gelatin.
Mechanical agitation: Mechanical agitation introduces heat and kinetic energy to the proteins, which breaks the bonds in the structure and denatures the protein. Most familiar example of denaturation of protein by mechanical agitation is the foaming that occurs during beating of egg whites.
Detergents: Very low concentrations of detergents can cause denaturation by disrupting the association of hydrophobic side chains.
Organic compounds: Polar solvents such as acetone or ethanol can interfere with hydrogen bonding by competing for bonding sites.
pH change: Excess H+ or OH- ions reacts with the basic or acidic side chains in amino
acid residues and disrupts salt bridges.
Inorganic salts: Sufficiently high concentrations of ions can disrupt salt bridges.
What did the afinsen experiement show and how did they come to this conclusion
The Chrsitian Afinsen experiement demonstrated that it is the primary structure of proteins which evnetually determine how it folds. He also showed that the native state of the protein is the most thermodynamically stable.
He showed this by isolating ribonuclease from a cell and adding two denaturing agents, 8.0M urea which would destabilize the non-covalent bonds such as hydrogen bonds, and beta-mercaptoethanol, which removes the 4 sulfide bridges. This denatured the enzyme, leaving just the linear sequence of amino acid, but once he removed them via dialysis, it regained its structure.
He then added the two again but only removed beta-mercaptoethanol. This then denatured the enzyme, and with the bmce removed, the sulfide bonds could form again, but they formed differently (“incorrectly), from the original ribonuclease, as the non-covalent bonds were still disrupted by the urea.
He then removed urea, allowing the non-covalent bonds to form again, which actually caused the protein to correct to its natural state, which demonstrated that the native state is the most thermodynamically stable state of the protein’s conformation.
Compare and contrast globular and fibrous proteins
Fibrous proteins are less numerous than globular proteins. Though they both play important roles, globular proteins have more functional roles in the body, such as transporting blood or lowering blood-glucose levels, while fibrous proteins have more structural roles, such as forming the structure of hair and nails.
Fibrous proteins have longer, simpler units of proteins, and are generally less flexible and more water insoluble due to high content of hydrophobic amino acids. There is little folding, and more like, parallel sequences next to one another. Globular proteins have several folds in their structures, mainly due to hydrophobic regions, which are regions where hydrophobic regions meet to get away from water.
One thing both types of proteins have in common is that hydrophobic side chains will often face the interior of the protein.
Explain Levinthal’s paradox
Levinthal’s paradox is that most proteins are able to fold into their native state within seconds-milliseconds, despite computations saying that if the protein as to assess every conformation, it would take an unreasonable amount of time. The paradox illustrates that proteins must only sample through limited conformations, or fold by “specific pathways”
What is chromatography
Chromatography is a purification technique where the substance being purified is dissolved in a solvent, and this together is called the mobile phase, which passes through some matrix called the stationary phase, which either takes contaminants out the mobile phase or leaves the desired product there
Contrast between ion-exchange, affinity, and gel-filtration chromatography
Ion exchange chromatography uses an ion exchanger to let only one type of charged molecules through the matrix. In size-exclusion/gel-filtration chromatography, the protein passes through a solution of amorphous beads, which then trap smaller molecules, but larger sized protein pass through crevices between the bead. In affinity chromatography, the protein passes through a matrix where the beads contain a ligand that binds to the protein of interest, which can the be removed afterwards by changing the pH of by adding a competitive inhibitor
How does SDS-PAGE work
SDS polyacrylamide gel electrophoresis (SDS-PAGE) is a gel electrophoresis technique used for protein separation. This technique uses a detergent, sodium dodecyl sulfide. Recall detergents will denature proteins via disruption of non-covalent bonds.
SDS carries a negative charge, and the cumulative negative charge renders the net charge of the protein insignificant
SDS also binds to every protein in roughly the same proportion, one molecule for every two amino acid residues
Therefore, every protein will have the same charge to mass ratio, which causes all proteins to migrate towards the cathode with a rate dependent on their sizes only
Smaller molecules will migrate faster through the gel than larger molecules
Β-mercaptoethanol may also be added to reduce any disulfide bonds
What is isoelectric focusing and how does it work
Isoelectric focusing is a technique used to obtain the isoelectric point of a substance. In this technique, a matrix is ampholytic, and therefore has a pH gradient along it. An amino acid is placed in and an electric field is applied, and the amino acid migrates through the different pHs of the matrix until it reaches its isoelectric point, where the protein is net neutral, and therefore the EF has no effect on it. Staining can be used to visualize this location.
