Term
| What limits the possible folding of proteins? |
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Definition
1) Planar nature of the peptide bond
2) Steric hindrance causes some angles of rotation of the peptide bond to be preferred (Ramachandran plot) |
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Term
| In the quaternary structure of a protein, what are oligomers hled by? |
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Definition
| Non-covalent linkages like hydrogen bonding or hydrophobic interactions. |
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Term
| Name three types of oligomers that can exist in the quaternary structure of a protein? |
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Definition
alpha 2: homodimer
alpha-Beta: heterodimer
alpha2-Beta2: heterotetramer (2 alpha and 2 Beta sheets) |
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Term
| What happens to the protein structure if you have an amino acid that isn't a helix former/breaker or B-turn? |
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Definition
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Term
| Give me a general definition for chromatography |
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Definition
| It's a method for separation of a mixture made up of molecules by passing them through a packed column of material. |
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Term
| Describe Gel Filtration Chromatography |
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Definition
Large proteins are excluded from the beads and they travel more rapidly than the small proteins, becuase each bead has small "caves" in them, so they get stuck in there.
Extraction based on molecular size! |
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Term
| Describe Ion-Exchange Chromatography |
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Definition
| Certain oppositely charged proteins are attracted to the bead and travel more slowly (or not at all) to proteins that are the same charge or uncharged. |
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Term
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Definition
Add a specific binding agent to extract the protein from the matrix.
Then elute by adjusting the pH or ionic strength after it binds. |
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Term
| Hydrophobic Chromatography |
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Definition
| The packed beads are hydrophobic so they denature the protein. The greater the amount of hydrophobic R groups on the protein, the stronger it sticks to the column. |
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Term
| Give me a general definition for Electrophoresis. |
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Definition
| A method for seperating mixtures of molecules based on their movement through an electric field. |
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Term
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Definition
| Proteins move from the negative to the positive electrode based on how much charge they carry, and are also effected on their molecular size. Small proteins travel faster, since they have less resistance to face as they pass through the medium. |
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Term
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Definition
Move from negative to positive electrode based only on their molecular size. You mix the detergent to coat the protein, and the bigger the protein the more negative it gets.
Larger proteins on the top, smaller ones on the bottom. |
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Term
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Definition
| Size doesn't matter; migrate based only on their charge. They move through an "ampholite media" and they move until they meet an ampholite molecule with exactly the same opposite charge so that the overall charge is neutral. |
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Term
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Definition
| A completed Isoelectric focusing gel is used to load an SDS PAGE gel. Combination of charge and size where large proteins on top, small at the bottom. Positve proteins on the left and negative on the right. |
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Term
| Give me a general definition for Centrifugation. |
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Definition
| Seperation of mixtures of molecules based on their density. |
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Term
| Density gradient ultracentrifugation |
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Definition
| Based on density and friction as they "fall" through the tube in a density gradient. The tube is filled with a density gradient medium and the proteins end up in gradient based on their density at a rate based on their size. |
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Term
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Definition
| Addition of organic solvents or high salt concentration causes the protein to precipitate at the bottom. |
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Term
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Definition
| A method to remove contaminating small molecules from samples of large proteins. A protein solution is contained in a bag w/ small molecules, while the surrounding material lacks small molecules. The bag is permeable to small molecules, so they migrate out of the bag, leaving the large proteins inside. |
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Term
| What is the key to purification? |
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Definition
| You must consider how the protein you want is different than the contaminating molecules. |
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