Chapter 3 Flashcards
The isoelectric point is the pH at which ________
the net charges of the molecule are zero
How is the isoelectric point determined?
Take the average of the pH’s where the charge is +1 and the charge is -1
A _____ bond forms between amino acids to form proteins
Peptide bond –> between the carboxylic acid and the amine
Why is GFP used to tag amino acids?
GFP glows, so you are able to see where the amino acid is going as it travels through the subject
The average molecular weight of an amino acid is ___
110
A protein with mainly positive charges will have a _____ pI while a protein with mainly negative charges will have a _____ pI
High
Low
Trypsin cleaves after ___ and ____ while chymotrypsin cleaves after ____, _____ and ____
Lys and Arg
Phe, Trp, Tyr
How would you describe the primary sequence of a protein?
It’s linear and acts as the very basic structure of a protein before it takes on a 3D shape. It’s made up of the AA residues
A protein can be called multisubunit when it consists of _____ or more polypeptide chains
Two
What technique can be used to separate/purify proteins based on solubility and how does it work?
Ammonium sulfate precipitation crystallization.
Adding Ammonium sulfate decreases the solubility of proteins without denaturing them. Each of the proteins will precipitate out of solution at different concentrations of Ammonium sulfate
What is ion exchange chromatography used for and how does it work?
IEC is used to purify/separate by charge. Ions with a different charge than the stationary phase will elute first, while those with the same charge as the stationary phase will stick longer
What is the difference between cation exchange and anion exchange in IEC?
In cation exchange, the stationary phase is negatively charged so that cations will pass through first. In anion exchange, the stationary phase is positively charged so that anions will elute first
The stationary phase is the OPPOSITE of the name
What does affinity chromatography purify/separate and how does it work?
Done for specific binding sites. `The stationary phase is designed to form covalent bonds with one chemical group in the solution. Once these new bonds form, the other proteins will elute from the column. Then the stationary phase will be out competed with another solution that causes the bonds to break, and the protein that was originally bound will elute from the column.
What is the purpose of dialysis for purification/separation?
Putting the proteins in a dialysis bag in dilute solution will encourage the small proteins to dissociate into solution, while the proteins that are too large to get through the porous bag will stay behind
What size proteins elute first in size exclusion chromatography?
The larger proteins elute first because the smaller proteins are trapped between the beads of the stationary phase, while the larger proteins are too large to fit into the spaces and flow right through.
What is the purpose of SDS in electrophoresis? (SDS-PAGE)
SDS uses hydrophobic interactions to bind with proteins. It helps to denature the protein and gives all molecules are overall negative charge, but cannot break disulfide bonds
What is used in electrophoresis to break disulfide bonds?
Betamercapthanol (BME), dithiothreitol (DTT) and/or heat
Unlike SEC, the _____ proteins reach the bottom first in electrophoresis
Smallest
What is the purpose of isoelectric focusing? (IE)
A procedure using electrophoresis that allows for the determination of pI. A pH gradient is established and the proteins filter out into the pH that matches their pI (Gradient being low pH at the top, high pH at the bottom)
What is the purpose of 2D electrophoresis?
It combines SDS-PAGE and IE so that proteins can be separated by both pI and molecular weight
What is the purpose of SDS-PAGE?
Uses SDS in electrophoresis to determine the molecular weight of proteins
What is Edman degradation and what is its limit in terms of AA length?
It can determine the primary sequence of a peptide by sequentially removing AA’s from the chain without damaging them. It only works for proteins <50 AA long
Edman degradation _____ work if there are disulfide bonds present. What is done to solve this?
Will not
The disulfide bonds have to be reduced with DTT or BME and free cystines are attached to methyls to prevent them from forming any new disulfide bonds
Why does Edman degradation only work for ~50 AA?
It is 98% effective but after 50 AA the efficacy drops off
Trypsin and chymotrypsin are examples of ____- enzymes that cleave at specific sites of proteins
Endopeptidases
How would you determine the location of disulfide bonds when cleaving a protein?
Cleave the protein like normal, leaving the disulfide bonds present
Denature the protein so that the disulfide bonds are broken, then cleave and compare the fragments with those of the first cleavage
How is mass spectrometry used to determine protein sequencing?
The molecules are first ionized in a collision chamber, and then compared on a ratio of mass/charge (m/z). From there, data analysis determines their identity by comparing theoretical and experimental m/z ratios
(Tandem) mass spectrometry can be used to determine both the _____ and ______ sequence
Mass and amino acid sequence
The shotgun proteomic approach is best used for large, complex proteins. How does it work?
The protein is identified using chromatography, tandem mass spec, and data analysis. It uses multiple techniques to form an understanding of a broad range of proteins
The idea is that if two protein sequences are similar in structure, they should also be similar in _____
Function
