Chapter 3: Amino Acids, Peptides, & Proteins Flashcards
What are proteins?
polymers of amino acids
What are the three components of an amino acid?
acidic carbonyl, basic amino, a-hydrogen
What form are amino acids commonly in?
L form
What amino acid is the ONLY one that is not chiral?
glycine
What absorbance range to aromatic amino groups usually react in?
270-280 nm
Why is cysteine an important amino acid?
can form disulfide bonds
What amino acids are positively charged?
lysine, arginine, histdine
What amino acids are negatively charged?
aspartate and glutamate
What is pK1?
pK value that deprotonates -COOH
What is pK2?
pK value that deprotonates -NH3+
What is pKr?
pK value that deprotonates the R group
What is pI?
the isoelectric point, pH value at which a particular molecule has no NET electric charge
Are amino acids positive or negative at a low pH?
positive
Are amino acids positive or negative and a high pH?
negative
What is the name for a peptide that has a net charge of zero?
a zwitterion
What type of reaction is a protein synthesis reaction?
dehydration reaction
Why is peptide hydrolysis favorable?
water is very abundant, there is a high activation energy for dehydration
What is a residue?
the name for an amino acid in a protein
What pK values do you pay attention to when determining pI?
first pK2, last pK1, all pKr values
What are the four general functions of amino acids?
hormones, neuropeptides, antibodies, protection
What is a cofactor?
functional components added to a protein
What is a coenzyme? What’s an example?
an organic cofactors such as NAD+
What is a prosthetic group? What are 3 examples)
covalently attached cofactors such as heme, lipids, and sugars
What are other names for separation by size (2)?
gel filtration chromatography; size exclusion
In gel filtration chromatography, which type of protein elutes first?
larger proteins
What is another word for separation by charge?
ion exchange chromoatography
What are the two types of ion exchange chromatography?
anion exchange and cation exchange
Which type of ion exchange uses positively charged beads?
anion exchange
Which type of ion exchange purifies positively-charged proteins?
cation exchange
How can you elute proteins trapped on an ion exchange chromatography?
with different salt concentration solutions
What is another word for separation by affinity?
affinity chromatography
What is usually the very first purification step?
affinity chromatography
What are the four steps to affinity chromatography?
1) add solution of ligand to column
2) add protein mixture to column
3) wash unwanted protein through the column
4) elute protein of interest with ligand solution
How can we still do affinity chromatography is a native ligand is unavailable?
recombination DNA technology!
What are two recombinant protein chromatographys?
GST/GSH and Ni-NTA
How does GST/GSH work?
- bind GST tag to protein
- GSH is anchored to the column
- protein (with GST) binds to GSH
- protein is eluted out with a high glutathione (GSH) solution
How does Ni-NTA work?
- NTA tag is coupled to an Ni ion and binds to the column
- protein (with His6) binds to the Ni ion
- elute protein out with imidazole
How is separation for analytical measurement done?
electrophoresis
How does an SDS page work?
- SDS binds to and unfolds all proteins, adding a negative charge to all proteins
- electric field (- to +) pulls smaller proteins farther through the gel
Why would you use DTT in an SDS PAGE?
DTT breaks disulfide bonds and makes everything a monomer
What are the two uses of an SDS PAGE?
1) to check the purity of a sample
2) to estimate molecular weight
If I know how many amino acids are in a protein, how can I determine molecular weight?
(aa x 110)/1000 = weight in kDA
If I know the molecular weight of an amino acid, how can I determine the number of amino acids?
weight x 9 = number of amino acids
What is the use of a nonreducing SDS PAGE?
indicates the presence of disulfide bonds
What is the use of a native PAGE?
can see protein-protein interaction
What is the use of an Isoelectric Focusing PAGE?
can determine the isoelectric point of a protein in its native conformation
How does isoelectric focusing work?
proteins stop traveling where their net charge is zero
What is 2D electrophoresis?
the combination of isoelectric focusing with 2D electrophoresis?
How can you quantify how many proteins are in a sample?
by using a spectrophotometer
How does western-blotting work?
1) sorts all proteins by size using SDS page gel
2) sandwich this gel with a membrane coated in buffer
3) soak the membrane in a primary antibody that binds to only our protein
4) add a secondary antibody that binds with the first antibody for visualizaiton
What biochemistry technology is the basis for COVID-19 tests and pregnancy tests?
ELISA
What are the two ways to do protein sequencing?
edman degradation and mass spectrometry
What are the constraints of the edgman degradation?
can only do 50 amino acids, can only identify protein with known sequence
How does mass spectrometry work (very generally)?
identifies the mass of a peptide (and thus the aa sequence)
Why is mass spectrometry wildly used over the edman degradation?
can sequence all amino acids, can be used to determine post-translational modifications
How can you identity a protein using mass spec? Explain.
using electron ionization! utailizes the mass to charge ration for protein identification
What are paralogs?
homologous proteins found within the same species
What are orthologs?
homologous proteins found in different species
What is a consensus sequence? What does it indicate?
a sequence that has not changed over the course of evolution, indicates that that protein is extremely important
What is a signature sequence?
a small set of amino acids that accurately identifies a specific set of proteins
