What do you want to know about your protein?
Polarity: Isoelectric focusing, amino acid sequence--> amount/ different and expensive
- Function in a physiological functional environment: where is it used and can we deduce its function
- Evolutionarily, is it a member of a fam?
Shape of protein: interaction/ if enzyme, active site--> make drug to interact with the active sites
1) amino acid analysis
protein isolated and purified--> want a list of amino acids and relative proteins--> Amino acid analysis
Easier than getting a seequence
What are the steps to amino acid analysis
1) put protein in glass bottle with 6 M HCl and hook tube to vacuum pump--> stick in heating block at close to 100 degrees Celsium
Required to chop up every peptide bond in the protein
In our mixture, what do we have? What do we do with it?
we have individual amino acids
We take our mix and pass through HPLC because it separates molecules that are very similar in size, structure, and characteristics
At end of HPLC is what?
Allows what? What is the problem with this? How is it resolved?
at end is a spectrophotometer, whcih allows us to see what comes out.
Problem: how many amino acids can we see with spec? Only two--> tryptophan and tyrosine
We covalently link ninhydrin to amino acids so we can see them
We then set the spec at a particular wavelength.
After setting the spec, what?
we pass amino acids through the HPLC column; amino acids are separated on teh basis of charge, etc.
They come out in a very specific order at a specific time.
What is the result of AAA?
Why is it useful?
Result: a series of peaks, the size is proportional to the amount present
It is useful because some proteins have unique compositions (ex: collagen)
mixture --> HPLC--> spec--> graph
Edman degradation does what?
breaks bonds one at a time
- - HPLC Colum
- - spectrophotometer
- - Chamber with little disk that protein goes into, causing the disk to bind at the C-terminus end of polypeptide with N-terminus floating up
What happens in Edman degradation?
in stepwise fashion, the amino acid at the terminal amino end is removed
o Phenyl isothiocyanate reacts with the uncharged terminal amino group of the peptide to form a phenylthiocaramoyl derivative. Then, under mildly acidic conditions, a cyclic derivative of the terminal amino acid is liberated, which leaves an intact peptide shortened by one amino acid
What do you when a bond is broken?
wash the amino acid, figure out which it is with the spectrophotometer, repeat
Summary of Edman Degradation
adjust conditions--> label--> cleave first amino acid--> wash through HPLC--> identify by spec reading--> one peak--> repeat
Time determines what it is
What happens as the rounds of ED increase?
the residual peaks may show up in the reading if you missed some. Eventually, there are so many peaks, you cannot distinguish the cleaved one from teh previous
As a result of this limitation of ED, what is ED restricted to?
only proteins with a certain number of amino acids
What is the solution for ED?
cut the protein into smaller fragments, separate them by HPLC
Then you need to know which fragment comes before the other. So, you cut the protein with a chemical or enzymatic reagent (use specific cleavage of proteins). And, do the amino acid sequence oer with the new polypeptides. Once this is done, you get the overlaps that are preesent and determine the sequence from there
What is the overall problem with ED?
expensive and time consuming and requires a lot of protein
Cheaper way of doing things?
clone genes and translate into amino acids or reverse transcription to determine the codons
How do you answer the question of where the protein is used?
How to generate antibodies against a protein?
injuect into host--> recognized as foreign--> antibodies form
Structure of antibodies
four chains held by disulfide linages. Two heavy and two light chains
What makes antibodies unique?
the two ends make it unique. It is the antigen-binding site created with amino acids that will bind to invaders with high degrees of affinity to epitopes on teh antigen
Explain the features of the antigen
Different structural features on the surface of the molecule that the antibody binds to = epitope
Forms different antibody for each epitope
makes multiple clones of each antibody that recognizes different epitopes on the same antigen
grabs teh antibody that binds to just the epitope that we need that is in teh antigen