BIOCHEMICAL TECHNIQUES TO PURIFY A VARIETY OF PROTEINS
The first step for protein purification is to obtain a solution of proteins usually by disrupting cells or tissues. The chopped material must be suspended in a buffer and large debris are removed by filtration through a cheesecloth. Proteins will be dissolved in the buffer solution. All this first obtention step must be done at a temperature of 0 to 4°C, to minimize protein degradation by the activity of proteases (enzymes that cleave peptide bonds).
The next step is a relatively crude separation called fractionation that makes use of the different solubilities proteins have in solutions of salts. Ammonium sulfate is a protein-stabilizing salt, it is mixed with the solution of proteins to precipitate the less soluble impurities. The target protein, more soluble is recovered by centrifugation and remains in the supernatant. More amonium sulfate is added untill the desired protein is precipitated.
Once the precipitated protein is suspended in a small volume the salty buffer can be exchanged by dialysis. The sample is placed in a cellophane sack that is a semipermeable membrane inside a large volume of the wanted buffer solution. The proteins are too big to escape the sack but the salt dilutes into the larger buffer by diffusion.
Several types of chromatography are classified according to the type of matrix:
Electrophoresis separates proteins based on their migration in an electrical field. In polyacrilamide gel electrophoresis (PAGE) the protein sample is placed in a highly cross-linked gel and an electric field is applied. The proteins migrates according to both their mass and charge.
THE SEQUENATOR!
The EDMAN degradation procedure determines the sequence of amino acid residues.
First, the peptide bonds (that's covalent) of the protein are cleaved by acid hydrolysis, typically using 6M HCl at 110°C for 16 to 72 hours.
Next the hydrolyzed mixture, or hydrolysate, is subjected to a chromatopraphic procedure during which each amino acid is separated and quantitated a process called amino acid analysis.
One method of amino acid analysis involves treatment of the protein hydrolysate with phenylisothiocyanate (PITC) at pH 9. to generate PTC-amino acid derivatives. The PTC-amino acid mixture is then subjected to HPLC in a column of finely divided silica to which short hydrocarbon chains have been attached. The amino acids are separated by the hydrophobic properties of their side chain. In each elution the concentration is determined by the absorbance at 254 nm (the peak absorbance of the PTC moiety)
Pehr Edman developed a technique that permits sequential removal and identification of one residue at a time from the N-terminus of a protein.
The Edman degradation procedure involves treating a protein or polypeptide with phenylisothiocyanate (PITC), also known as the Edman reagent at pH 9. PITC reacts under these conditions with the N-terminus of the chain to form a PITC-peptide. When the PITC-peptide is treated with an anhydrous acid, such as trifluoretic acid, the peptide bond of the N-terminus is selectively cleaved releasing the residue that needs to be treated again to finally be converted into a PTH-amino acid that can be identified chromatographically, usually by HPLC.
SEQUENATOR
FOLDING AND UNFOLDING OF PROTEINS
Folding and stabilization of globular proteins depend on a variety of interactions including; hydrophobic effect, hydrogen bridge, Van der Waals interactions and ionic interactions.
Although noncovalent interaction are individually weak, the sum of these interactions stabilizes the native shapes of proteins. Furthermore, the weakness of each non-covalent interaction gives proteins the flexibility to undergo small conformational changes needed for their functioning.
The hydrophobic effect is the principal driving force in protein folding. Then hydrogen bonds help to stabilize the native conformation of proteins. The carbonyl and amide groups of the polypeptide backbone, especially those in the interior of globular proteins, often form hydrogen bridges with each other to produce apha-helices and beta-sheets. The following hydrogen bonds can be formed beside the previously mentioned:
| From strongest to weakest: | |
|---|---|
| Hydroxyl-hydroxyl: | O-H:::::O-H |
| Hydroxyl-carbonyl: | O-H:::::O=C |
| Amide-carbonyl: | N-H::::::O=C |
| Amide-hydroxyl: | N-H:::::::O-H |
| Amide-imidazole nitrogen: | N-H::::::::N from a cycle |
SECONDARY, TERTIARY AND QUATERNARY STRUCTURE OF PROTEINS
There are four levels of protein folding:
-HELIX Theoretically, an
-helix might be right-handed or left-handed, but for L-amino acid residues, the left-handed conformation is destabilized by steric interference between carbonyl oxygens and side chains. Within an
-helix, each carbonyl oxygen residue "n" is hydrogen bonded to the -amino nitrogen of residue "n+4". 
An -helix -helix stabilizes this conformation, especially in hydrophobic regions within the interior of a protein where water molecules do not compete for hydrogen bonds. BETA SHEET
The B sheet consist of extended polypeptide chains (called B strands) stabilized by hydrogen bonds between carbonyl oxygens and amide hydrogens.
An anti-parallel beta hairpin SUPERSECONDARY STRUCTURES
Supersecondary structure, also called motifs, consist of various combinations of secondary structure. Supersecondary structure may have a particular function or simply occurs as part of a larger functional unit called domains.
Some common supersecondary structures are: The
-loop-,the beta--beta unit,the hairpin and the greek key that is like a double hairpin (one inside the other. 
-loop- is an example of a motif.
An barrel can serve as a channel through biological membranes 
This beta barrels is an example of a funtional domain. It is made of both sheets and helixes and beta--beta unit. barrel and the alpha-beta barrel. 
Funtional domain properties is given by its exposed amino acids side chains. This is the electrostatic image of an E.coli protein that shows a very static side and a non polar one. Go back to the main menu
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