28 Ali Yaghi Tamara Wahbeh Mamoun Ahram
This sheet is a continuation of protein purification methods. Isoelectric focusing Separation of proteins based on Isoelectric points(charge),and it is a horizontal separation. Proteins will move across two poles of negative and positive charges determined by certain ph levels, because they are charged. As proteins migrate through the gel, they encounter regions of different ph levels, so the charge on the protein changes. They will migrate to both sides of the dipole until they reach a region which represents its isoelectric point. Note: Isoelectric point: it is when the protein is charged but the total charge is zero (neutral). Neutral proteins don't move toward any side of the gel. Two-dimensional gel electrophoresis (2D-PAGE) It is a combination of the isoelectric point and SDS-PAGE. First we separate molecules according to their charge through the isoelectric point method, then, the SDS-PAGE method will follow for further separation according to size. 1 P a g e
1- Low ph, low molecular weight. 2- Low ph, high molecular weight. 3- High ph, low molecular weight. 4- High ph, high molecular weight. ELISA Enzyme-linked immunosorbent assay Identification + quantification It depends on the usage of immunoglobins (antibodies). At first antigen (sample) binds to antibody that is attached to a solid surface, then another antibody binds to a different location of the same antigen. The second antibody is the signaling molecule. If the antigen is bound to the first antibody, a signal (it could be color change) will be detected, otherwise the signal won't be shown. Other information that could be concluded by Elisa is how much antigen is found in the sample by comparing it to a known concentration of another sample, and comparing the intensity of each signal. 2 P a g e
Protein sequencing Knowing the sequence of amino acids in a protein. We can know it in 2 ways: 1- Edman Degradation 2- Cleavage methods a- Chemical digestion b- Endopeptidases c- Exopeptidases Edman Degradation This procedure involves a step-by-step cleavage of the N-terminal residue of a peptide, allowing for the identification of each cleaved residue This method utilizes phenylisothiocyanate (PITC) to react with the N-terminal residue (the first amino acid) and causes cleavage of it out of the peptide or protein. The resultant amino acid is hydrolyzed, liberated from the peptide, and identified by chromatographic procedures. - Advantage Since the remainder of the peptide is intact, the entire sequence of reactions can be repeated over and over to obtain the sequences of the peptide. The Edman degradation technique does not allow peptides more than 50 residues to be sequenced. If we want 3 P a g e
to know the sequence of amino acids in a protein which has 200 amino acids for example, we have to simplify it by using cleavage method(s), and then we use Edman degradation. Chemical digestion: In this step we use cyanogen bromide (CNBr), which cleaves the peptide at the site of the amino acid methionine. A protein that has 10 methionine residues will usually yield 11 peptides on cleavage with CNBr.If we know that a peptide has 10 methionines, and by using (CNBr), 10 segments were produced, then one methionine was at the end of peptide. Note: This reagent causes specific cleavage at the C-terminal side of methionine residues. Endopeptidases These are enzymes that cleave at specific sites within the primary sequence of proteins. 4 P a g e
Notes: We exclude proline because it makes the peptides unstable, where it cannot undergo cleavage using this method. The previous table is very important (memorize). Exopeptidases These are enzymes that cleave amino acids starting at the end of the peptide. They are two types: Aminopeptidases that cleave at the N-terminus Carboxypeptidases that cleave at the C-terminus Remember: we always start reading an amino acid sequence starting from the N- terminus end and ending at the C-terminus end. Note: These enzymes aren't specific for some amino acids, they have the ability to work on any peptide (similar to Edman degradation). Important questions: 5 P a g e
A sample of an unknown peptide was divided into two aliquots. One aliquot was treated with trypsin; the other was treated with cyanogen bromide. Given the following sequences (N-terminal to C-terminal) of the resulting fragments, deduce the sequence of the original peptide. Trypsin treatment Asn Thr Trp Met Ile Lys Gly Tyr Met Gln Phe Val Leu Gly Met Ser Arg Cyanogen bromide treatment Gln Phe Val Leu Gly Met Ile Lys Gly Tyr Met Ser Arg Asn Thr Trp Met Deduce the sequence of the original peptide. Answer: Val Leu Gly Met Ser Arg Asn Thr Trp Met Ile Lys Gly Tyr Met Gln Phe **A sample of a peptide of unknown sequence was treated with trypsin; another sample of the same peptide was treated with chymotrypsin. The sequences (N-terminal to C-terminal) of the smaller peptides produced by trypsin digestion were as follows: Met Val Ser Thr Lys Val Ile Trp Thr Leu Met Ile Leu Phe Asn Glu Ser Arg The sequences of the smaller peptides produced by chymotrypsin digestion were as follows: Asn Glu Ser Arg Val Ile Trp Thr Leu Met Ile Met Val Ser Thr Lys Leu Phe Deduce the sequence of the original peptide. Answer: Met Val Ser Thr Lys Leu Phe Asn Glu Ser Arg Val Ile Trp Thr Leu Met Ile 6 P a g e
Determining the 3D structure of a protein 1- Crystallography 2- Nuclear magnetic resonance (NMR) spectroscopy Crystallography We use a purified protein and convert it into a crystal at a very low temperature. Crystals are exposed to x-rays, which are scattered by the electrons of the molecule, therefore determining the geometry of the protein. NMR The structure of a protein is determined in the liquid state (physiological environment). It is also used to know the change that happened to the protein structure after binding to another molecule. Example: knowing that a Heme group in hemoglobin is dome shaped when it is in t state and knowing the 0.4 angstrom change in angle, which in turn affects the quaternary structure. 7 P a g e