Lecture 3: Amino Acids, Purification, and Analysis of Protein ...
Lecture 3: Amino Acids, Purification, and Analysis of Protein Composition, Sequence -‐ -‐
if the pH of the solution is less than the pKa value of the ionizable species = the amino acid will be protonated if the pH of the solution is more than the pKa = the ionizable species will be deprotonated there are 7 AMINO ACIDS with R groups that are ionizable
-‐ Titration Curve of Glycine -‐ there are no ionizable R groups in glycine -‐ glycine has 2 ionizable groups (the alpha carboxyl group and the alpha amino acid) -‐ 2 ionizable group, 2 buffering region, 2 molar equivalents -‐ at pH < 2 = 100% of the protonated form of glycine Titration Curve for Histidine -‐ histidine has 3 ionizable groups with pKas 2, 6 and 10 -‐ hisitdine has a pH of 6 -‐ 3 ionizable R groups, 3 pKas, 3 buffering regions -‐ If the solution’s pH < 6 = R group will be protonated -‐ Histidine has 3 ionizable g -‐ At a pH of 0, R group will have a positive charge and Histidine is 100% protonated -‐ pKa1 = pKa for the alpha carboxyl group -‐ pKa2 = pH of 6 (Histidine’s pH) -‐ pI of Histidine = 7.5 o pI takes the pKa value from either side where the species have a net charge of 0 o pI = (9+6)/2 = 7.5 o the average of the 2 pKa values around the species where net charge = 0 pKa Values of Amino Acids -‐ pKa of carboxyl group = ~ 2 -‐ pKa of alpha amino group = ~9 -‐ pKa of Arginine’s R group: 12.5 à a + charged amino acid -‐ pKa of Lysine’s R group: 10.5 à a + charged amino acid -‐ pKa of Aspartic Acid’s R group: 3.9 -‐ pKa of Cysteine’s R group: 8.3 -‐ pKa of Glutamic Acid’s R group: 4.3 -‐ pKa of Histidine’s R group: 6 o near physiological pH à critical role in acid-‐base catalysis (lose/gain protons) -‐ pKa of Tyrosine’s R group: 10 Titration Curve for Glutamic Acid -‐ pI = isoelectric point = (2+4)/2 = 3 -‐ acidic amino acid What is the pH of a glutamic acid solution if the alpha carboxyl is ¼ dissociated?
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pH = 2 + log10[1/4]/[3/4] = 2 + log10[1]/[3] = 2 + (-‐0.477) = 1.523 we took a pka of 2 because its asking about the alpha carboxyl group gamma carboxyl group pka = 4 note* when the group is ¼ dissociated, ¼ is dissociated and ¾ are not à thus the ration in the log term is ¼/¾ = 1/3
Titration Curve for Lysine -‐ pKa2 = R group’s pKa -‐ at a charge of 0 = pI value o use amino group pKas to calculate this -‐ amino group pKa = 9 -‐ R groups pKa = 10.5 -‐ pI = (9 + 10.5)/2 = 10 -‐ at a pH of 10, you have the isoelectric point -‐ protons are removed from the carboxyl group, then amino group, the R group accordingly General Rules for finding pI’s of Amino Acid 1) non-‐ionizable groups à pI = [pKa of alpha carbon group + pKa of alpha amino group]/2 -‐ [2+9]/2 = 7.5 2) acidic R groups (D/E) à pI = [pKa of alpha carbon group + pKa of alpha amino group]/2 -‐ (2+4)/2 = 3 3) basic R groups (H, R, K) à pI = [pKa of alpha carbon group + pKa of alpha amino group]/2 -‐ H’s pI = (9+6)/2 = 7.5 Peptide Bonds -‐ Read from the N terminal to the C terminal -‐ typically, the –ine/-‐ate at the end of the FIRST amino acid is changed to “-‐ly” -‐ keep the second name of the amino acid -‐ alanine + serine = alaylserine dipeptide ex. Aspartic acid + phenylalanine = aspartyphelalanine (known as aspartane) à à aspartic acid at the top, phenylalanine at the bottom Protein Purification 1) exploit minor differences in solubilities, net charges, sizes, binding specificities 2) need protein to retain its biological acidity 3) high yield 4) thousands of different proteins in starting mixture 5) many procedures for performed at 0-‐4C (to maintain biological activity of the protein) Differential Purification 1. homogenate is formed by disrupting the cell membrane 2. mixture is fractionated by centrifugation yielding a dense pellet of heavy material at the bottom of the centrifuge tube and a lighter supernatant on top 3. several fractions of decreasing density containing hundreds of different proteins are then assayed for the activity being purified
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pellets can then be salted out, liquid salted in
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proteins are least soluble when they are at their pI value o there is no net charge = no electrostatic effect = proteins do not repel each other = proteins will come together an coalesce = precipitates when there is a low range of pH = low solubility salt in/out depends on pH most proteins increases solubility when there is an increase in ionic strength o salt-‐in of proteins depends on the diminishment of electrostatic attractions between proteins by the presence of abundant salt ions o as salt concentration reaches high levels, protein is salted out of solution à precipitates o numerous salt ions compete with protein for water solvation, salt wins, protein becomes insoluble
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Dialysis -‐ semipermeable membrane separates small molecules -‐ removes a salt or other small molecules à goes out of membrane -‐ does not distinguish between proteins effectively though -‐ 4-‐5 proteins of interest will be left -‐ takes 1-‐2 days Column Chromatography – HPLC, Ion Exchange, Gel Filtration, Affinity
Pre-‐Column Modification there is matrix in the column (column differs depending on which property you want to exploit (ex. Charge, size, etc) -‐ use different columns to exploit different properties -‐ add a buffer so that different proteins can go through -‐ samples will be collected at a constant time interval (per test tube) -‐ add different buffers to separate different proteins -‐ will be colourless, you will have to measure the absorbance of colour -‐
Different Chromatographic Techniques for Protein Isolation 1) High Pressure using small packed columns with the solvent flow controlled by computer (HPLC) -‐ High Pressure Liquid Chromatography -‐ Exploits hydrophobic properties of the protein -‐ Silica beads that have hydrocarbons attached -‐ Hydrocarbons are hydrophobic à Hydrophobic proteins will be in the column longer -‐ Hydrophilic proteins come out first 2) Ion Exchange -‐ exploits charge -‐ can have columns that bind cations (cation exchanger) or those than bind anions 3) Gel Filtration -‐ Separate proteins based on size 4) Affinity -‐ most selective -‐ needs to know if protein has a specific binding affinity to some molecule
Ion Exchange -‐ proteins separated based on their net charge -‐ grey beads: positively charge proteins will bind, negatively charged proteins will not -‐ a cation exchanger -‐ positively charged proteins will stick in the column -‐ to take the positively charged proteins out of the column later, change the pH by adding a salt solution à salt ions compete with positively charged groups on the protein for binding to the column o straight competition -‐ Cation exchanger media – separates positively charged proteins o Dowex-‐50, CM cellulose, Chelex-‐100 -‐ Anion exchange media – separates negatively charged proteins o Dowex-‐1, DEAE cellulose Gel Filtration -‐ Separation of sizes of proteins -‐ large proteins come out first, small ones come out after -‐ sample is put into the top of the column consisting of porous beads made of an insolubly but highly hydrated polymer -‐ small molecules can enter the beads, but larges one cant -‐ small molecules are distributed in the aqueous solution both inside the beads and between them -‐ large molecules are located only in the solution between the beads à cannot enter the internal volume of the beads -‐ large moelcuels flow more rapidly through this column and emerge first, this is because smaller volume is accessible to them Affinity -‐ utilizes the high affinity of many proteins for specific chemical groups -‐ a protein may bind strongly to the column because of its highly affinity for glucose, whereas other proteins do not -‐ the protein can then be released from the column by adding concentration solution of glucose -‐ glucose solution displaces the column-‐attached glucose residues from binding sites of the protein -‐ simple competition to separate protein and glucose Gel Electrophoresis -‐ SDS-‐Page & MW Determination -‐ Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis -‐ Separates protein based on size (MW – molecular weight) -‐ SDS is a buffer added to the solution -‐ Look at the mobility pattern of protein in an electrical field à proteins that are large (large MW) takes longer to migrate to the end o Compare to gel filtration where large proteins come out first, here, large proteins move down last o Electrophoresis also differs from
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gel filtration as all of the molecules (regardless of size) will move through the same matric § Gel behaves as one bead of a gel-‐filtration column Determines the molecular weight of a protein à mobility of protein is inversely proportional to the logarithm of the protein’s molecular weight Formation of the polyacrylamide gel:
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a 3D mesh is formed by co-‐polymerizing activated monomer (blue) and cross-‐linker (red)
SDS-‐PAGE 1) likes to bind proteins in 1:1.4 ratio à negatively charge -‐ charge-‐to-‐mass ratio is gone -‐ each SDS contributes to 2 negative charges 2) non-‐covalent forces à denatures proteins à left with rod-‐like proteins (linear) -‐ SDS disrupts protein folding 3) Also a reducing agent à protein may have disulfide bond à this agent will break them (ex. Betamercaptoethanol) -‐ mixture is first dissolved into a solution of SDS (anionic detergent that disrupts nearly all noncovalent interactions in proteins -‐ anions of SDS bind to main chains at a ratio of about one SDS anion for every 2 amino acids residues RF Values -‐ rf value is calculated as the ratio of distance migrated by the molecule : distance migrated by the dye -‐ when you run protein on electrophoresis gel, smaller pieces move further, heavier pieces stay closer to the well -‐ take the well as the origin of each sample, measure how far the gel marker moved from it, and measure how far the protein sample moved divided by the distance the gel marker moved -‐ rf value is the (distance the protein moved)/(distance the dye marker moved) -‐ electrophoretic mobility of the protein is inversely proportional to the logarithm of the protein’s molecular weight How is the Amino Acid Analysis of Proteins Performed? -‐ acid hydrolysis liberates the amino acids of a protein -‐ note that some amino acids are partially or completely destroyed by acid hydrolysis -‐ chromatographic methods are used to separate amino acids -‐ the amino acid compositions of proteins are different -‐ the sequence of amino acids in a protein is distinctive -‐ both chemical and enzymatic methodologies are used in protein sequencing
Techniques for Determining Amino Acid Composition Acid Hydrolysis
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acromatic rings on amino acids allows us to measure absorbance -‐ attach PITC onto amino acid can now measure for the absorbance of each amino acid using standard chromatography:
some R groups are partially destroyed à tend to be Ser and Thr à may not be a good representation of their amino acid analysis Trp is totally destroyedà no W
-‐ Sequence Determination -‐ Identify the N-‐ and C-‐ terminal residues -‐ N-‐terminal analysis o Edman’s reagent o Phenylisothiocyanate – PITC reagent o Derivatives are phenylthiohydantoins o Or PTH derivatives Edman Degradation
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Edman’s reagent (phenylisothiocyanate) reacts with an alpha-‐amino group of an amino acid, followed by a cyclization to produce a phenylthiohydantoin (PTH) derivative Yields 100% Efficiency decreases at 30 residues – can only do 30 residues at a time Cleave peptides to smaller proteins à then subject them to Edmans TFA = Trifluroacetic acid (F3CCOOH) Before using Edman’s degradation, you also have to cleave disulfide bonds first o Causes sulfur to nucleophilically attack carbonyl carbon o Cleavage of the peptide bond Thiazoline derivatives are unstable PTH-‐amino acid: run through HPLC to determine which amino acid it is (elution time vs. absorbance)
Sequence Determination -‐ Identify N-‐ and C-‐terminal residues C-‐terminal analysis -‐ enzymatic analysis (carboxypeptidase)
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carboxypeptidase A cleaves any residue except Pro, Arg, Lys o any C-‐terminals that start with these, or any encounter on the sequence will terminate cleavage action carboxylpeptidase B (hog pancreas) only works on Arg and Lys also have to cleave disulfide bonds first
Sequence Determination -‐ Cleavage of Disulfide Bridges o Ex. Disulfide bond formed between cysteine to form cystine o Performic acid oxidation o Sulfhydryl reducing agents § Mercaptoethanol à in SDS-‐PAGE to break disulfide bonds § Dithiothreitol (DTT) or dithioerythritol (DTE) • Has sulfur group in them • Very reactive • Have to modify it when you break the disulfide bonds so that it doesn’t react § To prevent recombination, follow with an alkylating agent like iodoacetate à will modify S-‐H residues so they wouldn’t form disulfide bridges again Disulfide Bridges -‐ this is an oxidation reduction reaction -‐ The disulfide bridge is a feature of protein 3° structure -‐ Two cysteines that can be distant in primary sequence coming together to form disulfide bond -‐ beta-‐mercaptoethanol is used as the reducing agent -‐ disulfide bridges are reduced in cysteine residues à now are very reactive à block it with iodoacetate so that bonds don’t form again
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iodoacetic acid: alkylating agent the S-‐carboxymethyl derivative will have acetic acid attached to the sulfur to block disulfide bridge formation now its non-‐reactive now you can perform Edman’s degradation
Sequence Determination -‐ Fragmentation of the Chains (into 30 residues) Enzymatic Fragmentation -‐ Trypsin, chymotrypsin, clostripain, staphylococcal protease Chemical Fragmentation -‐ Cyanogen bromide
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Breaks at the C-‐terminal of Methionine residues (carboxyl side) End up with a modified C-‐terminal homoserine structure CNBr acts only on methionine residues CNBr is useful because proteins usually have only a few Met residues Be able to recognize the results
§ A peptide with a C-‐terminal homoserine lactone o CNBr is a highly selective reagent for cleavage of peptides at ONLY methionine residues 1) nucleophilic attack of the Met S atom on the –CN carbon atom & displacement of Br 2) nucleophilic attack by the Met carbonyl oxygen atom on the R group -‐ the cyclic derivative is unstable in aqueous solution 3) hydrolysis cleaves the Met peptide bond -‐ C-‐terminal homoserine lactone residues occur where Met residues once were
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the reaction of cyanogen bromide with a peptide à cleavage at Met residues à produces peptides with C-‐terminal homoserine lactone residues (where Met residues once were)
Sequence Determination -‐ Enzymatic Fragmentation o Trypsin – cleavage on the C-‐side of Lys, Arg § Trypsin cleaves basic amino acids (K, R) o Chymotrypsin – cleavage on the C-‐side of Phe, Tyr, Trp § Chymotrypsin cleaves aromatic amino acids o Clostripain – like trypsin, but attacks Arg more than Lys o Staphylococcal protease – cleaves acidic amino acids (D, E) § cleavage on the C-‐side of Glu, Asp (in phosphate buffer) § specific for Glu in acetate/bicarbonate buffer -‐ reconstructing the sequence (to figure which sequence came first) -‐ use 2 or more fragmentation agents in separate fragmentation experiments to know which sequence was first -‐ sequence all the peptides produced (usually by Edman degradation) -‐ compare and align overlapping peptide sequences to learn the sequence of the original polypeptide chain v Compare the cleavage by trypsin (cleave at C-‐term at R and K) and stalphylococcal protease (cleave at C-‐ term at D and E) on an unknown peptide: o Trypsin cleavage of unknown peptide gave: AEFSGITPK LVGK o Staphylococcal protease cleavage gave: FSGITPK LVGKAE à can now see F follows after E Ø Overlap of the 2 sets of fragments: LVGK AEFSGITPK (from trypsin) LVGAE FSGITPK (from staphylococcal protease) Ø Correct sequence: LVGKAEFSGITPK ü Chymotrypsin also cleaves at Leu, not only aromatic amino acids Sequence Determination -‐ in 1953, Sanger sequenced the 2 chains of insulin
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Sanger’s results established that all of the molecules of a given protein have the same sequence Proteins can be sequence in 2 ways: 1) real amino acid sequencing (Edmans, fragmentation) 2) sequencing the corresponding DNA in the gene
you cannot acccuratly predict the sequence if you only use DNA àDNA has introns
