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05. Protein Sequencing


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05. Protein Sequencing

  1. 1. How To Sequence A Protein W. Robert Midden Department of Chemistry Bowling Green State University
  2. 2. Protein Sequencing Preliminary Steps <ul><li>For multisubunit proteins, the individual protein chains must first be separated </li></ul><ul><li>Break interchain disulfide bonds, if necessary </li></ul><ul><li>Two reagents are commonly used: </li></ul><ul><ul><li>performic acid </li></ul></ul><ul><ul><li>mercaptoethanol </li></ul></ul>
  3. 3. 2-Mercaptoethanol <ul><li>2-mercaptoethanol reduces disulfides to sulfhydryls </li></ul><ul><li>But the sulfhydryls are easily oxidized back to the disulfide </li></ul>
  4. 4. Preventing Reversal <ul><li>to prevent oxidation the suflhydryls are alkylated with iodoacetic acid or acrylonitrile </li></ul>
  5. 5. Perfomic Acid <ul><li>Performic acid oxidizes cysteine to negatively charged cysteic acid </li></ul>
  6. 6. Reversal Prevented <ul><li>The repulsion of the negatively charged SO 3 - groups prevents reformation of the disulfide bond </li></ul><ul><li>Therefore alkylation is not necessary with performic acid </li></ul>
  7. 7. Protein Sequencing Preliminary Steps <ul><li>After breaking disulfide bonds, the chains are separated by disrupting noncovalent interchain interactions with pH extremes, 8 M urea, 6 M guanidium hydrochloride, or high salt </li></ul><ul><li>Then the individual protein chains are separated by electrophoresis or chromatography on the basis of size or charge </li></ul>
  8. 8. Determining Amino Acid Sequence <ul><li>Once each protein is purified the amino acid sequence is determined by: </li></ul><ul><li>1) determining the amino acid composition (how many of each amino acid are in the protein) </li></ul><ul><li>2) identifying the amino and carboxyl terminal amino acids </li></ul><ul><li>3) cleaving the protein into two or more sets of peptides using specific enzymatic or chemical reagents such as trypsin or cyanogen bromide </li></ul>
  9. 9. Determining Protein Sequence <ul><li>4) determining the amino acid sequence of each of the peptide fragments </li></ul><ul><li>5) determining the entire protein sequence from the sequences of overlapping peptide fragments </li></ul><ul><li>6) locating the position of disulfide bridges between cysteines </li></ul>
  10. 10. Determining Amino Acid Composition <ul><li>The amino acid composition is determined by: </li></ul><ul><li>Hydrolysis with 6N HCl for one to three days </li></ul><ul><li>Separating and quantifying individual amino acids by ion exchange HPLC using an amino acid analyzer </li></ul>
  11. 11. Determining the N-Terminal Amino Acid <ul><li>The N-terminal amino acid is determined using either chemical reagents or enzymes </li></ul><ul><li>Chemical reagents include: </li></ul><ul><li>Sanger’s reagent </li></ul><ul><li>dansyl chloride </li></ul><ul><li>Edman Degradation </li></ul>
  12. 12. Determining the N-Terminal Amino Acid <ul><li>Sanger’s reagent </li></ul><ul><li>Treat with dinitrofluorobenzene to form a dinitrophenyl (DNP) derivative of the amino-terminal amino acid </li></ul><ul><li>Acid hydrolysis </li></ul><ul><li>Extract the DNP-derivative from the acid hydrolysate with organic solvent </li></ul><ul><li>Identify the DNP-derivative by chromatography and comparison with standards </li></ul>
  13. 13. Determining the N-Terminal Amino Acid <ul><li>Dansyl chloride (dimethylaminonaphthylenesulfonyl chloride) </li></ul><ul><li>Forms a highly fluorescent derivative of the amino-terminal amino acid </li></ul><ul><li>Identified by chromatography and fluorescence detection after acid hydrolysis </li></ul><ul><li>Highly senstive </li></ul><ul><li>Best choice when the amount of protein is limited </li></ul>
  14. 14. Determining the N-Terminal Amino Acid <ul><li>Edman degradation </li></ul><ul><ul><li>phenylisothiocyanate (phenyl-N=C=S) adds to N-terminus then acid treatment cleaves the N-terminal amino acid as a PTH derivative </li></ul></ul><ul><ul><li>the remaining protein chain is intact and the cycle can be repeated </li></ul></ul><ul><ul><li>under ideal conditions the sequence of 30-60 amino acids can be determined </li></ul></ul><ul><li>Leucine aminopeptidase </li></ul><ul><ul><li>enzyme from hog kidney hydrolyzes the N-terminal peptide bond </li></ul></ul><ul><ul><li>best with nonpolar amino acids </li></ul></ul>
  15. 15. Determining the C-Terminal Amino Acid <ul><li>Hydrazinolysis </li></ul><ul><ul><li>hydrazine at 100°C cleaves all peptide bonds forming hydrazides except for the carboxyl terminal </li></ul></ul><ul><li>C-terminus reduced with LiAlH 4 </li></ul><ul><ul><li>forms amino alcohol at C-terminus </li></ul></ul><ul><li>Carboxypeptidases </li></ul><ul><ul><li>enzymatic removal of C-terminus </li></ul></ul><ul><ul><li>Carboxypeptidase A all except proline, arginine and lysine </li></ul></ul><ul><ul><li>Carboxypeptidase B only arginine and lysine </li></ul></ul><ul><ul><li>Carboxypeptidase C any amino acid </li></ul></ul><ul><ul><li>care required since rate of removal varies with the type of amino acid </li></ul></ul>
  16. 16. Peptide Fragments <ul><li>After determining the amino acid composition and the N & C-terminal amino acids, at least two different sets of protein fragments are needed for sequencing </li></ul>
  17. 17. Why Use Fragments? <ul><li>Why is the protein broken into fragments? Why isn’t the protein sequenced directly? </li></ul><ul><li>The sequencing methods currently available are only accurate for peptides up to about 20-30 amino acids, 60 under ideal conditions </li></ul>
  18. 18. Why 2 Sets of Fragments? <ul><li>Why can't the entire protein amino acid sequence be determined from a single set of peptide fragments obtained by cleavage with a single reagent? </li></ul><ul><li>There’s no way to determine how the fragments are connected with just one set </li></ul><ul><li>A second or third set of fragments are used to deduce how the fragments are connected by identification and comparison of overlapping sequnces </li></ul>
  19. 19. Protein Cleavage Reagents <ul><li>What types of reagents are best suited for preparing these sets of fragments? </li></ul><ul><li>Reagents that cleave the protein chain only at a few specific sites forming fragments that are less than 20-30 amino acids in length </li></ul>
  20. 20. Protein Cleavage Reagents <ul><li>Chemical or enzymatic reagents can be used to prepare protein fragments </li></ul><ul><li>The most commonly used reagents are: </li></ul><ul><li>cyanogen bromide </li></ul><ul><li>various enzymes including </li></ul><ul><ul><li>trypsin </li></ul></ul><ul><ul><li>chymotrypsin </li></ul></ul><ul><ul><li>clostripain </li></ul></ul><ul><ul><li>Staphylococcal protease </li></ul></ul><ul><ul><li>various endopeptidases </li></ul></ul>
  21. 21. Cyanogen Bromide <ul><li>At which amino acid in the protein sequence does the reagent, cyanogen bromide, cleave protein chains? </li></ul><ul><li>At internal methionines by reaction with the methionine sulfur as illustrated above </li></ul>
  22. 22. Trypsin & Chymotrypsin <ul><li>Where in the protein sequence do the enzymes, trypsin and chymotrypsin cleave protein chains? </li></ul><ul><li>trypsin cleaves at the carboxyl side of amino acids with positively charged side chains such as lysine and arginine </li></ul><ul><li>chymotrypsin cleaves at the carboxyl side of amino acids with aromatic side chains such as phenylalanine and tyrosine </li></ul>
  23. 23. Clostripain <ul><li>Where in the protein sequence does the enzyme, clostripain, cleave? </li></ul><ul><li>prefers positively charged amino acids, arginine even more than lysine </li></ul><ul><li>narrower specificity than tryptophan </li></ul><ul><li>which enzyme is likely to produce larger fragments? </li></ul>
  24. 24. Staphyloccal Protease <ul><li>Where in the protein sequence does the enzyme, Staphylococcal protease cleave? </li></ul><ul><li>carboxyl side of acidic amino acids in phosphate buffer </li></ul><ul><li>in acetate or bicarbonate buffer it is more specific and cleaves only glutamic acid </li></ul>
  25. 25. Endopeptidases <ul><li>The following endopeptidases are less specific than the enzymes metioned above </li></ul><ul><li>Pepsin, papain, subtilisin, thermolysin, elastase </li></ul><ul><ul><li>(papain is the active ingredient in meat tenderizer, soft contact cleansing solutions, some laundry detergents) </li></ul></ul><ul><li>These enzymes are most often used to further reduce the size of large tryptic or chymotryptic fragments </li></ul>
  26. 26. How are Peptide Fragments Separated? <ul><li>Usually by column chromatography, often HPLC </li></ul><ul><li>Separations are most often based on differences in polarity (reverse phase) or electric charge (ion exchange) </li></ul>
  27. 27. Edman Degradation <ul><li>Edman degradation is most often used to sequence the peptides </li></ul><ul><li>It removes one amino acid from the N-terminal end of the peptide during each cycle of the procedure </li></ul><ul><li>The removal of the N-terminal amino acid is accomplished using the reagent, phenylisothiocyanate </li></ul>
  28. 28. Edman Degradation <ul><li>Pheylisothiocyanate attaches to the N-terminal amino acid </li></ul><ul><li>The peptide amino nitrogen atom bonds to the PITC carbon </li></ul><ul><li>Sulfur then bonds to the peptide carboxyl carbon breaking the peptide bond </li></ul><ul><li>This cyclization forms a pheylthiohydantoin derivative which is removed from the peptide chain by treatment with anhydrous acid </li></ul><ul><li>Identified by extraction, treatment with aqueous acid and analysis by chromatography </li></ul>
  29. 29. Edman Degradation
  30. 30. Disulfide Bridges <ul><li>The location of disulfide bridges can be determined by diagonal electrophoresis </li></ul><ul><li>Fragments with intact disulfide bonds are electrophoresed in one dimension </li></ul><ul><li>Treated with fumes of performic acid to cleave disulfide bonds </li></ul><ul><li>Then electrophoresed in the second dimension </li></ul><ul><li>Fragments that had no disulfide bonds will be on the diagonal </li></ul><ul><li>Fragments that had disulfide bonds will migrate off diagonal due to altered mobility </li></ul>
  31. 31. Mass Spectroscopy <ul><li>Used for sequencing peptides </li></ul><ul><li>Peptides are fragmented in the mass spectrometer </li></ul><ul><li>The fragments are identified by their mass/charge ratio </li></ul><ul><li>Peptide mixtures can be analyzed using a temperature gradient </li></ul><ul><li>The temperature gradient causes variation in signals corresponding to different peptides </li></ul>
  32. 32. Protein Sequencing by DNA Sequencing <ul><li>In fact, while you have just learned how to sequece a protein by chemical and enzymatic degradation, protein sequences are now most often determined by translating the corresponding cloned genes </li></ul><ul><li>This latter process is usually easier and quicker once the gene corresponding to a given protein has been identified </li></ul>
  33. 33. Sequence Databases <ul><li>International databases of protein sequences are maintained </li></ul><ul><li>Many of these databases are accessible via the internet </li></ul><ul><li>Examples: </li></ul><ul><li>GenBank </li></ul><ul><li>Protein Identification Resource (PIR) </li></ul><ul><li>European Molecular Biology Data Library (EMBL) </li></ul>