lecture3 - Protein Structure and Func/on Lecture 3:...

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Unformatted text preview: Protein Structure and Func/on Lecture 3: Amino acid sequencing Outline •  Classical methods –  Analyzing mixtures of amino acids –  Hydrolyzing pep/des/proteins –  Sequencing polypep/des/proteins •  Mass spectrometry •  Sample Problems Post ­column ninhydrin deriva/za/on AA mixture O OH OH Ion ­exchange chromatography Ninhydrin •  •  •  •  •  O •  •  •  •  Reacts with free amines Sensitivity makes it useful in forensics Visualize fingerprints in crime scenes Works with porous materials Detect: Purple (570 nm) Yellow (440 nm), Pro Detection limit 10 pmol, 50 pmol (Pro) Linear response in range of 20–500 pmol Need 1ug of protein before hydrolysis Fingerprint detec/on Sample elu/on profile •  •  •  (Ala, Arg, Asp, 2 Gly, Phe) Sulfonated polystyrene resin (e.g, Dowex ­50) Only ra/os of amino acids obtained From Biochemistry, L. Stryer Pre ­column PITC deriva/za/on AA mixture Phenylisothiocyanate R O H2N + NCS O PITC Basic pH R S Reverse ­phase HPLC N H O N H O Acid •  •  •  •  Detect at 254 nm Detection limit 1 pmol Linear response in range of 20–500 pmol Need 500 ng of protein before hydrolysis S NH N O R H Phenylthiohydantoin (PTH) amino acid Pre ­column FMOC deriva/za/on AA mixture Fluorenylmethyloxycarbonyl chloride O FMOC Cl O R + O H2N O Reverse ­phase HPLC O •  •  •  •  Fluorescent Excite at 260 nm Detect at 313 nm Detection limit 1 fmol R O N H O O FMOC amino acid Pep/de bond hydrolysis + N H3N O 6N HCl" N N R2 H +H N 3 R1 O OH H H + – O N R4 O H R5 O H R3 O H R1 +H N 3 R2 O O OH + etc." •  Trp is destroyed (oxidized) and not detected. Protec/ng Tryptophan •  Add mercaptan an/oxidants to HCl –  thioglycolic acid, thiodiglycolic acid •  Use mercaptomethanesulfonic acid instead of HCl Asn and Gln hydrolysis O Asn N H2 +H N 6N HCl OH +H N O 3 O NH4+ OH N H2 O O OH 6N HCl +H N + 3 OH Gln Asp O O 3 +H N O Glu + NH4+ 3 OH OH •  Tes/ng for the presence of ammonia shows if the amide was originally present. BTI and Asn/Gln •  Bis(1,1 ­trifluoracetoxy)iodobenzene converts the side chain amides to amines. •  Compare chromatogram peaks for Asp/Glu posi/on with and without BTI treatment. Biochemical J., 39, 507 Amino ­terminal labeling Fluorodinitrobenzene (FDNB), Sanger’s reagent, reacts with free uncharged amino group HO O2N F + H2N CH3 NO2 O N Gly-Asp-Phe-Arg-Gly H O– HCO3– Dinitrophenyl (DNP) derivative NO2 O2N HO N H CH3 O N Gly-Asp-Phe-Arg-Gly H O– Hydrolysis, 6N HCl DNP-amino acid, Identify by chromatography O2N NO2 Asp HO N H CH3 O – Gly Phe Arg Rest of the peptide is completely hydrolyzed. Gly ε ­amino labeling of an internal lysine O2N HN NH2 O2N F NO2 + Xaa NO2 Yaa N H O Xaa Yaa N H O O2N HN 6N HCl NO2 ε-DNP-Lysine +NH OH 3 O Note: if lysine is N-terminal, both the backbone and side chain amino groups get labeled. O ­DNP ­Tyr O2N HO O2N F NO2 + NO2 Xaa Xaa Yaa N H NO2 OH +H N 3 Ο-DNP-Tyrosine O Yaa N H O O O2N 6N HCl O O Note: if Tyr is N-terminal, both the backbone amino and side chain hydroxy groups get labeled. DNP ­His O2N H N O2N F + NO2 N Xa a NO2 N Ya a N H O N Xa a Ya a N H O O2N N 6N HCl NO2 DNP-Histidine N OH +H N 3 O Note: if His is N-terminal, both the backbone amino and side chain imidazole groups get labeled. Par/al hydrolysis •  Par/al hydrolysis of DNP ­Gly ­Asp ­Phe ­Arg ­Gly can yield a mixture of DNP ­tagged pep/des –  DNP ­Gly –  DNP ­Gly ­Asp –  DNP ­Gly ­Asp ­Phe –  DNP ­Gly ­Asp ­Phe ­Arg •  Frac/ona/on of the mixture followed by full hydrolysis and analysis of each component yields the sequence. Biochemical J., 45, 563. Enzyma/c cleavage + DNP labeling •  Example: Gly ­Asp ­Phe ­Arg ­Gly •  Chymotrypsin cleaves a]er Phe,Tyr or Trp •  Strategy: –  Treat pep/de with chymotrypsin –  React products with FDNB –  Analyze composi/on of each DNP ­labeled pep/de •  Results: –  –  –  –  Mixture of Gly ­Asp ­Phe and Arg ­Gly Mixture of DNP ­Gly ­Asp ­Phe and DNP ­Arg ­Gly (DNP ­Gly,Asp,Phe) and (DNP ­Arg,Gly) Deduce sequence to be Gly ­Asp ­Phe ­Arg ­Gly Biochemical J., 49, 463. Biochemical J., 49, 481. Specific pep/de cleavage •  Chemical cleavage –  Cyanogen bromide Met ­X –  Hydroxylamine Asp ­Gly –  2 ­Nitro ­5 ­thiocyanobenzoate X ­Cys •  Enzyma/c cleavage –  Trypsin Lys ­X, Arg ­X, X ≠ Pro –  Chymotrypsin Phe ­X, Tyr ­X, Trp ­X, X ≠ Pro –  Clostripain Arg ­X –  Staphylococcal protease Asp ­X and Glu ­X –  Staphylococcal V8 protease Glu ­X –  Carboxypep/dase A all C ­term except Arg, Lys, Pro –  Carboxypep/dase B only C ­term Arg or Lys •  Neither carboxypep/dase can cleave X ­Pro CNBr reac/on CN S Met NC + S CH3 CNBr S + CH3 Met + Br - CH3 R O NH + CH NH R O CH NH CH NH + CH + H3C S CN OH H2O O NH CH O + homoserine lactone" R H2N CH H2O O– NH CH O homoserine" Specific pep/de cleavage •  Chemical cleavage –  Cyanogen bromide Met ­X –  Hydroxylamine Asp ­Gly –  2 ­Nitro ­5 ­thiocyanobenzoate X ­Cys •  Enzyma/c cleavage –  Trypsin Lys ­X, Arg ­X, X ≠ Pro –  Chymotrypsin Phe ­X, Tyr ­X, Trp ­X, X ≠ Pro –  Clostripain Arg ­X –  Staphylococcal protease Asp ­X and Glu ­X –  Staphylococcal V8 protease Glu ­X –  Carboxypep/dase A all C ­term except Arg, Lys, Pro –  Carboxypep/dase B only C ­term Arg or Lys •  Neither carboxypep/dase can cleave X ­Pro Free N-terminal group required Edman degrada/on HO Phenyl isothiocynate S C N + H2N CH3 • Allows iterative sequencing. • However, incomplete and side reactions limit number of cycles. Phenylthiocarbamyl (PTC) peptide • In an automated sequencer with 98% yield, up to 70 residues can be sequenced from ~10 pmol of protein. N Gly-Asp-Phe-Arg-Gly H O– Basic pH S HO N N H CH3 O N Gly-Asp-Phe-Arg-Gly H O– Rest of the New peptide remains Trifluoroacetic acid N-terminal intact group regenerated (TFA) S Phenylthiohydantoin (PTH) amino acid O H N N O CH3 H + O H2N Gly-Asp-Phe-Arg-Gly O– Edman degrada/on, cont’d •  Requires free N ­terminal amino •  Itera/ve process –  New N ­terminal fragment generated –  Rest of polypep/de remains intact –  However, errors may accumulate at each stage, e.g., incomplete reac/on Sequencing strategy •  Divide and conquer –  Cleave the protein into overlapping smaller fragments –  Sequence the fragments –  Reassemble the sequence Overlapping pep/des example •  Diges/on with Trypsin yields two pep/des: –  Ala ­Ala ­Trp ­Gly ­Lys and Thr ­Asn ­Val ­Lys –  Order of the two fragments in the full ­length pep/de is unkown •  Diges/on with Chymotrypsin yields: –  Thr ­Asn ­Val ­Lys ­Ala ­Ala ­Trp as one of the pep/des •  The original pep/de must be Thr-Asn-Val-Lys-Ala-Ala-Trp-Gly-Lys Trypsin cleavage Chymotrypsin cleavage Example 2 •  Tryp/c diges/on gives: AGSFSTR VIWGD GMYR •  Chymotrypsin diges/on gives: RVIWGD STRGMY AGSF AGSFSTRGMYRVIWGD AGSFSTRGMYRVIWGD Disulfide bonds •  Cys – Cys bond, intra ­chain or interchain H X O S" N Y S" S S X' Y' N H S" S" O •  S ­S bond can be broken with a reducing agent. Reduc/on of S ­S bond HS HO + HO HO RSS S R S HO SH + 2 RSH Dithiothreitol (DTT)" RSSR + HS OH mercaptoethanol" HO S S OH + 2 RSH Other Cys reac/ons CH2 S + ICH2CO2 Iodoacetate Cys CH2 S CH2 CO2 + Carboxymethyl-Cys Performic acid oxidizes Cys or Cys-Cys to cysteic acid CH2 SH CH2 SO3H Both reactions prevent re-formation of disulfide bonds. I C ­terminal sequencing? Mass spectrometry for sequencing •  Alterna/ve to or in conjunc/on with Edman degrada/on •  Measure masses of pep/de fragments –  Compare with database of sequences –  For de novo sequences, analysis of masses and mass differences between mass spec peaks leads to possible sequences •  Use heuris/cs and computer algorithms to automate the analysis •  Possible ambiguity (e.g., K and Q have nearly similar masses) •  Mass spec is more sensi/ve (low fmol) but Edman degrada/on is more accurate (for > 2 pmol) Sample mass spectrum Analysis of sample MS peaks Sample problems http://bri.nrc.ca/ccb/bioc450 ...
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