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Bioch/BIMS 503 Lecture 1 Structure and Properties of Amino Acids and the Peptide Backbone

Bioch/BIMS 503 Lecture 1 Structure and Properties of Amino Acids and the Peptide Backbone. August 26, 2008 Robert Nakamoto Mol. Physiology & Biol Physics Tel: 982-0279, rkn3c@virginia.edu Snyder 380 (Fontaine). Major topics –. Names, abbreviations, general structure of amino acids

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Bioch/BIMS 503 Lecture 1 Structure and Properties of Amino Acids and the Peptide Backbone

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  1. Bioch/BIMS 503 Lecture 1 Structure and Properties of Amino Acids and the Peptide Backbone August 26, 2008 Robert Nakamoto Mol. Physiology & Biol Physics Tel: 982-0279, rkn3c@virginia.edu Snyder 380 (Fontaine)

  2. Major topics – • Names, abbreviations, general structure of amino acids • Amino acid chemical classes (polar, hydrophobic, acidic, basic, aromatic, S-containing) • Amino acid structural classes/affinity • Amino acid evolutionary classes • pK - Henderson-Hasselbach equation • Structure of the peptide bond • Proteomics – MS protein sequencing

  3. Further Reading – • Lehninger, Chapter 3 pp 75-86, 102-106 • MvHA, Chapter 5, pp 126-142 • Brandon & Tooze, Ch. 1 • Aebersold R, Mann M. (2003) Mass spectrometry-based proteomics. Nature. 422:198-207 PMID:12634793

  4. Hierarchies of protein structure primary structure MVDFYYLPGSSPCRSVIMTAKAVGVELNKK secondary structure a-helix b-strand super-secondary structure aa bab bb ternary fold aaaa 4-helix bundles babab Rossman fold bbb bbbb b-meander Greek key ternary structure Does this structural hierarchy reflect the folding process? Secondary structure first, or last?

  5. Examples of protein folds and complexes:Many bacterial toxin proteins undergo conformational changes that insert into host cell membrane: for example, Anthrax toxin protein/Protective protein complex From Santelli et al., 2004, Nature 430, 905-908

  6. Currently 10,340 protein fold families in Pfam [http://pfam.sanger.ac.uk/] Pfam is a comprehensive collection of protein domains and families,represented as multiple sequence alignments and as profile hiddenMarkov models. Generally does not include membrane proteins. What defines the 3-dimensional fold of a protein?

  7. Structure and properties ofAmino-acids Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Glutamine Gln Q Serine Ser S Glutamic acid Glu E Threonine Thr T Glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V Asp/Asn Asx B Glu/Gln Glx Z

  8. L-glyceraldehyde D-glyceraldehyde Amino-acid Chirality

  9. R Ca H N CO Amino-acid Chiralitythe “CORN” rule R H O N C O

  10. Figure 5.3: The amino acids found in proteins. CYCLIC AMINO ACID AROMATIC AMINO ACIDS

  11. Leucine a b H a

  12. Alanine b R R H a CO N N CO H

  13. Proline - a cyclic amino-acid R R b Pro H a N N CO CO b R R Ala H a CO N N CO

  14. Classifications of amino-acids Molecular weight Number of codon(s) Bulkiness Polarity / Zimmerman Polarity / Grantham Refractivity Recognition factors Hphob. / Eisenberg et al. Hphob. OMH / Sweet et al. Hphob. / Hopp & Woods Hphob. / Kyte & Doolittle Hphob. / Manavalan et al. Hphob. / Abraham & Leo Hphob. / Black Hphob. / Bull & Breese Hphob. / Fauchere et al. Hphob. / Guy Hphob. / Janin Hphob. / Miyazawa et al. Hphob. / Rao & Argos Hphob. / Roseman Hphob. / Wolfenden et al. Hphob. / Welling & al Hphob. HPLC / Wilson & al Hphob. HPLC / Parker & al Hphob. HPLC pH3.4 / Cowan Hphob. HPLC pH7.5 / Cowan Hphob. / Rf mobility HPLC / HFBA retention HPLC / TFA retention HPLC / retention pH 2.1 HPLC / retention pH 7.4 % buried residues % accessible residues Hphob. / Chothia Hphob. / Rose & al Ratio hetero end/side Average area buried Average flexibility alpha-helix / Chou & Fasman beta-sheet / Chou & Fasman beta-turn / Chou & Fasman alpha-helix / Deleage & Roux beta-sheet / Deleage & Roux beta-turn / Deleage & Roux Coil / Deleage & Roux alpha-helix / Levitt beta-sheet / Levitt beta-turn / Levitt Total beta-strand Antiparallel beta-strand Parallel beta-strand A.A. composition A.A. comp. in Swiss-Prot Relative mutability • Abundance • Hydrophobicity • Mutability • Structural preference • Charge properties us.expasy.org/cgi-bin/protscale.pl

  15. Amino acid frequencies in proteins + Ala A 0.0780 Arg R 0.0512 Asn N 0.0448 Asp D 0.0536 - Cys C 0.0192 Gln Q 0.0426 + Glu E 0.0629 + Gly G 0.0737 - His H 0.0219 Ile I 0.0514 + Leu L 0.0901 Lys K 0.0574 - Met M 0.0224 - Phe F 0.0385 Pro P 0.0520 + Ser S 0.0711 Thr T 0.0584 - Trp W 0.0132 - Tyr Y 0.0321 + Val V 0.0644

  16. Amino acid Hydropathicity/Hydrophobicity Hopp T.P., Woods K.R. (1981) PNAS. 78:3824-3828. Kyte J., Doolittle R.F. (1982). J. Mol. Biol. 157:105-132 D. M. Engelman, T. A. Steitz, A. Goldman, (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 321 Hopp/ Woods Kyte/ Doolittle GES Arg: 3.0 Lys: 3.0 Asp: 3.0 Glu: 3.0 Ser: 0.3 Gln: 0.2 Asn: 0.2 Pro: 0.0 Gly: 0.0 Thr: -0.4 His: -0.5 Ala: -0.5 Cys: -1.0 Met: -1.3 Val: -1.5 Leu: -1.8 Ile: -1.8 Tyr: -2.3 Phe: -2.5 Trp: -3.4 Arg: -4.5 Lys: -3.9 Asp: -3.5 Glu: -3.5 Gln: -3.5 Asn: -3.5 His: -3.2 Pro: -1.6 Tyr: -1.3 Trp: -0.9 Ser: -0.8 Thr: -0.7 Gly: -0.4 Ala: 1.8 Met: 1.9 Cys: 2.5 Phe: 2.8 Leu: 3.8 Val: 4.2 Ile: 4.5 Arg: 12.3 Asp: 9.2 Lys: 8.8 Glu: 8.2 Asn: 4.8 Gln: 4.1 His: 3.0 Tyr: 0.7 Pro: 0.2 Ser: -0.6 Gly: -1.0 Thr: -1.2 Ala: -1.6 Trp: -1.9 Cys: -2.0 Val: -2.6 Leu: -2.8 Ile: -3.1 Met: -3.4 Phe: -3.7

  17. Amino-acid classes from evolution/mutation Given a set of (closely) related protein sequences... GSTM1_HUMAN MPMILGYWDIRGLAHAIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLNEKFKLGLD GSTM2_HUMAN MPMTLGYWNIRGLAHSIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLNEKFKLGLD GSTM4_HUMAN MPMILGYWDIRGLAHAIRLLLEYTDSSYEEKKYTMGGAPDYDRSQWLNEKFKLGLD GSTM5_HUMAN MPMTLGYWDIRGLAHAIRLLLEYTDSSYVEKKYTMGDAPDYDRSQWLNEKFKLGLD GTM1_MOUSE MPMILGYWNVRGLTHPIRMLLEYTDSSYDEKRYTMGDAPDFDRSQWLNEKFKLGLD GTM2_MOUSE MPMTLGYWDIRGLAHAIRLLLEYTDTSYEDKKYTMGDAPDYDRSQWLSEKFKLGLD GTM3_MOUSE MPMTLGYWNTRGLTHSIRLLLEYTDSSYEEKRYVMGDAPNFDRSQWLSEKFNLGLD GTM4_MOUSE MSMVLGYWDIRGLAHAIRMLLEFTDTSYEEKRYICGEAPDYDRSQWLDVKFKLDLD GTM3_RABIT MPMTLGYWDVRGLALPIRMLLEYTDTSYEEKKYTMGDAPNYDQSKWLSEKFTLGLD … how often is one amino-acid replaced by another?

  18. Relative mutability of amino acids (Ala=100) Ala: 100.0 Arg: 65.0 Asn: 134.0 Asp: 106.0 Cys: 20.0 Gln: 93.0 Glu: 102.0 Gly: 49.0 His: 66.0 Ile: 96.0 Leu: 40.0 Lys: 56.0 Met: 94.0 Phe: 41.0 Pro: 56.0 Ser: 120.0 Thr: 97.0 Trp: 18.0 Tyr: 41.0 Val: 74.0 Dayhoff M.O., Schwartz R.M., Orcutt B.C.(1978) In "Atlas of Protein Sequence and Structure", Vol.5, Suppl.3

  19. Mutation frequencies after 1% change X 100,000 A 98754 R 30 98974 N 23 19 98720 D 42 8 269 98954 C 11 22 7 2 99432 Q 23 125 35 20 4 98955 E 65 18 36 470 3 198 99055 G 130 99 59 95 43 19 87 99350 H 6 75 89 25 16 136 6 5 98864 I 20 12 25 6 9 5 6 3 9 98729 L 28 35 11 6 21 66 9 6 51 209 99330 K 21 376 153 15 4 170 105 16 27 12 8 99100 M 13 10 7 4 7 10 4 3 8 113 92 15 98818 F 6 2 4 2 31 2 2 2 16 35 99 2 17 99360 P 98 37 8 8 7 83 9 13 58 5 52 11 8 9 99270 S 257 69 342 41 152 37 21 137 50 27 40 32 20 63 194 98556 T 275 37 135 23 25 30 19 20 27 142 15 60 131 7 69 276 98665 W 1 18 1 1 16 3 1 8 1 1 7 1 3 8 1 5 2 99686 Y 3 6 22 15 67 8 2 3 182 10 8 3 6 171 3 20 7 23 99392 V 194 12 11 20 41 13 29 31 8 627 118 9 212 41 15 25 74 17 11 98761 A R N D C Q E G H I L K M F P S T W Y V Jones D.T., Taylor W.R. and Thornton J.M. (1992) CABIOS 8:275-282

  20. The PAM250 matrixPAM: Point Accepted Mutation Cys 12 Ser 0 2 Thr -2 1 3 Pro -1 1 0 6 Ala -2 1 1 1 2 Gly -3 1 0 -1 1 5 Asn -4 1 0 -1 0 0 2 Asp -5 0 0 -1 0 1 2 4 Glu -5 0 0 -1 0 0 1 3 4 Gln -5 -1 -1 0 0 -1 1 2 2 4 His -3 -1 -1 0 -1 -2 2 1 1 3 6 Arg -4 0 -1 0 -2 -3 0 -1 -1 1 2 6 Lys -5 0 0 -1 -1 -2 1 0 0 1 0 3 5 Met -5 -2 -1 -2 -1 -3 -2 -3 -2 -1 -2 0 0 6 Ile -2 -1 0 -2 -1 -3 -2 -2 -2 -2 -2 -2 -2 2 5 Leu -6 -3 -2 -3 -2 -4 -3 -4 -3 -2 -2 -3 -3 4 2 6 Val -2 -1 0 -1 0 -1 -2 -2 -2 -2 -2 -2 -2 2 4 2 4 Phe -4 -3 -3 -5 -4 -5 -4 -6 -5 -5 -2 -4 -5 0 1 2 -1 9 Tyr 0 -3 -3 -5 -3 -5 -2 -4 -4 -4 0 -4 -4 -2 -1 -1 -2 7 10 Trp -8 -2 -5 -6 -6 -7 -4 -7 -7 -5 -3 2 -3 -4 -5 -2 -6 0 0 17 C S T P A G N D E Q H R K M I L V F Y W

  21. Solvent Exposed Area (SEA) The data for this table was calculated from data taken from 55 proteins in the Brookhaven data base, coming from 9 molecular families: globins, immunoglobins, cytochromes c, serine proteases, subtilisins, calcium binding proteins, acid proteases, toxins and virus capsid proteins. Red entries are found on the surface of a proteins on > 70% of occurrences and blue entries are found inside of a protein of < 20% of occurrences. The only clear trend in this table is that some residues, such as R and K, locate themselves so that they have access to the solvent. The so-called hydrophobic residues, such as L and F, show no clear trend: they are found near the solvent as often as they are found buried. Probability that a particular residue will be positioned in real proteins so that its solvent exposed area meets the particular criterion in the columns title. http://www.cmbi.kun.nl/swift/future/aainfo/access.htm

  22. For all amino acids, there are two modes of ionization depending on the pH of the aqueous medium: (1) uncharged at low pH, –1 at high pH (acid), or (2) +1 at low pH, uncharged at high pH (base). From the Henderson-Hasselbalch equation: 90% or 99% of the functional group is deprotonated (or protonated) when the pH is 1 or 2 pH units above (below) the pK. Ionization of Amino Acids in water

  23. pK2=9.6 pK1=2.3 zwitterion (net charge 0) cation anion The ionic properties of amino acids reflect the ionization of the COO–, NH3+, and R-groups When subjected to changes in pH, amino acids change from the protonated form with net positive charge in strongly acidic solution to the unprotonated form with net negative charge in strongly basic solution. During this transition, the amino acid will pass through a state with no net charge. The pH at which this occurs is the isoelectric point or pI. pI can be calculated from pKa values. For zwitteronic and acidic amino acids, pI = 1/2(pK1+pK2). For basic amino acids, pI = 1/2(pK2+pK3).

  24. Ionic characteristics of amino-acids pK2=9.2 pK2=6.0 pK1=1.8 Overall, the aa in solution is positively charged at pH < pI

  25. pKa values of common amino acids

  26. The planar nature of the peptide bond MvHA Fig. 5.8 MvHA Fig. 5.12

  27. Limited rotation around the peptide bond – cis- and trans-proline The 19 amino-acids other than proline strongly prefer (>99.7%) to have the Ca–carbons in the trans- configuration. Proline shows a weaker preference, with about 5% of Xaa-Pro in the cis- configuration. Pro

  28. Classic “Edman” sequencing PTC-conjugation to N-terminal amino-acid Cleave N-terminal peptide bond Identify PTH amino-acid Repeat 20 - 30 cycles Sequencing with Mass-Spectrometry isolate protein (or use mixture of proteins) cleave with trypsin (proteins don’t “fly”) separate on HPLC separate peptides in MS(1) fragment peptides in collision cell separate peptide fragments in MS(2) Strategies for Protein Sequencing (Proteomics)

  29. Protein primary structure can be determined by chemical methods and from gene sequences Edman degradation

  30. Time-of-flight mass spectrometry measures the mass of proteins and peptides Positive ESI-MS m/z spectrum of lysozyme. Most protein analysis done by Electrospray Ionisation (ESI) or Matrix Assisted Laser Desorption Ionisation (MALDI) http://www.healthsystem.virginia.edu/internet/biomolec/

  31. Figure 1 Generic mass spectrometry (MS)-based proteomics experiment. The typical proteomics experiment consists of five stages. In stage 1, the proteins to be analysed are isolated from cell lysate or tissues by biochemical fractionation or affinity selection. This often includes a final step of one-dimensional gel electrophoresis, and defines the 'sub-proteome' to be analysed. MS of whole proteins is less sensitive than peptide MS and the mass of the intact protein by itself is insufficient for identification. Therefore, proteins are degraded enzymatically to peptides in stage 2, usually by trypsin, leading to peptides with C-terminally protonated amino acids, providing an advantage in subsequent peptide sequencing. In stage 3, the peptides are separated by one or more steps of high-pressure liquid chromatography in very fine capillaries and eluted into an electrospray ion source where they are nebulized in small, highly charged droplets. After evaporation, multiply protonated peptides enter the mass spectrometer and, in stage 4, a mass spectrum of the peptides eluting at this time point is taken (MS1 spectrum, or 'normal mass spectrum'). The computer generates a prioritized list of these peptides for fragmentation and a series of tandem mass spectrometric or 'MS/MS' experiments ensues (stage 5). These consist of isolation of a given peptide ion, fragmentation by energetic collision with gas, and recording of the tandem or MS/MS spectrum. The MS and MS/MS spectra are typically acquired for about one second each and stored for matching against protein sequence databases. The outcome of the experiment is the identity of the peptides and therefore the proteins making up the purified protein population. Aebersold R, Mann M. (2003) Nature. 422:198

  32. FIG. 3. Tandem mass (MS/MS) spectra resulting from analysis of a single spot on a 2D gel. The first quadrupole selected a single mass-to-charge ratio ( m/z) of 687.2(A) or 592.6(B), while the collision cell was filled with argon gas, and a voltage which caused the peptide to undergo fragmentation by CID was applied. The third quadrupole scanned the mass range from 50to 1,400m/z. The computer program Sequest (8) was utilized to match MS/MS spectra to amino acid sequence by database searching. Both spectra matched peptides from the same protein, S57593 (yeast hypothetical protein YMR226C). Five other peptides from the same analysis were matched to the same protein. Gygi SP, et al. (1999) Mol Cell Biol. 19:1720

  33. Search human protein (International Protein Index) database 20242509 residues in 65082 sequences FASTS (4.00 July 2001 (ajm)) function [MD20 matrix (18:-29)] ktup: 1 join: 58, gap-pen: -12/-2, width: 16 Scan time: 13.183 The best scores are: initn init1 bits E(65082) IPI00015759.1|SP:Q07244|NP:NP_112552 Het ( 463) 523 218 523 133 7.2e-36 3 46 IPI00063875.1|NP:NP_112553;NP_002131|TR: ( 464) 523 218 523 133 6.2e-36 3 46 IPI00059339.2|XP:XP_062032|ENSENSP000002 ( 482) 330 135 330 67 4.1e-16 3 46 IPI00076129.1|XP:XP_087643 similar to he ( 161) 188 188 188 50 7.7e-11 1 19 >>IPI00015759.1|SP:Q07244|NP:NP_112552 Heterogeneous nuc (463 aa) initn: 523 init1: 218 opt: 523 bits: 132.7 E(): 7.2e-36 Smith-Waterman score: 523; 100.000% identity in 46 aa overlap (1-46:149-396) 10 gi|108 LLIHQSLAGGIIGVK--------------- ::::::::::::::: IPI000 ATSQLPLESDAVECLNYQHYKGSDFDCELRLLIHQSLAGGIIGVKGAKIKELRENTQTTI 120 130 140 150 160 170 20 gi|108 -----------------------------IILDLISESPIK------------------- :::::::::::: IPI000 KLFQECCPHSTDRVVLIGGKPDRVVECIKIILDLISESPIKGRAQPYDPNFYDETYDYGG 180 190 200 210 220 230 30 40 gi|108 -------------------GSYGDLGGPIITTQVTIPK ::::::::::::::::::: IPI000 MAYEPQGGSGYDYSYAGGRGSYGDLGGPIITTQVTIPKDLAGSIIGKGGQRIKQIRHESG 360 370 380 390 400 410

  34. Figure 3 Schematic representation of methods for stable-isotope protein labelling for quantitative proteomics. a, Proteins are labelled metabolically by culturing cells in media that are isotopically enriched (for example, containing 15N salts, or 13C-labelled amino acids) or isotopically depleted. b, Proteins are labelled at specific sites with isotopically encoded reagents. The reagents can also contain affinity tags, allowing for the selective isolation of the labelled peptides after protein digestion. The use of chemistries of different specificity enables selective tagging of classes of proteins containing specific functional groups. c, Proteins are isotopically tagged by means of enzyme-catalysed incorporation of 18O from 18O water during proteolysis. Each peptide generated by the enzymatic reaction carried out in heavy water is labelled at the carboxy terminal. In each case, labelled proteins or peptides are combined, separated and analysed by mass spectrometry and/or tandem mass spectrometry for the purpose of identifying the proteins contained in the sample and determining their relative abundance. The patterns of isotopic mass differences generated by each method are indicated schematically. The mass difference of peptide pairs generated by metabolic labelling is dependent on the amino acid composition of the peptide and is therefore variable. The mass difference generated by enzymatic 18O incorporation is either 4 Da or 2 Da, making quantitation difficult. The mass difference generated by chemical tagging is one or multiple times the mass difference encoded in the reagent used. Aebersold R, Mann M. (2003) Nature. 422:198-207

  35. Correlation between Protein and mRNA Abundance in Yeast – Conclusions • Correlation between mRNA and protein levels insufficient to predict protein expression levels (but good for very abundant proteins) • 20-fold change in protein with little change in mRNA • no change in protein with 30-fold change in mRNA • codon bias does not predict protein or mRNA levels (but abundant proteins have biased codons)

  36. Review questions – • List the 20 amino acids, with their 1-letter and 3-letter abbreviations. • What are some of the most common amino-acids? Least common? • Which amino acids contain hydroxyl groups that can be phosphorylated? (Why is this important?) • Which amino-acids contain aromatic rings? • Which amino-acids are more likely to be on the outside of proteins? On the inside? Why? • Which amino-acid is likely to change its charge state with pH changes within the physiological range (pH 6.5 – 8.0)? Why? • Outline the steps required for MS/MS protein identification • Which MS/MS protein sequencing techniques require a comprehensive protein sequence database?

  37. Questions from previous exams – • Pick an acidic or basic amino-acid. (a) name the amino-acid; (b) draw the charge-structure of the amino-acid for each of the charge-states that it can assume (the actual covalent structure need not be correct, focus on the ionizable groups); (c) suggest an approximate pK for each of the ionizable groups. (d) Indicate the most abundant charge-state at pH 7.0. • The carboxyl group of amino acid alanine has a pKa value of 2.4 . In order to have 99% of the alanine in its COO form, what must the numerical relation be between the pH of the solution and the pKa of the carboxyl group of alanine. • Pick 5 amino acids including some that are more common and some that are less common. Construct a "PAM" amino-acid similarity matrix using those 5 amino acids, using +5 or +3 for identities, +1 for "conserved" amino acids (amino acids with similar properties), and -2 or -5 for non-conservative amino acids.

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