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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, [email protected] Snyder 380 (Fontaine) Major topics – • Names, abbreviations, general structure of amino acids • Amino acid chemical classes (polar, hydrophobic, acidic, basic, aromatic, Scontaining) • Amino acid structural classes/affinity • Amino acid evolutionary classes • pK - Henderson-Hasselbach equation • Structure of the peptide bond • Proteomics – MS protein sequencing 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 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? 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 QuickTime™ and a decompressor are needed to see this picture. QuickTime™ and a decompressor are needed to see this picture. QuickTime™ and a decompressor are needed to see this picture. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. Currently 10,340 protein fold families in Pfam [http://pfam.sanger.ac.uk/] QuickTi me™ and a decompressor are needed to see thi s pi ctur e. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. Pfam is a comprehensive collection of protein domains and families, represented as multiple sequence alignments and as profile hidden Markov models. Generally does not include membrane proteins. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. What defines the 3-dimensional fold of a protein? QuickTi me™ and a decompressor are needed to see thi s pi ctur e. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. QuickTi me™ and a decompressor are needed to see thi s pi ctur e. Structure and properties of Amino-acids Alanine Arginine Asparagine Aspartic acid Cysteine Glutamine Glutamic acid Glycine Histidine Isoleucine Ala Arg Asn Asp Cys Gln Glu Gly His Ile A R N D C Q E G H I Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Leu Lys Met Phe Pro Ser Thr Trp Tyr Val L K M F P S T W Y V Asp/Asn Asx B Glu/Gln Glx Z Amino-acid Chirality L-glyceraldehyde D-glyceraldehyde Amino-acid Chirality the “CORN” rule R R H CO Ca O N H C O N Figure 5.3: The amino acids found in proteins. CYCLIC AMINO ACID AROMATIC AMINO ACIDS Leucine a b H a Alanine b R R H a CO N CO H N Proline - a cyclic amino-acid b R Pro R H a CO N N CO b R R Ala H a CO N CO N Classifications of amino-acids • • • • Abundance Hydrophobicity Mutability Structural preference • Charge properties us.expasy.org/cgi-bin/protscale.pl Molecular weight Bulkiness Polarity / Grantham Recognition factors Hphob. OMH / Sweet et al. Hphob. / Kyte & Doolittle Hphob. / Abraham & Leo Hphob. / Bull & Breese Hphob. / Guy Hphob. / Miyazawa et al. Hphob. / Roseman Hphob. / Welling & al al Hphob. HPLC / Parker & al Cowan Hphob. HPLC pH7.5 / Cowan HPLC / HFBA retention HPLC / retention pH 2.1 % buried residues Hphob. / Chothia Ratio hetero end/side Average flexibility Fasman beta-sheet / Chou & Fasman alpha-helix / Deleage & Roux Roux beta-turn / Deleage & Roux alpha-helix / Levitt beta-turn / Levitt Antiparallel beta-strand A.A. composition Relative mutability Number of codon(s) Polarity / Zimmerman Refractivity Hphob. / Eisenberg et al. Hphob. / Hopp & Woods Hphob. / Manavalan et al. Hphob. / Black Hphob. / Fauchere et al. Hphob. / Janin Hphob. / Rao & Argos Hphob. / Wolfenden et al. Hphob. HPLC / Wilson & Hphob. HPLC pH3.4 / Hphob. / Rf mobility HPLC / TFA retention HPLC / retention pH 7.4 % accessible residues Hphob. / Rose & al Average area buried alpha-helix / Chou & beta-turn / Chou & Fasman beta-sheet / Deleage & Coil / Deleage & Roux beta-sheet / Levitt Total beta-strand Parallel beta-strand A.A. comp. in Swiss-Prot Amino acid frequencies in proteins + Ala Arg Asn Asp - Cys Gln + Glu + Gly - His Ile + Leu Lys - Met - Phe Pro + Ser Thr - Trp - Tyr + Val A R N D C Q E G H I L K M F P S T W Y V 0.0780 0.0512 0.0448 0.0536 0.0192 0.0426 0.0629 0.0737 0.0219 0.0514 0.0901 0.0574 0.0224 0.0385 0.0520 0.0711 0.0584 0.0132 0.0321 0.0644 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 Arg: Lys: Asp: Glu: Ser: Gln: Asn: Pro: Gly: Thr: His: Ala: Cys: Met: Val: Leu: Ile: Tyr: Phe: Trp: 3.0 3.0 3.0 3.0 0.3 0.2 0.2 0.0 0.0 -0.4 -0.5 -0.5 -1.0 -1.3 -1.5 -1.8 -1.8 -2.3 -2.5 -3.4 Kyte/ Doolittle Arg: Lys: Asp: Glu: Gln: Asn: His: Pro: Tyr: Trp: Ser: Thr: Gly: Ala: Met: Cys: Phe: Leu: Val: Ile: -4.5 -3.9 -3.5 -3.5 -3.5 -3.5 -3.2 -1.6 -1.3 -0.9 -0.8 -0.7 -0.4 1.8 1.9 2.5 2.8 3.8 4.2 4.5 GES Arg: Asp: Lys: Glu: Asn: Gln: His: Tyr: Pro: Ser: Gly: Thr: Ala: Trp: Cys: Val: Leu: Ile: Met: Phe: 12.3 9.2 8.8 8.2 4.8 4.1 3.0 0.7 0.2 -0.6 -1.0 -1.2 -1.6 -1.9 -2.0 -2.6 -2.8 -3.1 -3.4 -3.7 Amino-acid classes from evolution/mutation Given a set of (closely) related protein sequences... GSTM1_HUMAN GSTM2_HUMAN GSTM4_HUMAN GSTM5_HUMAN GTM1_MOUSE GTM2_MOUSE GTM3_MOUSE GTM4_MOUSE GTM3_RABIT MPMILGYWDIRGLAHAIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLNEKFKLGLD MPMTLGYWNIRGLAHSIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLNEKFKLGLD MPMILGYWDIRGLAHAIRLLLEYTDSSYEEKKYTMGGAPDYDRSQWLNEKFKLGLD MPMTLGYWDIRGLAHAIRLLLEYTDSSYVEKKYTMGDAPDYDRSQWLNEKFKLGLD MPMILGYWNVRGLTHPIRMLLEYTDSSYDEKRYTMGDAPDFDRSQWLNEKFKLGLD MPMTLGYWDIRGLAHAIRLLLEYTDTSYEDKKYTMGDAPDYDRSQWLSEKFKLGLD MPMTLGYWNTRGLTHSIRLLLEYTDSSYEEKRYVMGDAPNFDRSQWLSEKFNLGLD MSMVLGYWDIRGLAHAIRMLLEFTDTSYEEKRYICGEAPDYDRSQWLDVKFKLDLD MPMTLGYWDVRGLALPIRMLLEYTDTSYEEKKYTMGDAPNYDQSKWLSEKFTLGLD … how often is one amino-acid replaced by another? Relative mutability of amino acids (Ala=100) Ala: 100.0 Arg: Asn: Asp: Cys: Gln: Glu: Gly: His: Ile: Leu: Lys: Met: Phe: Pro: Ser: Thr: Trp: Tyr: Val: 65.0 134.0 106.0 20.0 93.0 102.0 49.0 66.0 96.0 40.0 56.0 94.0 41.0 56.0 120.0 97.0 18.0 41.0 74.0 Dayhoff M.O., Schwartz R.M., Orcutt B.C.(1978) In "Atlas of Protein Sequence and Structure", Vol.5, Suppl.3 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 The PAM250 matrix PAM: Point Accepted Mutation Cys Ser Thr Pro Ala Gly Asn Asp Glu Gln His Arg Lys Met Ile Leu Val Phe Tyr Trp 12 0 -2 -1 -2 -3 -4 -5 -5 -5 -3 -4 -5 -5 -2 -6 -2 -4 0 -8 C 2 1 1 1 1 1 0 0 -1 -1 0 0 -2 -1 -3 -1 -3 -3 -2 S 3 0 1 0 0 0 0 -1 -1 -1 0 -1 0 -2 0 -3 -3 -5 T 6 1 -1 -1 -1 -1 0 0 0 -1 -2 -2 -3 -1 -5 -5 -6 P 2 1 0 0 0 0 -1 -2 -1 -1 -1 -2 0 -4 -3 -6 A 5 0 1 0 -1 -2 -3 -2 -3 -3 -4 -1 -5 -5 -7 G 2 2 1 1 2 0 1 -2 -2 -3 -2 -4 -2 -4 N 4 3 2 1 -1 0 -3 -2 -4 -2 -6 -4 -7 D 4 2 1 -1 0 -2 -2 -3 -2 -5 -4 -7 E 4 3 1 1 -1 -2 -2 -2 -5 -4 -5 Q 6 2 0 -2 -2 -2 -2 -2 0 -3 H 6 3 0 -2 -3 -2 -4 -4 2 R 5 0 6 -2 2 5 -3 4 2 6 -2 2 4 2 4 -5 0 1 2 -1 -4 -2 -1 -1 -2 -3 -4 -5 -2 -6 K M I L V 9 7 10 0 0 17 F Y W 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. > 30 A 2 < 10 A 2 30 > SEA > 10 A2 S 0.70 0.20 0.10 T 0.71 0.16 0.13 A 0.48 0.35 0.17 G 0.51 0.36 0.13 P 0.78 0.13 0.09 C 0.32 0.54 0.14 D 0.81 0.09 0.10 E 0.93 0.04 0.03 Q 0.81 0.10 0.09 N 0.82 0.10 0.08 L 0.41 0.49 0.10 I 0.39 0.47 0.14 V 0.40 0.50 0.10 M 0.44 0.20 0.36 F 0.42 0.42 0.16 Y 0.67 0.20 0.13 W 0.49 0.44 0.07 K 0.93 0.02 0.05 R 0.84 0.05 0.11 H 0.66 0.19 0.15 http://www.cmbi.kun.nl/swift/future/aainfo/access.htm Ionization of Amino Acids in water 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: [base ] pH pK a log [acid] [base ] log pK a pH [acid] [base ] 10 pKa pH [acid] 90% or 99% of the functional group is deprotonated (or protonated) when the pH is 1 or 2 pH units above (below) the pK. 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). pK2=9.6 pK1=2.3 cation zwitterion (net charge 0) anion Ionic characteristics of amino-acids A pK2=9.2 Zw pK2=6.0 pK1=1.8 C22 C1 pH=7.4 pK2=6.0 pH=7.4 pK2=7.0 pH=6.8 pK2=6.0 [C1 ] 3.8% 28.2% 13.6% [Zw] 94.7% 70.4% 86.0% 1 1 pI ( pK 2 pK 3 ) (6.0 9.2) 7.6 2 2 Overall, the aa in solution is positively charged at pH < pI pKa values of common amino acids Amino Acid Alanine Arginine Asparagine Aspartic Acid Cysteine Glutamic Acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine a-COOH pKa 2.4 2.2 2.0 2.1 1.7 2.2 2.2 2.3 1.8 2.4 2.4 2.2 2.3 1.8 2.1 2.2 2.4 2.4 2.2 2.3 a-NH3+ pKa 9.7 9.0 8.8 9.8 10.8 9.7 9.1 9.6 9.2 9.7 9.6 9.0 9.2 9.1 10.6 9.2 10.4 9.4 9.1 9.6 R group pKa 12.5 3.9 8.3 4.3 6.0 10.5 ~13 ~13 10.1 The planar nature of the peptide bond MvHA Fig. 5.8 MvHA Fig. 5.12 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 Strategies for Protein Sequencing (Proteomics) • Classic “Edman” sequencing – PTC-conjugation to Nterminal amino-acid – Cleave N-terminal peptide bond – Identify PTH amino-acid – Repeat 20 - 30 cycles • Sequencing with MassSpectrometry – 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) Protein primary structure can be determined by chemical methods and from gene sequences Edman degradation 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/ 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 onedimensional 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 highpressure 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 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 50 to 1,400 m/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 Gygi matched SP, et al. (1999) Biol. 19:1720 were toMol theCell same protein. 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: IPI00015759.1|SP:Q07244|NP:NP_112552 Het IPI00063875.1|NP:NP_112553;NP_002131|TR: IPI00059339.2|XP:XP_062032|ENSENSP000002 IPI00076129.1|XP:XP_087643 similar to he ( ( ( ( 463) 464) 482) 161) initn 523 523 330 188 init1 bits E(65082) 218 523 133 7.2e-36 218 523 133 6.2e-36 135 330 67 4.1e-16 188 188 50 7.7e-11 3 3 3 1 >>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 46 46 46 19 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 enzymecatalysed 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 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) Review questions – 1. List the 20 amino acids, with their 1-letter and 3letter abbreviations. 2. What are some of the most common amino-acids? Least common? 3. Which amino acids contain hydroxyl groups that can be phosphorylated? (Why is this important?) 4. Which amino-acids contain aromatic rings? 5. Which amino-acids are more likely to be on the outside of proteins? On the inside? Why? 6. Which amino-acid is likely to change its charge state with pH changes within the physiological range (pH 6.5 – 8.0)? Why? 7. Outline the steps required for MS/MS protein identification 8. Which MS/MS protein sequencing techniques require a comprehensive protein sequence database? Questions from previous exams – 1. 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. 2. 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. 3. 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.