Bioch/BIMS 503 Lecture 1 Structure and Properties of
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, S-containing) 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 super-secondary
structure -helix -strand ternary fold 4-helix bundles Rossman fold-meander Greek key ternary structure Does this structural hierarchy reflect the folding process? Secondary structure first, or last? 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.
QuickTime and a QuickTime and a QuickTime and a QuickTime and a QuickTime and a decompressor decompressor decompressor decompressor decompressor are needed to see thisare picture. needed to see thisare picture. needed to see thisare picture. needed to see thisare picture. needed to see this Currently 10,340 protein fold families in Pfam [http://pfam.sanger.ac.uk/] QuickTime and a decompressor are needed to see this picture. 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. QuickTime and a decompressor are needed to see this picture.
QuickTime and a decompressor are needed to see this QuickTime and a decompressor are needed to see this What defines the 3-dimensional fold of a protein? QuickTime and a QuickTime and a QuickTime and a QuickTime and a QuickTime and a decompressor decompressor decompressor decompressor decompressor are needed to see thisare picture. needed to see thisare picture. needed to see thisare picture. needed to see thisare picture. needed to see this Structure and properties of Amino-acids Alanine Ala Arginine Arg
Asparagine Asn Aspartic acid Phenylalanine Cysteine Cys Glutamine Gln Glutamic acid Thr Glycine Gly Histidine His Isoleucine Ile A R N Asp Phe C Q Glu T G H I Leucine Lysine Methionine D F Proline Serine E Tryptophan Trp Tyrosine Tyr Valine Val W Y V
Asp/Asn B Glu/Gln Z Asx Leu Lys Met L K M Pro P Ser S Threonine Glx Amino-acid Chirality L-glyceraldehyde Dglyceraldehyd e Amino-acid Chirality the CORN rule R R H CO C O N
C O H N Figure 5.3: The amino acids found in proteins. CYCLIC AMINO ACID AROMATIC AMINO ACIDS Leucine H Alanine R R H CO N CO H N Proline - a cyclic aminoacid R
R Pro H N CO N CO R R Ala H CO N CO N Classifications of aminoacids
Abundance Hydrophobicity Mutability Structural preference Charge properties 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 .expasy.org/cgi-bin/protscale.pl 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. 78:38243828. 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
-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. > 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 The only clear trend in this table is I 0.39 0.47 that some residues, such as R and K, V 0.40 0.50 locate themselves so that they have M
0.44 0.20 access to the solvent. The so-called F 0.42 0.42 hydrophobic residues, such as L and F, Y 0.67 0.20 show no clear trend: they are found near the solvent as often as they are found W 0.49 0.44 buried. Probability that a particular residue K 0.93 0.02 will be positioned in real proteins so R 0.84 0.05 that its solvent exposed area meets the H 0.66 0.19 particular criterion in the columns title. http://www.cmbi.kun.nl/swift/future/aainfo/access.htm 0.14 0.10 0.36 0.16 0.13 0.07 0.05 0.11 0.15 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 [basepH ] (base). pH = pK a + log [acid] From the Henderson-Hasselbalch [base ] equation: log [acid] = pK a pH [base ] =10 pK a 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+, andsubjected R-groups When 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 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+ ] [Zw] + C1 pH=7.4
pK2=6. 0 pH=7.4 pK2=7. 0 pH=6.8 pK2=6. 0 3.8% 28.2% 13.6% 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 pKa values of common amino acids Amino Acid -COOH pKa -NH3+ pKa Alanine Arginine Asparagine
1.8 9.1 9.6 9.2 9.7 9.6 9.0 9.2 9.1 2.1 2.2 2.4 2.4 2.2 10.6 9.2 10.4 9.4 9.1 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 transproline The 19 amino-acids other than proline strongly prefer (>99.7%) to have the Ccarbons in the trans- configuration. Proline shows a weaker preference, with about 5% of Xaa-Pro in the cisconfiguration. Pro Strategies for Protein Sequencing (Proteomics) Classic Edman sequencing PTC-conjugation to N-terminal aminoacid 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 dont 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 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 Aebersold M. (2003) spectrum'). R, The Mann computer generates 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 Gygi SP, et al. (1999) Mol Cell peptides from the same Biol. 19:1720 analysis were matched to the 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
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 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 Aebersold R, Mann M. (2003) Nature. 422:198207 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 3-letter 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 aminoacid; (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.
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