蛋白質體學. Proteomics Amino acids, Peptides and Proteins 陳威戎 & 21

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1 蛋白質體學 Proteomics 2015 Amino acids, Peptides and Proteins 陳威戎 & 21

2 Outline 1. Amino Acids 2. Peptides and Proteins 3. Covalent Structure of Proteins

3 Amino Acids Proteins are polymers of amino acids, with each amino acid residues joined to its neighbor by a specific covalent bond. Twenty different amino acids are commonly found in proteins. First: asparagine (1806) ; last: threonine (1938). Names derived from the sources: Asparagine asparagus Glutamate wheat gluten Tyrosine cheese Glycine sweet taste

4 Amino acids share common structural features

5 Two conventions used to identify the carbons in an amino acid The α-carbon atom is a chiral center.

6 L-Form Amino Acid Structure Carboxylic group COO - Amino group + H N 3 a H R group H = Glycine CH 3 = Alanine Juang RH (2007) BCbasics

7 Juang RH (2007) BCbasics = Basic -C-C-C-C-NH 3 + Central line Non-polar Polar Northwest line -C-C-C-N-C-N -C-C C N N + N + Arg R Lys K -H -CH 3 -C-OH -C- His H Gly G -C-C OH Chung-San line -C- Ser S Thr T -OH Hydroxy Aromatic Trp W Tyr Phe F Ala Y A South line -C- N Cys C Met M Sulfur -C-CONH 2 Asn N Asp D -C-COOH Val C C -C -C-SH Amino Acid Subway Map V -C-C-S-C Nan-Kan line -C-C-CONH 2 Gln Q Glu E -C-C-COOH Ile C -C-C-C Circular line Pro I P Amide Acidic Aliphatic Leu L C -C-C-C C C C HN C-COOH a Imino, Circular

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10 Absorption of ultraviolet light by aromatic amino acids

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12 Reversible formation of the disulfide bond

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15 Classification of Amino Acids by Polarity NON- POLAR POLAR Acidic Neutral Basic Asp Glu Ala Val Tyr Ile Asn Ser Cys Leu Gln Gly Thr His Arg Lys Met Phe Trp Pro Polar or non-polar, it is the bases of the amino acid properties. Juang RH (2007) Biochemistry

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18 Uncommon amino acids also have important functions

19 Uncommon amino acids also have important functions

20 Hydride, Hydrogen and Proton hydride - 1s - - Proton Hydrogen atom 1 H Juang RH (2007) BCbasics

21 Proton Is Adsorbed or Desorbed Proton: abundant and small, affects the charge of a molecule Amino lone pair electrons N H H High pka H + Low H + N H H Carboxylic C O O H Low pka High C O O H + Ampholyte contains both positive and negative groups on its molecule Juang RH (2007) BCbasics

22 Amino acids can act as acids and bases

23 Acidic environment Neutral environment Alkaline environment pk 2 ~ 9 NH 2 H + R - C - H COOH NH 2 H + R - C - H COO - NH 2 R - C - H COO - pk 1 ~ Isoelectric point Juang RH (2007) BCbasics

24 Amino Acids Have Buffering Effect ph 12 9 pk NH 2 H + H-C-R Isoelectric point = COO - pk 1 + pk 2 2 pi pk 1 0 [OH] Juang RH (2007) BCbasics

25 Environment ph vs Protein Charge Isoelectric point, pi + Buffer ph Net Charge of a Protein - Juang RH (2007) BCbasics

26 pka of Amino Acid Residues Residues on amino acids can release or accept protons a R His Cys Tyr a R a R -COOH -COO - -COOH -COO - His -Imidazole H + -Imidazole -SH Cys -S - -OH -O - Tyr -NH + 3 -NH 2 -NH + 3 R -NH 2 a + H + + H + + H + + H + + H + + H + + H + pka = 1.8~2.4 pka = 3.9~4.3 pka = 6.0 pka = 8.3 pka = 10 pka = 8.8~11 pka = 10~12.5 Smaller pka releases proton easier Only His has the residue with a neutral pka (imidazole) pka of a carboxylic or amino groups is lower than pka of the R residues Juang RH (2007) BCbasics

27 pka of Amino Acids Amino acids -COOH -NH 2 -R Gly G Ala A Val V Leu L Ile I Ser S Thr T Met M Phe F Trp W Asn N Gln Q Pro P Asp D Glu E His H Cys C Tyr Y Lys K Arg R ph pk 2 pk 1 pk 3 pk 2 pk 1 two pka three pka? pi pk 1 + pk 2 2? pi? [OH - ] Juang RH (2004) BCbasics

28 H first HOOC-CH 2 -C-COOH NH 3 + second H HOOC-CH 2 -C-COO - NH pk 1 = pk 2 = 3.9 Aspartic acid Isoelectric point is the average of the two pka flanking the zero net-charged form = 3.0 Isoelectric point H - OOC-CH 2 -C-COO - NH 3 + third -1 pk 3 = 9.8 pk 3 pk H - OOC-CH 2 -C-COO - NH 2-2 pk 1 +1 [OH] Juang RH (2004) BCbasics

29 Titration curves predict the charge of amino acids Isoelectric point, Isoelectric ph, pi

30 Amino acids differ in their acid-base properties

31 Amino acids differ in their acid-base properties

32 Quiz 1: Net electric charge and pi of a peptide A peptide has the sequence : Glu-His-Trp-Ser-Gly-Leu-Arg-Pro-Gly 1. What is the net charge of this peptide at ph 3, 8, and 11? 2. Estimate the pi for this peptide.

33 Peptides are chains of amino acids Formation of a peptide bond by condensation

34 Peptides are chains of amino acids The pentapeptide serylglycyltyrosylalanylleucine, or Ser-Gly-Tyr-Ala-Leu

35 Biologically active peptides and polypeptides occur in a vast range of sizes

36 Peptides have characteristic amino acid compositions

37 Some proteins contain chemical groups other than amino acids

38 There are several levels of protein structure

39 Proteins can be separated and purified Crude extract Fractionation Ammonium sulfate (salting out) Dialysis Column chromatography

40 Proteins can be separated and characterized by electrophoresis

41 The amino acid sequences of millions of proteins have been determined Frederick Sanger

42 Short polypeptides are sequenced using automated procedures

43 Large proteins must be sequenced in smaller segments 1. Breaking disulfide bonds 2. Cleaving the polypeptide chain: proteases 3. Sequencing of peptides 4. Ordering peptide fragments 5. Locating disulfide bonds

44 Breaking disulfide bonds in proteins

45 Cleaving the polypeptide chain: proteases

46 Cleaving proteins and sequencing and ordering the peptide fragments

47 Amino acid sequences can also be deduced by other methods 1. New methods based on mass spectrometry permit sequencing of short polypeptides (20-30 a.a.) in just a few minutes. 2. Development of rapid DNA sequencing methods.

48 Quiz 2: Sequence determination of a nonapeptide 1. A nonapeptide was determined to have the following amino acid composition: (Lys) 2, (Gly) 2, (Phe) 2, His, Leu, Met. 2. The native peptide was incubated with 1-fluoro-2,4-dinitrobenzene (FDNB) and then hydrolyzed; 2,4-dinitrophenylhistidine was identified by HPLC. 3. When the native peptide was exposed to cyanogen bromide (CNBr), an octapeptide and free glycine were recovered. 4. Incubation of the native peptide with trypsin gave a pentapeptide, a tripeptide, and free Lys. 2,4-Dinitrophenyl-histidine was recovered from the pentapeptide, and 2,4-dinitrophenylphenylalanine was recovered from the tripeptide. 5. Digestion with the enzyme pepsin produced a dipeptide, a tripeptide, and a tetrapeptide. The tetrapeptide was composed of (Lys) 2, Phe, and Gly. The native sequence was determined to be: