蛋白質體學 Proteomics 2015 Amino acids, Peptides and Proteins 陳威戎 2015. 09. 14 & 21
Outline 1. Amino Acids 2. Peptides and Proteins 3. Covalent Structure of Proteins
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
Amino acids share common structural features
Two conventions used to identify the carbons in an amino acid The α-carbon atom is a chiral center.
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
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
Absorption of ultraviolet light by aromatic amino acids
Reversible formation of the disulfide bond
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
Uncommon amino acids also have important functions
Uncommon amino acids also have important functions
Hydride, Hydrogen and Proton hydride - 1s - - Proton + + + - - Hydrogen atom 1 H 1.008 Juang RH (2007) BCbasics
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
Amino acids can act as acids and bases
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 ~ 2 5.5 +1 0-1 Isoelectric point Juang RH (2007) BCbasics
Amino Acids Have Buffering Effect ph 12 9 pk 2 6 3 NH 2 H + H-C-R Isoelectric point = COO - pk 1 + pk 2 2 pi pk 1 0 [OH] Juang RH (2007) BCbasics
Environment ph vs Protein Charge Isoelectric point, pi + Buffer ph 10 9 8 7 6 5 4 3 0 - Net Charge of a Protein - Juang RH (2007) BCbasics
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
pka of Amino Acids Amino acids -COOH -NH 2 -R Gly G 2.34 9.60 Ala A 2.34 9.69 Val V 2.32 9.62 Leu L 2.36 9.68 Ile I 2.36 9.68 Ser S 2.21 9.15 Thr T 2.63 10.4 Met M 2.28 9.21 Phe F 1.83 9.13 Trp W 2.38 9.39 Asn N 2.02 8.80 Gln Q 2.17 9.13 Pro P 1.99 10.6 Asp D 2.09 9.82 3.86 Glu E 2.19 9.67 4.25 His H 1.82 9.17 6.0 Cys C 1.71 10.8 8.33 Tyr Y 2.20 9.11 10.07 Lys K 2.18 8.95 10.53 Arg R 2.17 9.04 12.48 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
H first HOOC-CH 2 -C-COOH NH 3 + second H HOOC-CH 2 -C-COO - NH 3 + +1 pk 1 = 2.1 0 pk 2 = 3.9 Aspartic acid Isoelectric point is the average of the two pka flanking the zero net-charged form 2.1 + 3.9 2 = 3.0 Isoelectric point H - OOC-CH 2 -C-COO - NH 3 + third -1 pk 3 = 9.8 pk 3 pk 2 0-2 -1 H - OOC-CH 2 -C-COO - NH 2-2 pk 1 +1 [OH] Juang RH (2004) BCbasics
Titration curves predict the charge of amino acids Isoelectric point, Isoelectric ph, pi
Amino acids differ in their acid-base properties
Amino acids differ in their acid-base properties
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.
Peptides are chains of amino acids Formation of a peptide bond by condensation
Peptides are chains of amino acids The pentapeptide serylglycyltyrosylalanylleucine, or Ser-Gly-Tyr-Ala-Leu
Biologically active peptides and polypeptides occur in a vast range of sizes
Peptides have characteristic amino acid compositions
Some proteins contain chemical groups other than amino acids
There are several levels of protein structure
Proteins can be separated and purified Crude extract Fractionation Ammonium sulfate (salting out) Dialysis Column chromatography
Proteins can be separated and characterized by electrophoresis
The amino acid sequences of millions of proteins have been determined Frederick Sanger
Short polypeptides are sequenced using automated procedures
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
Breaking disulfide bonds in proteins
Cleaving the polypeptide chain: proteases
Cleaving proteins and sequencing and ordering the peptide fragments
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.
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: