chapter 3: amino acids, peptides, and proteins
DESCRIPTION
Chapter 3: Amino Acids, Peptides, and Proteins. Dr. Rita P.-Y. Chen ( 陳佩燁 ) Assistant Research Fellow Institute of Biological Chemistry Academia Sinica. Amino acid. Chiral center Optically active: rotate plane-polarized light Amino acid in protein: L stereoisomer. - PowerPoint PPT PresentationTRANSCRIPT
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Dr. Rita P.-Y. Chen (陳佩燁 )Assistant Research Fellow
Institute of Biological Chemistry
Academia Sinica
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Amino acid
•Chiral center•Optically active: rotate plane-polarized light•Amino acid in protein: L stereoisomer
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Absolute configuration: D,L systemAbsolute configuration: D,L system
Not all L-amino acids are levorotatory (rotating polarized light to the left)
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Specifying onfiguration: RS system L-amino acid has S configuration
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aliphatic amino acid structuresaliphatic amino acid structures
CH CH3
CH2
CH3
Isoleucine (I) (Ile)
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Side Chains with Alcohol GroupsSide Chains with Alcohol Groups• Serine (Ser, S) and Threonine (Thr, T) have
uncharged polar side chains
Catalytic role, phosphorylation, o-linked glycosylation, hydrogen bond
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Structures of aspartate, glutamate, Structures of aspartate, glutamate, asparagine and glutamineasparagine and glutamine
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Structures of histidine, lysine and Structures of histidine, lysine and argininearginine
-Guanido groupimidazole group
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Methionine and cysteineMethionine and cysteine
First a.a. Catalytic role, disulfide bond
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Fig 3.4 Formation of cystineFig 3.4 Formation of cystine
Disulfide bond
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Aromatic amino acid structuresAromatic amino acid structures
phosphorylation
Indole group
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Proline has a nitrogen in the Proline has a nitrogen in the aliphatic ring systemaliphatic ring system
• Proline (Pro, P) - has a three carbon side chain bonded to the -amino nitrogen
• The heterocyclic pyrrolidine ring restricts the geometry of polypeptides
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Uncommon a.a.Uncommon a.a.
Plant cell wallCollagen
Collagen
myosin
ProthrombinCa2+ binding protein
elastin
21th a.a.Added during protein synthesisUGA codonglutathione peroxidases
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Other amino acids (not constituents Other amino acids (not constituents of proteins) : metaboliteof proteins) : metabolite
Key intermediate in biosynthesis of Arg and in urea cycle
p843
Urea
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補充教材補充教材
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Amino acid can act as acids and basesAmino acid can act as acids and bases
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較易解離
較易解離
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The characteristic pH at which the net electric charge is zero is called the isoelectric point or isoelectric pH, designated pI.For glycine, which has no ionizable group in its sidechain, the isoelectric point is simply the arithmetic meanof the two pKa values:
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pI = (2.19+4.25)/2 = 3.22
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pI = (6+9.17)/2 = 7.59
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Peptides and ProteinsPeptides and Proteins
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SGYAL
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+2 +1 0 -1
2.34
9.6
4.25
10.53
pI = (4.25+9.6)/2 =6.93
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• Artificial Sweetener, ex. in Diet Coke• 200 times sweeter than sugar• D-form a.a. substitution is bitter
• 苯酮尿症(Phenylketonuria) 患者不可使用 , use Alatame instead
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36Fig. 3-13, p.75
催產素
血管收縮素
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GlutathioneGlutathione
• an important water-phase antioxidant and essential cofactor for antioxidant enzymes
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Protein size is variedProtein size is varied
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Polypeptides have Polypeptides have characteristic amino characteristic amino acid compositionsacid compositions
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Some proteins have chemical groups Some proteins have chemical groups other than a.a.other than a.a.
• Non a.a. part – prosthetic group
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Protein purificationProtein purification
• Fractionation: protein solubility depends on temperature, pH, salt
• Dialysis• Ultrafiltration: N2 purge, centrifugation• Column chromatography• Electrophoresis
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Column chromatographyColumn chromatography
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• Cation exchange chromatography:
• Protein carries postive charge (cation)
• Buffer pH must be lower than protein pI
• No sample volume limit
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• Size exclusion chromatography
• Big protein runs faster
• Sample volume is limited
• Column is usually long
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• Affinity chromatography
• Separate proteins by their binding specificities
• No sample volume limit
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Electrophoresis: (1) SDS-PAGEElectrophoresis: (1) SDS-PAGE
• Proteins migrate according to their size and shape
• One SDS bind for every two residues
• Protein is denatured, subunits will be separated
cathode
Anode
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Coomassive blue staining
4 subunits
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Electrophoresis: (2) Isoelectric focusingElectrophoresis: (2) Isoelectric focusing
• Determine protein pI
• Use ampholytes to create get with pH gradient
• Proteins stop migration when pH = pI
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Two-dimensional electrophoresisTwo-dimensional electrophoresis
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Protein sequencingProtein sequencing
• Protein function depends on its sequence• 20 -30 % proteins are polymorphic• Most proteins contain crucial regions that
are essential to their function and whose sequence is therefore conserved.
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1953, Frederick Sanger worked out the sequence of insulin
1958 Nobel Prize in Chemistry1980 Nobel Prize in Chemistry: DNA sequencing
b. 1918
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Only identify the first a.a.
Pehr Edman:Edman degradation
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• Breaking disulfide bond• Cleaving the polypeptide chain• Sequencing of peptides• Ordering peptide fragments• Locating disulfide bond
Large proteins must be sequenced in Large proteins must be sequenced in smaller segmentsmaller segment
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Breaking disulfide bond
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Cleaving the polypeptide chainCleaving the polypeptide chain
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Locating disulfide bondLocating disulfide bond
• Do the same thing except breaking disulfide bond
• See which peptide fragments are missing or which peptide fragment (longer) appears
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Other methods to solve protein Other methods to solve protein sequencesequence
• Translate from DNA:Genome, proteome• Mass spectrometry
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The Nobel Prize in Chemistry 1984 --for his development of methodology
for chemical synthesis on a solid matrix
Robert Bruce MerrifieldRockefeller University
1921-2006
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Homologous proteinsHomologous proteins
• Paralog: homologous genes within a single species that diverged by gene duplication.
• Ortholog: genes in different species that derive from a common ancestor. Orthologous genes may or may not have the same function.
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Molecular evolutionMolecular evolution• 1960s Zuckerkandle and Pauling use nucleotide and
protein sequence to explore evolution• 1970s Carl Woese used ribosomal RNA sequence
(archaebacteria is different from other bacteria)• Not every protein is a good target (choose protein with
essential function ex. cellular metabolism EF-1)• Lateral gene transfer ex. Antibiotic-resistent gene• At some position, only particular amino substitutions can
be tolerated• Electronic search, multiple sequence alignment• Gap, penalty
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Determining how closely related the proteins are – Blosum (blocks substitution matrix)
•Based on short conserved blocks•Unique chemical properties – higher score•Each substitution has a score, based on its frequency
Blosum 62 table(62% identity)
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Signature sequence – useful Signature sequence – useful sequence segment in taxonomysequence segment in taxonomy
• Sequence in signature sequence might be quite distinct
EF-1/EF-Tu family
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Evolutionary treeEvolutionary tree
• Length of line is proportional to the number of a.a. substitution• From the sum of length, we can know how close two species are• From different proteins, we can obtain different evolutionary trees
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