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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

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

較易解離

較易解離

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:

0

pI = (2.19+4.25)/2 = 3.22

0

pI = (6+9.17)/2 = 7.59

Peptides and ProteinsPeptides and Proteins

SGYAL

+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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催產素

血管收縮素

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GlutathioneGlutathione

• an important water-phase antioxidant and essential cofactor for antioxidant enzymes

Protein size is variedProtein size is varied

Polypeptides have Polypeptides have characteristic amino characteristic amino acid compositionsacid compositions

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

Column chromatographyColumn chromatography

• Cation exchange chromatography:

• Protein carries postive charge (cation)

• Buffer pH must be lower than protein pI

• No sample volume limit

• Size exclusion chromatography

• Big protein runs faster

• Sample volume is limited

• Column is usually long

• Affinity chromatography

• Separate proteins by their binding specificities

• No sample volume limit

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

Coomassive blue staining

4 subunits

Electrophoresis: (2) Isoelectric focusingElectrophoresis: (2) Isoelectric focusing

• Determine protein pI

• Use ampholytes to create get with pH gradient

• Proteins stop migration when pH = pI

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.

1953, Frederick Sanger worked out the sequence of insulin

1958 Nobel Prize in Chemistry1980 Nobel Prize in Chemistry: DNA sequencing

b. 1918

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

Breaking disulfide bond

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

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

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)

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

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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