Transcript Protein carries postive charge (cation)

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 planepolarized light
•Amino acid in protein: L
stereoisomer
Absolute configuration: D,L system
Not all L-amino acids are levorotatory (rotating polarized light to the left)
Specifying onfiguration: RS system
L-amino acid has S configuration
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aliphatic amino acid structures
CH
Isoleucine (I)
(Ile)
CH3
CH2
CH3
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Side 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,
asparagine and glutamine
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Structures of histidine, lysine and
arginine
imidazole group
d-Guanido group
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Methionine and cysteine
First a.a.
Catalytic role, disulfide bond
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Fig 3.4 Formation of cystine
Disulfide bond
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Aromatic amino acid structures
Indole group
phosphorylation
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Proline has a nitrogen in the
aliphatic ring system
• Proline (Pro, P) - has a three
carbon side chain bonded to
the a-amino nitrogen
• The heterocyclic pyrrolidine
ring restricts the geometry of
polypeptides
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p
t
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Uncommon a.a.
Prothrombin
Ca2+ binding protein
Plant cell wall
Collagen
Collagen
elastin
myosin
21th a.a.
Added during protein synthesis
UGA codon
glutathione peroxidases
Other amino acids (not constituents
of proteins) : metabolite
Key intermediate in
biosynthesis of Arg and
in urea cycle
Urea
p843
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補充教材
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Amino acid can act as acids and bases
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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 side
chain, the isoelectric point is
simply the arithmetic mean
of the two pKa values:
0
pI = (2.19+4.25)/2 = 3.22
0
pI = (6+9.17)/2 = 7.59
Peptides and Proteins
SGYAL
9.6
4.25
+2
+1
0
-1
pI = (4.25+9.6)/2 =6.93
10.53
2.34
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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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Fig. 3-13, p.75
Glutathione
• an important water-phase antioxidant and
essential cofactor for antioxidant enzymes
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Protein size is varied
Polypeptides have
characteristic amino
acid compositions
Some proteins have chemical groups
other than a.a.
• Non a.a. part – prosthetic group
Protein purification
• Fractionation: protein solubility depends on
temperature, pH, salt
• Dialysis
• Ultrafiltration: N2 purge, centrifugation
• Column chromatography
• Electrophoresis
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Column 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-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 focusing
• Determine
protein pI
• Use ampholytes
to create get
with pH
gradient
• Proteins stop
migration when
pH = pI
Two-dimensional electrophoresis
Protein 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 Chemistry
1980 Nobel Prize in Chemistry:
DNA sequencing
Only identify the first a.a.
Pehr Edman:
Edman degradation
Large proteins must be sequenced in
smaller segment
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Breaking disulfide bond
Cleaving the polypeptide chain
Sequencing of peptides
Ordering peptide fragments
Locating disulfide bond
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Breaking disulfide bond
Cleaving the polypeptide chain
Locating 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
sequence
• 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 Merrifield
Rockefeller University
1921-2006
Homologous 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 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-1a)
• 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)
Blosum 62 table
(62% identity)
•Based on short conserved blocks
•Unique chemical properties – higher score
•Each substitution has a score, based on its frequency
Signature sequence – useful
sequence segment in taxonomy
EF-1a/EF-Tu family
• Sequence in signature sequence might be
quite distinct
Evolutionary 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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