Download 8/18/2015 1 BCMB 3100

8/18/2015
Chapter 3 (with parts of 4 and 5)
Amino Acids and Primary Structures of Proteins
Pink: make up ~ 97% mass of organisms
Purple: five essential ions
Light blue: most common trace elements
Dark blue: less common trace elements
BCMB 3100 - Chapter 3 (part1)
• Diversity of protein function
• Complete definition of amino acids
• Memorize complete structure of 20 common
amino acids!!!
• pKa’s of  amino and  carboxyl groups
• Amino acids with ionizable side groups
• Titration curve
PROTEIN STRUCTURE AND FUNCTION
Mulder (1800s):_______________________
__________________
Berzelius (1838): ____________________
from Greek proteios "of first rank"
Proteins recognize and _______ many
different types of molecules & ________
most of the chemical ___________
necessary for life.
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Examples of Protein Function
Examples of Protein Function
Enzymatic catalysis:
enzymes increase reaction rates by
__________; nearly all known
enzymes are ____________
Mechanical support: _________(tensile
strength to skin and bones)
Transport & storage:
many small molecules and ions are
transported by _________;
examples: _____________________
Generation & transmission of nerve
impulses: example: ____________________
Coordinated motion:
examples: myosin and actin (muscle
movement)
Immune protection: ____________
Control of growth and differentiation:
___________________
(repressors,
activators, transcription factors,
hormones, regulation of translation)
WHAT ARE PROTEINS?
Proteins are macromolecules made up of
_________________
(20 amino acids)
_______________: consist of an __________,
a _______________, a _______________ and a
distinctive _________bonded to a carbon
atom. This carbon is called the __________
because it is adjacent to the carboxyl
group.
BCMB 3100 - Lecture 3
• Diversity of protein function
• Complete definition of amino acids
• Memorize complete structure of 20
common amino acids!!!
• pKa’s of  amino and  carboxyal groups
• Amino acids with ionizable side groups
• Titration curves & pI
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Amino acids in solution will be in a
_____________. The amino group and/or
the carboxyl group will be charged
depending upon the pH. The R group may
also be charged.
At neutral pH amino acids are
predominantly _______________________
• Under normal cellular conditions amino
acids are _____________ (dipolar ions):
Amino group =
-NH3+
Carboxyl group =
COOH → COOCOO
NH3+  C  H

R
 COO-: pKa 1.8-2.5
 NH+: pKa 8.7-10.7
Fischer
Projection
?
You MUST
know this!!!
-COO-
Two representations of an amino acid at neutral pH
(a) Structure
General Structure of the ionized from of an amino acid
______________________ (asymmetric) due
to the tetrahedral array of 4 different
groups around the -carbon (glycine is an
exception).
Thus all amino acids except glycine can
exist as ______________ : two __________
that are nonsuperimposable mirror images
of each other.
(b) Ball-and stick model
Enantiomers of amino acids are called D
(right-handed) or L (left-handed)
L and D refer to absolute configuration
L-amino acids are the only constituents of
_____________
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Mirror-image pairs of amino acids
20 different amino acids are found in proteins
___________________ (side chains) that differ
in size, shape, charge, hydrogen-bonding
capacity & chemical reactivity  20 different
amino acids found in proteins of all organisms
from bacteria to humans
The amino acid alphabet is at least __________
years old
The diversity of protein structure & function
is due to the sequence and number of amino
acids found in a protein (__________________)
It is essential that a biochemist commit to
memory the structure of the 20 amino acids!
Know this!! (structure, & 1 & 3 letter code)
Structures of the 20 Common Amino Acids
Page 38
Page 40
Four _________ amino acid structures
• You must learn the one and three letter
abbreviations
• You must know the properties of their side
chains (R groups)
• Classes: Aliphatic, Aromatic, Sulfur-containing,
Alcohols, Bases, Acids and Amides
• You must be able to draw the structure of the
20 amino acids in their correct ionized form at
any given pH
-C not chiral
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Stereoisomers of Isoleucine
Isoleucine [I] (Ile)
Page 38
Page 40
Aliphatic amino acid
Proline has a nitrogen in
the aliphatic ring system
Page 38
Page 40
• Ile has 2 chiral carbons, 4 possible stereoisomers
***
• Proline (Pro, P) - has a three
carbon side chain bonded to
the -amino nitrogen
• The heterocyclic pyrrolidine
ring restricts the geometry of
polypeptides
[I]
(Ile)
____________ R Groups
Aromatic amino acid structures
Page 38
Page 40
• Side chains have aromatic groups
Phenylalanine (Phe, F) - benzene ring (OD 260 nm)
Tyrosine (Tyr, Y) - phenol ring (OD 280 nm)
pKa = 10.5
Tryptophan (Trp, W) - bicyclic indole group (OD 280 nm)
OD at 280 nm is important for identifying
proteins during protein purification.
benzene ring
(OD 260 nm)
phenol ring
(OD 280 nm)
indole group
(OD 280 nm)
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Methionine and Cysteine
________________ R Groups
Page 38
Page 40
Page 39
Page 41
• Methionine (Met, M) - (-CH2CH2SCH3)
• Cysteine (Cys, C) - (-CH2SH)
pKa = 8.4
• Two cysteine side chains can be cross-linked by
forming a disulfide bridge (-CH2-S-S-CH2-)
• Disulfide bridges may stabilize the threedimensional structures of proteins
Formation of cystine
Side Chains with _______________
Serine (Ser, S) and Threonine (Thr, T) have
uncharged polar side chains
Page 39
Page 41
Two Cys side chains can be cross-linked by forming a
disulfide bridge (-CH2-S-S-CH2-); disulfide bridges may
stabilize 3D protein structure
Oxidation: loss of an electron
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________ R Groups
• Histidine (His, H) - imidazole
Structures of histidine, lysine and arginine
Page 40
Page 42
pKa = 6.0
• Lysine (Lys, K) - alkylamino group
• Arginine (Arg, R) - guanidino group
pKa = 10.5
• Side chains are nitrogenous bases which are
substantially positively charged at pH 7 (true
for K & R)
______ R Groups and ______ Derivatives
imidazole
pKa = 12.5
alkylamino
group
guanidino
group
Structures of aspartate, glutamate,
asparagine and glutamine
Page 41
Page 43
• Aspartate (Asp, D) and Glutamate (Glu, E)
are dicarboxylic acids, and are negatively
charged at pH 7
• Asparagine (Asn, N) and Glutamine (Gln, Q)
are uncharged but highly polar
pKa = 3.9
pKa = 4.1
dicarboxylic acids
uncharged but highly polar
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_______________ of Amino Acid Side Chains
• ___________ : the relative hydrophobicity of
each amino acid
• The larger the __________, the greater the
tendency of an amino acid to prefer a
hydrophobic environment
___________scale for
amino acid residues
(Free-energy change for transfer of an
amino acid from interior of a lipid
bilayer to water)
The larger the hydropathy, the greater
the tendency of an amino acid to
prefer a hydrophobic environment
• Hydropathy affects protein folding:
* hydrophobic side chains tend to be in the _____
* hydrophilic residues tend to be on the ________
BCMB 3100 - Lecture 3
• Diversity of protein function
• Complete definition of amino acids
• Memorize complete structure of 20 common
amino acids!!!
• pKa’s of  amino and  carboxyal groups
• Amino acids with ionizable side groups
• Titration curves & pI
Hydropathy: the relative
hydrophobicity of each amino
acid
Amino
acid
Free-energy change
for transfer (kjmol-1)
Highly hydrophobic
Isoleucine
Phenylalanine
Valine
Leucine
Methionine
Less hydrophobic
Tryptophan
Alanine
Glycine
Cysteine
Tyrosine
Proline
Threonine
Serine
Highly hydrophilic
Histidine
Glutamate
Asparagine
Glutamine
Apartate
Lysine
Arginine
3.1
2.5
2.3
2.2
1.1
1.5*
1.0
0.67
0.17
0.08
-0.29
-0.75
-1.1
-1.7
-2.6
-2.7
-2.9
-3.0
-4.6
-7.5
Structures of the 20 Common Amino Acids
• You must learn the one and three letter abbreviations
• You must know the properties of their side chains (R
groups)
• Classes: Aliphatic, Aromatic, Sulfurcontaining, Alcohols, Bases, Acids and
Amides
• You must be able to draw the structure of the 20
amino acids in their correct ionized form at any given
pH
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Figure 3.2, Page 37/39
Amino acids in solution will be in a charged state. The
-___________ and/or the -___________ will be
charged depending upon the pH. The _________ may
also be charged. At neutral pH amino acids are
predominantly dipolar ions (_____________).
( COOH ↔ COO- + H+ )
(NH3+ ↔ NH2 + H+ )
NH3+
COO
CH

R
Note:
pH dependance
of the ionization
state
 COO-: pKa 1.8-2.5
 NH+: pKa 8.7-10.7
Titration curve for alanine
Ionization of Amino Acids
• Ionizable groups in amino acids: (1) -carboxyl,
(2) -amino, (3) some side chains
• Titration curves
are used to
determine pKa
values
• Each ionizable group has a specific pKa
• pK1 = 2.4
AH
B + H+
• pK2 = 9.9
• For a solution pH below the pKa, the protonated form
predominates (AH)
• pIAla = isoelectric
point
• For a solution pH above the pKa, the unprotonated
(conjugate base) form predominates (B)
(pI = pH when net
charge is zero)
pI = (2.4+9.9) / 2 = 6.15
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pKa values of amino acids
All amino acids contain two ionizable groups:
-carboxyl (pKa 1.8-2.5)
and protonated -amino group (pKa 8.7-10.7)
(Table 3.1, pg 41 gives some specific pKa values)
BUT FOR THIS COURSE use pKa’s from the notes.
The R group of 7 amino acids are also ionizable. The
pKa of these R groups can range from 3.9 to 12.5. You
must memorize the pKa’s of the side chains of these
seven amino acids!
α
pKa values of
amino acid
ionizable groups
BCMB 3100 - Lecture 3
• Diversity of protein function
• Complete definition of amino acids
• Memorize complete structure of 20
common amino acids!!!
• pKa’s of  amino and  carboxyal groups
• Amino acids with ionizable side groups
• Titration curves & pI
α
Table 3.1
pKa values of
amino acid
ionizable groups
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Ionization of Histidine
Deprotonation of imidazolium ring
(a) Titration curve
of histidine
pK1 = 1.8
pK2 = 6.0
pK3 = 9.3
pKa=1.8
pKa=9.3
pI = pH at which
net charge is zero
To determine the pI: pick the
pKa above & below the point
where the charge is zero and
average those 2 pKa’s
Ionization of Histidine
Ionization of the protonated -carboxyl of glutamate
(a) Titration curve
of histidine
pK1 = 1.8
pK2 = 6.0
pK3 = 9.3
pI = pH at which
net charge is zero
pI = (6 + 9.3)/2 =7.65
To determine the pI: pick
the pKa above & below
point where charge is zero
and average those 2 pKa’s
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Deprotonation of the guanidinium group of Arg
BCMB 3100 – Chapter 3 (part 2)
The 20 amino acid are divided into 7 groups
• Summary of amino acids
• Polypeptides: definition, structure, and direction
• Peptide bond
• Disulfide bonds
• Protein purification
• Methods to determine amino acid composition,
cleavage of proteins, protein sequencing
• Diversity of proteins
•_________: Gly, Ala, Val, Leu, Ile, Pro (G A V L I P)
•_________: Phe, Tyr, Trp (F Y W)
• ____________________: Met, Cys (M C)
• __________: Ser, Thr (S T)
• ________: His, Lys, Arg (H K R)
• ________: Asp, Glu (D E)
•_________: Asn, Gln (N Q)
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Formation of a dipeptide
Amino acids are linked by ___________ to form
polypeptide chains
 carboxyl of one amino acid is joined to -amino group
of another amino acid by a peptide bond (amide bond)
with concomitant loss of H2O
Many amino acids are joined by peptide bonds to form
an ____________ polypeptide
H2O
NH3+ 
H O
 
C  C  O
R
+
NH3+ 
H O
 
C  C  O
R
NH3+ 
H O
H O
 
 
C  C  N  C  C  O


R
H R
_________ : amino acid unit in a polypeptide
By convention the direction of a polypeptide is written
starting with amino end
H2 O
H2 O
H2 O
aa + aa + aa + aa  NH3+-aa-aa-aa-aa-COO-
Figure 4.1
Peptide bond between two amino acids
Most polypeptides contain between 50 and 2000 amino
acids
Average M.W. for an amino acid is 110 daltons so M.W.
of most proteins is 5500 to __________ daltons. (One
dalton equals one atomic mass unit; kilodalton = 1000
daltons). Most proteins have M.W. of 5.5-220 kd.
Some proteins contain disulfide bonds that cross-link
between cysteine residues by the oxidation of cysteine.
Intracellular proteins often lack disulfides while
extracellular proteins often have them.
Formation of peptide bonds eliminates the ionizable
-carboxyl and  -amino groups of the free amino acids
except for those at the amino and carboxy termini
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Figure 4.2
Challenge of the Week
to be given out on 8-25-08
Work with your group of 4 people and find at least one
example of a mutation in humans, or in industryrelevant plants, animals or microbes. Present, on a single,
one-side, typed page, the amino acid mutated, the phenotype of
the effect on the organism, the molecular reason that the
mutation causes the effect(s), and the effect that this mutation
has on/for humans.
Hand in a single, one-sided, typed sheet of paper with ALL
group members names (first and last name correctly spelled) as
well as a single sentence behind each name describing their
contribution to the answer. These will be collected ONLY at
the Breakout Session on September 1.
Aspartame, an artificial sweetener
Aspartame, an artificial sweetener
• Aspartame is a
dipeptide methyl ester
(aspartylphenylalanine
methyl ester)
• Aspartame is a
dipeptide methyl ester
(aspartylphenylalanine
methyl ester)
• About 200 times
sweeter than table
sugar
• About 200 times
sweeter than table
sugar
• Used in diet drinks
• Used in diet drinks
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Cleaving, blocking disulfide bonds
-mercaptoethanol
Figure 4.5
See also
Figure 4.4
Figure 4.5 Amino acid sequence of bovine insulin.
This mature processed form of insulin exemplifies
intra-peptide disulfide bonds (within the same chain) and
inter-peptide disulfide bonds (between chains)
PROTEIN PURIFICATION
General strategy
Tissue  disrupt  crude fractionation  selected fractionation
To purify large amounts of proteins one requires:
Proteins can be separated by:
_____________ : gel electrophoresis, gel filtration
chromatography, dialysis, centrifugation
____________: salting out
_________: ion-exchange chromatography, isoelectric focusing
_________________: hydrophobic interaction chromatography,
reverse phase chromatography
_______________: affinity chromatography, antibodies
2. A __________to separate desired protein from "all" other
proteins and to keep the protein "active“ during the process
Purified proteins can be analyzed by:
____________: Edman degradation, (proteolysis), Mass Spectrometry
____________: X-ray crystallography, NMR
___________: automated solid phase
Dialysis often used to remove salts: separation of protein
from small molecules through a semipermeable membrane
(cellulose)
1. An _______ for the protein (enzyme, antibody, etc.)
3. Example strategy:
Salting-out: the "specific" precipitation of a given protein
at a specific high-salt concentration
Ion exchange  Gel-filtration  Affinity chromatography
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Figure 5.3
Different types of chromatography (1)
_____________________: proteins passed over a column filled
with a hydrated porous beads made of a carbohydrate or
polyacrylamide polymer (large molecules exit (elute) first)
Gel-filtration chromatography
Fig 5.4
Different types of chromatography (2)
__________________: separation of proteins over a column
filled with charged polymer beads (+ charged beads = anionexchange chromatography; - charged beads = cation
exchange chromatography.) Positively charged proteins bind
to beads of negative charge & vice versa. Bound proteins are
eluted with salt. Non-charged proteins and proteins of similar
charge to resin will elute first.
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Fig 5.5
Ion-exchange chromatography:
Different types of chromatography (3)
_________________________: proteins are passed through a
column of beads containing a covalently bound high affinity
group for the protein of interest. Bound protein is eluted by
free high affinity group.
Affinity chromatography:
Fig 5.6
Different types of chromatography (4)
______________________________________of protein:
hydrophobic interaction chromatography (HIC) and reversephase chromatography (RPC) are both based on interactions
between hydrophobic patches on the surface of a protein and
hydrophobicity of ligands (e.g. alkyl groups) covalently
attached to a gel matrix. In RPC, proteins can bind very
strongly to the gel and require non-polar solvents for their
elution. In HIC protein binding is promoted by inclusion of
salt in the solvent and elution of proteins is caused by
decreasing or removing salt from the solvent.
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Column Chromatography
_________________: movement of charged solutes through a
gel in response to an electric field
_____________________________________________:
chemically inert; polymerized acrylamide matrix of
controlled pore size; allows separation of proteins based on
mass and charge
(a) Separation of a
protein mixture
_______________: (sodium dodecyl sulfate, page 73): anionic
detergent used for polyacrylamide gel electrophoresis. It
complexes with proteins (1 SDS/2 amino acids)  denatured
protein of negative charge proportional to protein mass.
Note: reducing agents (mercaptoethanol, dithiothreitol) are
also added to reduce disulfide bonds. Mobility of many
proteins under these conditions is linearly proportional to
the log of their mass. Smaller proteins migrate faster.
(b) Detection of
eluting protein
peaks
Polyacrylamide gel electrophoresis (PAGE):
Fig 5.8
(a) SDS-PAGE Electrophoresis
(b) Protein banding pattern after run
Comassie Blue
or
Silver staining
SDS
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Isoelectric focusing:
Fig 5.10
Proteins can also be separated by electrophoresis
based on their native charge.
_______ (isoelectric point): pH at which net
protein charge is zero
__________________: electrophoresis of proteins
(w/o SDS) in a pH gradient to a position in the gel
at which pH = pI. pH gradient formed by
polyampholytes (small multi-charged polymers of
many pIs).
Combined SDS-PAGE and
Isoelectric focusing: 2D-PAGE
Fig 5.11
Purification Table
Fig 5.13
(see also Fig. 5.9)
In western blotting or immunoblotting, proteins are separated in an SDS‐PAGE gel, transferred to a polymer, and then stained with a fluorescent antibody.
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Fig 5.23
Amino Acid Composition (1)
1. Determine amino acid composition
a. Peptide
hydrolysis

6 N HCl
100°C, 24 hr
free amino acids
b. Amino acid composition of hydrolysates determined
by automated cation-exchange chromatography
(amino acid analyzer). Column contains solid
granules of sulfonated polystyrene. Amino acids
reacted with ninhydrin (yields colored product) &
detected by O.D.
Acid-catalyzed hydrolysis of a peptide
Amino Acid Composition (2)
c. Another method for aa composition analysis is to treat
protein hydrolysate with phenylisothiocyanate (PITC) at
pH 9.0 to yield PITC-aa derivatives, separate by HPLC
via hydrophobic attraction of aa side chains to
hydrocarbon matrix of column and quantitate by OD
254 nm (due to PTC moiety).
(The first pure protein analyzed for a.a. composition was
-lactoglobulin. It took several years of work. Today
Problem: Asn → Asp; Asn + Asp = Asx or B
Gln → Glu; Gln + Glu = Glx or Z
Loose some: Ser, Thr, Tyr;
Loose most Trp
amino acid analyzers allows composition
analysis within 2-4 hours with samples as small
as 1 pmole!!!)
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Amino acid treated with PITC
Chromatogram from HPLC-separated PTCamino acids
phenylisothiocyanate
Note: acid treatment converts Asn→Asp; Gln→Glu; Trp is destroyed,
some loss of Ser, Thr, Tyr.
B = (Asn→Asp) + Asp;
Fig 5.25
Z = (Gln→Glu) + Glu
2. Determine amino-terminal residue
_________ devised the first method to label Nterminal residue by reacting 2,4dinitrophenylbenzene with -amino group 
yellow derivative. Subsequent hydrolysis (6N HCl)
hydrolyzes away all other amino acids. N-terminal
residue derivative identified by chromatography
Today Dabsyl chloride (colored derivative) or
Dansyl chloride ( fluorescent derivative) are used.
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3. Determination of amino acid sequence by
Edman Degradation (________________) (1950)
Edman Degradation is now done on sequenators
(automated, liquid phase sequenator   analysis by
HPLC (one cycle 2 hr)
Edman degradation procedure
Phenylisothiocyanate
(Edman reagent)
pH = 9.0
Gas-phase sequenator: detection of pmole amounts (single
SDS-PAGE band)
Frederick Sanger determined the first complete sequence
of a protein (insulin) in 1953 (51 amino acid long)
Phenylthiocarbamoyl-peptide
Note: Amino acid sequence of small proteins and peptides
is now commonly determined by mass spectrometry
(e.g. electrospray MS, MALDI MS).
Edman degradation procedure (cont)
F3CCOOH
Edman degradation procedure (cont)
Polypeptide chain
with n-1 amino acids
Aqueous acid
Polypeptide chain
with n-1 amino acids
Aqueous acid
Returned to alkaline conditions
for reaction with additional
phenylisothiocyanate in the
next cycle of Edman degradation
Returned to alkaline conditions
for reaction with additional
phenylisothiocyanate in the
next cycle of Edman degradation
PTH-
Submit remaining
polypeptide to another
round of Edman
Degradation
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Edman Degradation Summarized
Fig 5.26
Protein Cleavage
Edman degradation limited to polypeptides of 50 a.a.
____________________:
Cyanogen bromide (CNBr): splits on carbonyl side of Met
_____________________:
________: protease cleaves on carbonyl side of Arg and Lys
_____________: protease cleaves on carbonyl side of bulky
hydrophobic and aromatic amino acids
Protein cleavage by BrCN
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Cleavage, sequencing an oligopeptide
______________must be removed from proteins by
reduction & alkylation before sequencing.
Reducing agent
(iodoacetate)
DTT
ICH2COOR-S-S-R  R-SH HS-R   
R-S-CH2-COODNA recombinant technology  DNA sequence of
nascent protein
Limitation of the amino acid sequence only from DNA
code: it is only the sequence of the nascent protein.
See also Fig 5.27
_______________: direct polypeptide product of
translation (no modification)
Cleaving, blocking disulfide bonds
human
& chimp
Cyt c are
identical
•Protein amino
acid sequences
can be deduced
from the sequence
of nucleotides in
the corresponding
gene
•Closely related species contain proteins
with very similar amino acid sequences
•Differences reflect evolutionary change
from a common ancestral protein
sequence
Phylogenetic tree for Cytochrome c
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Polypeptide chain nomenclature
• Amino acid “residues” compose peptide chains
• Peptide chains are numbered from the N (amino)
terminus to the C (carboxyl) terminus
• Example: (N) Gly-Arg-Phe-Ala-Lys (C)
(or GRFAK)
• Formation of peptide bonds eliminates the
ionizable -carboxyl and -amino groups of the
free amino acids
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