Lecture 5: Peptides
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Quiz available for pickup Jahn 119
Tutoring for biochemistry (tutoring@esf.edu)
More amino acid chemistry
Primary structure of polypeptides
Peptide synthesis
Nomenclature
• Glx can be Glu or Gln
• Asx can be Asp or Asn
• Polypeptide chains are always described from the N-terminus to
the C-terminus
Nomenclature
• Nonhydrogen atoms of the amino acid side chain are named in
sequence with the Greek alphabet
Peptide bonds
•
Proteins are sometimes called polypeptides since they contain many peptide bonds
R1 O
+
H3N
C
OH
C
+
H
H
R2 O
N
C
O-
H
H
+
H3N
C
R1 O
R2 O
C
N
C
H
H
H
C
C
O-
+ H 2O
Structural character of amide groups
• Understanding the chemical character of the amide is
important since the peptide bond is an amide bond.
• These characteristics are true for the amide containing
amino acids as well (Asn, Gln)
• Amides will not ionize but will undergo resonance
-O
O
R
C
NH2
R
C
Resonance forms
NH2
+
Amide has partial charge & double bond
• We can also look at the partial charge and double bond of an amide
as shown below.
• Since the free electrons of the N atom are tied up in forming the
partial double bond, the N atom can not accept a proton (H+).
• This N also has a partial positive charge which will repel protons
and prevent them from binding to the nitrogen (thus no ionization).


O
R
C

NH2
Amide character in the peptide bond
• Since the peptide bond is also an amide it also undergoes
resonance.
+
H3N
R1 O
R2 O
C
N
C
H
H
H
C
C
O-
• Therefore, peptides are rigid due to resonance around the amide
bond, having ≈ 40% double-bond character.
• This restricts the rotation due to delocalization of electrons and
overlap of the O-C-N  orbitals.
Amide character in the peptide bond
• The double bond character results in a planar form around the
peptide bond.
Structural hierarchy in proteins
• Primary structure (1º structure)-for a protein is the
amino acid sequence of its polypeptide chain(s).
• Secondary structure (2º structure)-the local spatial
arrangement of a polypeptide’s backbone atoms without
regard to the conformations of their side chains.
• Tertiary structure (3º structure)-refers to the 3dimensional structure of an entire polypeptide (close to
secondary structure).
• Quaternary structure (4º structure)-The spatial
arrangement of a protein’s subunits
– Most protein is made up of two or more polypeptide chains
(subunits) associated through noncovalent interactions.
Structural hierarchy in proteins
Primary structure (1º structure) of
proteins
• Primary structure (1º structure)-for a protein is the amino acid
sequence of its polypeptide chain(s).
• Amino acid sequence of a protein determines
– three-dimensional conformation.
– Resulting functional specificity (molecular mechanism of action)
• Sequence comparisons among analogous proteins are important in
comparing how proteins function and have indicated evolutionary
relationships among proteins
• Amino acid sequence analyses have important clinical applications
because many diseases are caused by mutations that lead to an
amino acid change in a protein.
• Therefore, amino acid sequence analysis is an important tool for
research.
General approach for the analysis of the
amino acid sequence of a protein
•
•
•
•
•
•
Purify protein to homogeneity
Break disulfide bonds
Determine the aa composition
Identify the N-terminal sequence
Identify the C-terminal sequence
Break the polypeptide into fragments by internal
cleavage (Trypsin, chymotrypsin, pepsin, CNBr).
• Determine the amino acid sequence of each fragment.
• Repeat using different enzymes or CNBr.
• Overlap and align fragments.
Breaking disulfide bonds
•
•
Recall that cysteine (Cys-SH HS-Cys) can convert to cystine (Cys-S-S-Cys)
in the presence of air (oxidation) and will convert back if reduced.
We can also prevent the formation of the disulfide bond by modifying the
SH group of Cys.
+
H3N
H
H
ox.
-OOC
C
+
H3N
Cysteine
CH2 SH
red. OOC
C
+
H3N
CH2 C
CH2 S-S
Cystine
H
COO-
+
H3N
Cysteine reactions
H
2
HS
CH2 CH2 OH
+
-OOC
-mercaptoethanol
C
+
H3N
CH2 C
CH2 S-S
Cystine
H
2 -OOC C
+
H3N
CH2 SH
Cysteine
+
S-CH2-CH2-OH
S-CH2-CH2-OH
H
COO-
Cysteine reactions
HS
CH2-CH-CH-CH2 SH
OH OH
-OOC
+
H3N
CH2 SH
Cysteine
C
+
H3N
H
2 -OOC C
H
+
Dithiothreitol
Dithioerythritol
Cleland’s reagent
+
+
H3N
CH2 C
CH2 S-S
Cystine
HO
S
HO
S
H
COO-
Cysteine reactions
H
ICH2COOIodoacetate
+ -OOC
C
+
H3N
R-group
CH2 SH
Cysteine
H
-OOC
C
+
H3N
CH2 S CH2COO-
+
Carboxymethylcysteine
HI
General approach for the analysis of the
amino acid sequence of a protein
•
•
•
•
•
•
Purify protein to homogeneity
Break disulfide bonds
Determine the aa composition
Identify the N-terminal sequence
Identify the C-terminal sequence
Break the polypeptide into fragments by internal
cleavage (Trypsin, chymotrypsin, pepsin, CNBr).
• Determine the amino acid sequence of each fragment.
• Repeat using different enzymes or CNBr.
• Overlap and align fragments.
N-terminus identification
• Sanger’s reagent - (fluorodintrobenzene) FDNB
• Dansylation - (1-dimethyl-amino-naphthalene-5-sulfonyl
chloride) Dansyl Chloride
• Edman degradation
– Invented by Pehr Edman
– Phenylisothiocyanate (PITC, Edman’s Reagent)
Sanger’s reagent (fluorodintrobenzene) FDNB
O 2N
F
FDNB
The reaction with FDNB is an aromatic
nucleophillic substitution reaction.
O 2N
..
R1 O
R2 O
O
N
C
C
C
C
H N
+
NO2
H H
HF
base
H H
polypeptide
O
O
R
R
1
2
H
O
N
C
C
C
C
N
NO2
H
H H
Sanger’s reagent will also react with other amino groups (epsilon amino group in-lysine). But only one alpha amino group will be
labeled by this reagent. Aromatic amino groups are more stable than the peptide bond.
Reaction with Dansyl Chloride
H3C
N
O
H3C
S
Cl
+
O
Dansyl Chloride
H3C
R2 O
O
N
C
C
C
C
N
H
H H
H H
HCl
base
H3C
N
..
R1 O
polypeptide
O
H R1 O
S
N C C N C C O-
O
H
R2 O
H H
From this we know the N-terminal amino acid and the amino acid composition but not the sequence.
N-terminus identification
• Sanger’s reagent - (fluorodintrobenzene) FDNB
• Dansylation - (1-dimethyl-amino-naphthalene-5-sulfonyl
chloride) Dansyl Chloride
• Edman degradation
– Invented by Pehr Edman
– Phenylisothiocyanate (PITC, Edman’s Reagent)
Amino acid composition of proteins
• Amino acid analysis yields a protein’s amino acid
composition (amounts of each amino acid in the protein).
• Free amino acids can be obtained from proteins by
strong acid hydrolysis:
Protein
6 N HCl
Amino acids
100 ºC, 24 h,
in vacuo
• 3 of the standard aas are lost during acid hydrolysis
treatment:
Asn
Gln
Trp
Asp Amides go
Glu to acids
Decomposed
Edman degradation
I. Condensation
Mild base
II. Cyclization
III. Conversion
H+
Weak acid
Possible to repeat
up to 60 times using
an amino acid
analyzer
Edman degradation
• Allows the determination of the N-terminal residue
identification.
• Also allows us to determine the amino acid sequence of
a polypeptide chain from the N-terminus inward by
subjecting the polypeptide to repeated cycles of the
Edman degradation and after every cycle identifying the
newly liberated PTH-amino acid.
Carboxy terminus identification
• No reliable chemical procedure comparable to Edman
degradation for the sequential end group analysis from
the carboxy terminus of a polypeptide.
• C-termini can be determined by hydrazine cleavage
Exopeptidases cleave the ends of
polypeptides
• Exopeptidases recognize the ends of peptides and can
be used for end group analysis
• Carboxypeptidases are exopeptidases that recognize
the carboxy terminal amino acids.
– Carboxypeptidase A recognizes all aas except Arg/Lys/Pro; Rn-1
cannot be Pro
– Carboxypeptidase B recognizes Arg/Lys; Rn-1 cannot be Pro
• Aminopeptidases are exopeptidases that recognize the
amino terminal amino acids.
• See table 7-1 in your text.
Figure 7-5a The hypothetical rate of the
carboxypeptidase-catalyzed release of amino acids. (a)
All bonds cleaved at the same rate.
Page 165
Figure 7-5b The hypothetical rate of the
carboxypeptidase-catalyzed release of amino acids. (b)
Ser slow, Tyr fast, and Leu intermediate.
Hydrazine cleavage
R1 O
R2 O
N C C N C C O
H H
H H
+
polypeptide
R1 O
NH2-NH2
hydrazine
90 ºC, 20-100 h, in the
presence of mildly acidic ion
exchange resin
Aminoacyl hydrazides
R2 O
+
H3N C C NH-NH2
R0 O +
H
+
H3N C C NH-NH2
H
+
+
H3N
C C OH
Free carboxy terminal
amino acid
1. Amino acids are pre- or
postcolumn derivatized with
dansyl chloride, Edman’s
reagent, or o-phtalaldehyde
(OPA) + 2-mercaptoethanol
form fluorescent adducts.
2. The aas are identified are
identified according to their
retention times on HPLC
3. Amounts of aas present are
determined fluorescent
intensities.
4. Sensitive: can detect less
than 1 pmol of each amino
acid.
OPA-amino acid analysis using reverse-phase HPLC
*note OPA does not react with proline so another reagent must be used
(FMOC)
Specific Peptide Cleavage Reactions
• Polypeptides longer than 40 to 100 residues cannot be
directly sequenced.
• Therefore these larger polypeptides must be cleaved
into smaller fragments that are small enough to be
sequenced.
Endopeptidases cleave polypeptides
internally
• Endopeptidases catalyze the hydrolysis of internal peptide bonds
• Trypsin cleaves specifically after (C-side)positively charged amino
acids; Arg or Lys (basic aas)
• Chymotrypsin cleaves specifically after (C-side) Trp, Phe, Tyr
(aromatic aas) and slowly at Leu, Met, Asn, His.
• Pepsin cleaves before (N-side) Trp, Phe, Tyr, Met, Leu and all
others under acidic conditions.
• Thermolysin cleaves before (N-side) Leu, Ile, Phe, Trp, Tyr, Val
and sometimes for all others.
• There are others.
• See table 7-2 in your text.
Methionine and CNBr-Internal Cleavages
CH3
CH3
Cyanogen bromide
S:
C
CH2
H
CH
N
N
CH2
Br
CH2
O N
+S C
BrO
C
CH2
O N CH
H
O
C
N CH C O
N CH C O
H R2
H R2
O
O
Methionine and CNBr-Internal Cleavages
Methyl thiocyanate
Peptidyl homoserine lactone
CH3
S C
CH2
N
H2O
+
CH2
O N CH
H
O N CH
CH2
O
C
H
CH2
O
O
+
C
N CH C O
H R2
O
Aminoacyl peptide
+
H3N CH C O
R2
O
Figure 7-7
The amino acid sequence of a polypeptide chain.
To make trypsin even more versatile you
can modify side chains of amino acids
Lys specific reaction to hide basicity
See p. 170 in your book-especially the reactions with
citraconic anhydride so trypsin won’t cleave at Lys
residues.
Also on p. 170 conversion of Cys side group with 2bromoethylamine to make a basic group to cleave at
Cys with trypsin.
Lysine reactions
H
O
+
R’ C
H
-OOC
+
CH
CH
CH
NH
C CH2
2
2
2
3
+
H3N
Lysine
aldehyde
H
-OOC
O
C CH2 CH2 CH2 CH2 N C
+
H3N
+
H
Schiff base
H2O
+
H+
Lysine reactions
O
H2C C
O
H
+
-OOC
+
CH
CH
CH
NH
C CH2
2
2
2
3
+
H3N
H2C C
Lysine
O
Succinic
anhydride
H
C CH2 CH2 CH2 CH2 N C CH2 CH2 C
+
H3N
O
O
-OOC
O-
+ 2H+
Determining primary structure of
polypeptides
Deduce the amino acid sequence of a simple polypeptide
from the following results:
A. Acid hydrolysis: (Ala2, Arg, Lys2, Met, Phe, Ser2)
B. Carboxypeptidase A: (Ala)
C. Trypsin: (Ala,Arg), (Lys,Phe,Ser), (Lys), (Ala, Met, Ser)
D. CNBr: (Ala, Arg, Lys2, Met, Phe, Ser), (Ala, Ser)
E. Thermolysin: (Ala, Arg, Ser), (Ala, Lys2, Met, Phe, Ser)
Where do we start?
First, from A. (acid hydrolysis) we know how many amino
acids are in the polypeptide: 9
Second from B. (carboxypeptidase A), we know the last
amino acid is one of the Ala.
1 - 2 - 3 - 4 - 5 - 6 - 7 - 8 -Ala
We know trypsin cleaves at the carboxy side of basic aas
(Lys and Arg)
Trypsin: (Ala,Arg), (Lys,Phe,Ser), (Lys), (Ala, Met, Ser),
so we can rearrange the amino acids as follows:
Ala-Arg, either Phe-Ser-Lys or Ser-Phe-Lys, Arg-Lys or
Lys-Lys, and either Lys-(Ala, Met, Ser) or Arg-(Ala,
Met, Ser).
For CNBr, we got two fragments (Ala, Arg, Lys2, Met,
Phe, Ser) and (Ala, Ser). We know that cleavage
occurs on the carboxy side of Met. So we know that
Met-(Ser-Ala) or Met-(Ala-Ser).
1 - 2 - 3 - 4 - 5 - 6 - 7 - 8 -Ala
For thermolysin, we know it cleaves N-terminal to Ile,
Met, Phe, Trp, Tyr, Val. So (Ala, Arg, Ser) are before
Met
From trypsin: Ala-Arg, Phe-Ser-Lys or Ser-Phe-Lys, ArgLys or Lys-Lys, and either Lys-(Ala, Met, Ser) or Arg(Ala, Met, Ser).
WE know that one Ala is the carboxy terminal amino acid,
so Ala-Arg cannot be the carboxy terminus. Therefore,
the only other possibility is the last sequence (Ala, Met,
Ser) where Ala is the carboxy terminal amino acid. So
the order at the carboxy terminus is basic aa-Met-SerAla or basic aa-Ser-Met-Ala
For CNBr, we know that cleavage occurs on the carboxy
side of Met. So, combined with the trypsin result we
get basic aa-Met (Ser-Ala).
1 - 2 - 3 - 4 - 5 - basic aa - Met - Ser -Ala
For thermolysin, we know it cleaves N-terminal to Ile,
Met, Phe, Trp, Tyr, Val. So (Ala, Arg, Ser) are before
Met or Phe.
We know from the CNBr cleavage that the Met must be
before Ser-Ala, so for the (Ala, Lys2, Met, Phe, Ser)
Phe must be the 1st aa in this sequence. We also
know that a basic aa precedes Met from the trypsin
experiment. Since the only basic aas in this fragment
are Lys, the order must be : Phe-Lys-Lys-Met-Ser-Ala
1 - 2 - 3 - Phe - Lys - Lys - Met - Ser -Ala
Remember for thermolysin, we know it cleaves N-terminal
to Ile, Met, Phe, Trp, Tyr, Val. So (Ala, Arg, Ser) are
before Met or Phe.
We know from the trypsin digest that Ala-Arg are in a
specified order so the final sequence must be Ala-ArgSer
Ala - Arg - Ser - Phe - Lys - Lys - Met - Ser -Ala