Download Primary Structure Determination (Sanger)

Primary Structure Determination
(Sanger)
The primary structure is the amino acid sequence plus any disulfide links.
• 1. Determine what amino acids are present
and
their molar ratios.
• 2. Cleave the peptide into smaller fragments,
and determine the amino acid composition of
these smaller fragments.
• 3. Identify the N-terminus and C-terminus in
the
parent peptide and in each fragment.
• 4. Organize the information so that the
sequences of small fragments can be
overlapped to reveal the full sequence.
Amino Acid Analysis
• Acid-hydrolysis of the peptide (6 M HCl, 24
hr) gives a mixture of amino acids.
• The mixture is separated by ion-exchange
chromatography, which depends on the
differences in pI among the various amino
acids.
• Amino acids are detected using ninhydrin.
• Automated method; requires only 10-5 to
10-7 g of peptide.
Partial Hydrolysis of Peptides and
Proteins
• Cleaving some, but not all, of the peptide
bonds gives smaller fragments. These smaller
fragments are then separated and the amino
acids present in each fragment determined.
• Enzyme-catalyzed cleavage is the preferred
method for partial hydrolysis.
• The enzymes that catalyze the hydrolysis of
peptide bonds are called peptidases,
proteases, or proteolytic enzymes.
Trypsin
Trypsin is selective for cleaving the peptide bond
to the carboxyl group of lysine or arginine.
O
O
O
NHCHC
NHCHC
R
R'
lysine or arginine
NHCHC
R"
Chymotrypsin
Chymotrypsin is selective for cleaving the peptide
bond to the carboxyl group of amino acids with
an aromatic side chain.
O
O
O
NHCHC
NHCHC
R
R'
phenylalanine, tyrosine, tryptophan
NHCHC
R"
Carboxypeptidase
Carboxypeptidase is selective for cleaving
the peptide bond to the C-terminal amino acid.
O
O
+
H3NCHC
R
O
–
protein
C
NHCHCO
R
End Group Analysis
• Amino sequence is ambiguous unless we know
whether to read it left-to-right or right-to-left.
• We need to know what the N-terminal and Cterminal amino acids are.
• The C-terminal amino acid can be determined
by carboxypeptidase-catalyzed hydrolysis.
• Several chemical methods have been
developed for identifying the N-terminus.
They depend on the fact that the amino N at
the terminus is more nucleophilic than any of
the amide nitrogens.
Sanger's Method
• The key reagent in Sanger's method for
identifying the N-terminus is 1-fluoro-2,4dinitrobenzene.
• 1-Fluoro-2,4-dinitrobenzene is very reactive
toward nucleophilic aromatic substitution
NO2
O2N
F
Sanger's Method
• 1-Fluoro-2,4-dinitrobenzene reacts with the
amino nitrogen of the N-terminal amino acid.
NO2
O2N
O
O
F + H2NCHC
NHCHC
NHCH2C
O2N
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
–
NHCHCO
CH3
CH(CH3)2 CH2C6H5
NO2
O
O
O
–
NHCHCO
CH3
Sanger's Method
• Acid hydrolysis cleaves all of the peptide bonds
leaving a mixture of amino acids, only one of
which (the N-terminus) bears a 2,4-DNP group.
NO2
O
O
O
O
+
+
+
NHCHCOH + H3NCHCO– + H3NCH2CO– + H3NCHCO–
O2N
CH(CH3)2
CH3
CH2C6H5
H3O+
NO2
O2N
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
O
–
NHCHCO
CH3
B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALY
YLVC
VCGERGF
GFFYTPK
YTPKA
FVNQHLCGSHLVGALYLVCGERGFFYTPKA
Edman Degradation
• 1.
Method for determining Nterminal amino acid.
• 2. Can be done sequentially one residue at
a time on the same sample. Usually one
can determine the first 20 or so amino
acids from the N-terminus by this method.
• 3. 10-10 g of sample is sufficient.
4. Has been automated.
N
C
5. Uses phenyl isothiocyanate.
S
Edman Degradation
+
O
C6H5N
C
+
H3NCHC
S
peptide
NH
R
• Phenyl isothiocyanate reacts with the amino
nitrogen of the N-terminal amino acid.
S
O
C6H5NHCNHCHC
NH
R
peptide
Edman Degradation
S
O
C6H5NHCNHCHC
peptide
NH
R
HCl
S
C6H5NH
C
O
C
+
+
N
CH
R
H3N
peptide
Peptide Bond Formation
• Random peptide bond formation in a mixture of
phenylalanine and glycine, for example, will give:
Phe—Phe Gly—Gly Phe—Gly Gly—Phe
Limit the number of possibilities by "protecting" the nitrogen of one amino acid and
the carboxyl group of the other.
N-Protected
phenylalanine
C-Protected
glycine
O
X
NHCHCOH
O
H2NCH2C
CH2C6H5
Only Phe- Gly is formed
Y
Protect Amino Groups as
Amides
• Amino groups are normally protected by
converting them to amides.
• Benzyloxycarbonyl (C6H5CH2O—) is a
common protecting group. It is abbreviated
as Z.
• Z-protection is carried out by treating an
amino acid with benzyloxycarbonyl
chloride.
Protect Amino Groups as
Amides
O
O
+
CH2OCCl
+
H3NCHCO
–
CH2C6H5
1. NaOH, H2O
2. H+
O
CH2OC
O
NHCHCOH
(82-87%)
Z-Phe
CH2C6H5
Removing Z-Protection
• An advantage of the benzyloxycarbonyl
protecting group is that it is easily removed
by:
• a) hydrogenation (H2/Pd)
• b) cleavage with HBr in acetic acid
The tert-Butoxycarbonyl
Protecting Group
O
(CH3)3COC
O
NHCHCOH
CH2C6H5
is abbreviated as:
O
BocNHCHCOH
CH2C6H5
or Boc-Phe
HBr Cleavage of Boc-Protecting
Group
O
(CH3)3COC
O
NHCHCNHCH2CO2CH2CH3
CH2C6H5
HBr
O
H3C
C
H3C
CH2
CO2
+
H3NCHCNHCH2CO2CH2CH3
CH2C6H5
Br
–
(86%)
Protect Carboxyl Groups as
Esters
• Carboxyl groups are normally protected as
esters.
• Deprotection of methyl and ethyl esters is
by hydrolysis in base.
• Benzyl esters can be cleaved by
hydrogenation. (H2/Pd)
Forming Peptide Bonds
• The two major methods are:
• 1. coupling of suitably protected amino
acids using N,N'-dicyclohexylcarbodiimide
(DCCI)
• 2. via an active ester of the N-terminal
amino acid.
DCCI-Promoted Coupling
O
O
ZNHCHCOH
+
H2NCH2COCH2CH3
CH2C6H5
DCCI, chloroform
O
ZNHCHC
CH2C6H5
O
NHCH2COCH2CH3
(83%)
DCCI-Promoted Coupling
• The species formed by addition of the Zprotected amino acid to DCCI is similar in
structure to an acid anhydride and acts as an
acylating agent.
• Attack by the amine function of the carboxylprotected amino acid on the carbonyl group
leads to nucleophilic acyl substitution.
C6H11N
NC6H11
C
H
+
O
O
C6H11N
C
OCCHNHZ
ZNHCHCOH
C6H11N
CH2C6H5
CH2C6H5
Mechanism of DCCI-Promoted
Coupling
O
O
H
C6H11N
C
O
+
ZNHCHC
NHCH2COCH2CH3
CH2C6H5
C6H11NH
O
H2NCH2COCH2CH3
H
C6H11N
C6H11N
O
C
OCCHNHZ
CH2C6H5
The Active Ester Method
A p-nitrophenyl ester is an example of an "active ester."
O
O
NO2
ZNHCHCO
+
H2NCH2COCH2CH3
CH2C6H5
chloroform
O
ZNHCHC
CH2C6H5
O
NHCH2COCH2CH3
(78%)
+ HO
NO2
Solid-Phase (Merrifield)
Synthesis
• In solid-phase synthesis, the starting material
is bonded to an inert solid support.
• Reactants are added in solution.
• Reaction occurs at the interface between the
solid and the solution. Because the starting
material is bonded to the solid, any product
from the starting material remains bonded as
well.
• Purification involves simply washing the
byproducts from the solid support.
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH2Cl
• The side chain chloromethyl group is a
benzylic halide, reactive toward
nucleophilic substitution (SN2).
CH
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
–
BocNHCHCO
R
CH
CH2Cl
CH2
CH
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
BocNHCHCO
• Next, the Boc
protecting group is
removed with HCl.
R
CH
CH2
CH2
CH
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
H2NCHCO
• DCCI-promoted
coupling adds the
second amino acid
R
CH
CH2
CH2
CH
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
BocNHCHC
R'
CH
O
CH2
CH
CH2
NHCHCO
R
• Remove the Boc
protecting group.
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
H2NCHC
R'
CH
O
CH2
CH
CH2
NHCHCO
R
• Add the next amino
acid and repeat.
The Merrifield Procedure
CH2
CH
CH2
O
CH
CH2
O
+
H3N peptide C NHCHC
R'
CH
O
CH2
CH
CH2
NHCHCO
R
• Remove the peptide
from the resin with
HBr in CF3CO2H
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
CH
CH2Br
O
O
+
H3N peptide C NHCHC
R'
O
–
NHCHCO
R
CH2
CH
The Merrifield Method
• Merrifield automated his solid-phase method.
• Synthesized a nonapeptide (bradykinin) in 1962 in
8 days in 68% yield.
• Synthesized ribonuclease (124 amino acids) in
1969.
369 reactions; 11,391 steps
• Nobel Prize in chemistry: 1984
Levels of Protein Structure
• Primary structure = the amino acid sequence
plus disulfide links
• Secondary structure = conformational
relationship between nearest neighbor amino
acids
•
•
•
– pleated  sheet
–  helix
planar geometry of peptide bond
anti conformation of main chain
hydrogen bonds between N—H and O=C
Pleated  Sheet
•
•
•
•
Adjacent chains are antiparallel.
Hydrogen bonds between chains.
small side chains
 Sheet is flexible, but resists stretching.
 Helix
• Shown is an  helix of a
protein in which all of the
amino acids are L-alanine.
• Helix is right-handed with
3.6 amino acids per turn.
• Hydrogen bonds are
within a single chain.
Tertiary Structure
• Refers to overall shape (how the chain is
folded)
• Fibrous proteins (hair, tendons, wool) have
elongated shapes
• Globular proteins are approximately spherical
•
most enzymes are globular proteins
•
an example is carboxypeptidase
Protein Quaternary Structure
• Some proteins are assemblies of two or
more chains. The way in which these chains
are organized is called the quaternary
structure.
• Hemoglobin, for example, consists of 4
subunits.
• There are 2  chains (identical) and 2 
chains (also identical).
• Each subunit contains one heme and each
protein is about the size of myoglobin.