Primary Structure Determination (Sanger) 1.Determine what amino acids are present and their molar...
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Transcript of Primary Structure Determination (Sanger) 1.Determine what amino acids are present and their molar...
Primary Structure Determination (Sanger)
• 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.
The primary structure is the amino acid sequence plus any disulfide links.
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.
TrypsinTrypsin is selective for cleaving the peptide bond to the carboxyl group of lysine or arginine.
NHCHC
O
R'
NHCHC
O
R"
NHCHC
O
R
lysine or arginine
ChymotrypsinChymotrypsin is selective for cleaving the peptidebond to the carboxyl group of amino acids withan aromatic side chain.
NHCHC
O
R'
NHCHC
O
R"
NHCHC
O
R
phenylalanine, tyrosine, tryptophan
Carboxypeptidase
proteinH3NCHC
O
R
+NHCHCO
O
R
–C
O
Carboxypeptidase is selective for cleavingthe peptide bond to the C-terminal amino acid.
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 C-terminal 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-2,4-dinitrobenzene.
• 1-Fluoro-2,4-dinitrobenzene is very reactive toward nucleophilic aromatic substitution
FO2N
NO2
Sanger's Method
• 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid.
FO2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
H2NCHC
O OOO
CH(CH3)2
–+
O2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
NHCHC
O OOO
CH(CH3)2
–
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.
O2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
NHCHC
O OOO
CH(CH3)2
–
H3O+
O
O2N
NO2
NHCHCOH
CH(CH3)2
H3NCHCO–
CH3
+H3NCH2CO–
O O
+
O
H3NCHCO–
CH2C6H5
++ +
+
B Chain of Bovine InsulinFVNQHLCGSHL
SHLVLVGA
VGAL
ALY
YLVCVCGERGF
GFFYTPK
YTPKA
FVNQHLCGSHLVGALYLVCGERGFFYTPKA
Edman Degradation• 1. Method for determining N-terminal
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.5. Uses phenyl isothiocyanate.
N C S
Edman Degradation
peptideH3NCHC
O
R
+NH
C6H5N C S
+
peptideC6H5NHCNHCHC
O
R
NH
S
• Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid.
Edman Degradation
peptideC6H5NHCNHCHC
O
R
NH
S
HCl
peptideH3N
++
C6H5NH C
S
C
N CH
R
O
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-Protectedphenylalanine
C-Protectedglycine
NHCHCOH
CH2C6H5
O
X H2NCH2C
O
Y
Only Phe- Gly is formed
• 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
Protect Amino Groups as Amides
CH2OCCl
O
+ H3NCHCO
CH2C6H5
O
–+
1. NaOH, H2O
2. H+
NHCHCOH
CH2C6H5
O
CH2OC
O
(82-87%)
Z-Phe
• 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
Removing Z-Protection
The tert-Butoxycarbonyl Protecting Group
NHCHCOH
CH2C6H5
O
(CH3)3COC
O
is abbreviated as:
BocNHCHCOH
CH2C6H5
O
or Boc-Phe
HBr Cleavage of Boc-Protecting Group
NHCHCNHCH2CO2CH2CH3
CH2C6H5
O
(CH3)3COC
O
HBr
H3NCHCNHCH2CO2CH2CH3
CH2C6H5
O
CO2
(86%)
+
Br–
CH2C
H3C
H3C
• 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)
Protect Carboxyl Groups as Esters
• 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.
Forming Peptide Bonds
DCCI-Promoted Coupling
ZNHCHCOH
CH2C6H5
O
+ H2NCH2COCH2CH3
O
DCCI, chloroform
ZNHCHC
CH2C6H5
O
NHCH2COCH2CH3
O
(83%)
DCCI-Promoted Coupling
CH2C6H5
O
C6H11N C
C6H11N
H
OCCHNHZ
• The species formed by addition of the Z-protected 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 carboxyl-protected amino acid on the carbonyl group leads to nucleophilic acyl substitution.
ZNHCHCOH
CH2C6H5
O
C6H11N C NC6H11
+
Mechanism of DCCI-Promoted Coupling
H2NCH2COCH2CH3
O
C6H11N C
C6H11NH
H
O + ZNHCHC
CH2C6H5
O
NHCH2COCH2CH3
O
CH2C6H5
O
C6H11N C
C6H11N
H
OCCHNHZ
The Active Ester Method
ZNHCHCO
CH2C6H5
O
+ H2NCH2COCH2CH3
O
NO2
chloroform
ZNHCHC
CH2C6H5
O
NHCH2COCH2CH3
O
(78%)
+ HO
NO2
A p-nitrophenyl ester is an example of an "active ester."
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 SupportCH2 CH2 CH2 CH2
CH CH CH CH
CH2Cl
• The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (SN2).
The Merrifield ProcedureCH2 CH2 CH2 CH2
CH CH CH CH
CH2Cl
BocNHCHCO
R
O–
The Merrifield Procedure
BocNHCHCO
R
O
CH2 CH2 CH2 CH2CH CH CH CH
CH2
• Next, the Boc protecting group is removed with HCl.
The Merrifield Procedure
H2NCHCO
R
CH2 CH2 CH2 CH2CH CH CH CH
CH2O
• DCCI-promoted coupling adds the second amino acid
The Merrifield Procedure
NHCHCO
R
O
CH2 CH2 CH2 CH2CH CH CH CH
CH2
BocNHCHC
R'
O
• Remove the Boc protecting group.
The Merrifield ProcedureCH2 CH2 CH2 CH2
CH CH CH CH
CH2
NHCHCO
R
O
H2NCHC
R'
O
• Add the next amino acid and repeat.
The Merrifield Procedure
• Remove the peptide from the resin with HBr in CF3CO2H
CH2 CH2 CH2 CH2CH CH CH CH
CH2
NHCHCO
R
O
NHCHC
R'
O
C
O+
H3N peptide
The Merrifield Procedure
CH2 CH2 CH2 CH2CH CH CH CH
CH2Br
NHCHCO
R
O
NHCHC
R'
O
C
O+
H3N peptide–
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.