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.