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 to 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. NHCHC O R' OR" O R lysine or arginine

Chymotrypsin Chymotrypsin is selective for cleaving the peptide bond to the carboxyl group of amino acids with an aromatic side chain. NHCHC O R' OR" O R phenylalanine, tyrosine, tryptophan

Carboxypeptidase protein H 3 NCHC OR + NHCHCO OR – CO Carboxypeptidase is selective for cleaving the 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 F O2NO2NO2NO2N NO 2

Sanger's Method 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid. F O2NO2NO2NO2N NO 2 NHCH 2 C NHCHCO CH 3 NHCHC CH 2 C 6 H 5 H 2 NCHC OOO O CH(CH 3 ) 2 – + O2NO2NO2NO2N NO 2 NHCH 2 C NHCHCO CH 3 NHCHC CH 2 C 6 H 5 NHCHC OOO O CH(CH 3 ) 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. O2NO2NO2NO2N NO 2 NHCH 2 C NHCHCO CH 3 NHCHC CH 2 C 6 H 5 NHCHC OOO O CH(CH 3 ) 2 – H3O+H3O+H3O+H3O+O O2NO2NO2NO2N NO 2 NHCHCOH CH(CH 3 ) 2 H 3 NCHCO – CH 3 + H 3 NCH 2 CO – OO+ O H 3 NCHCO – CH 2 C 6 H

B Chain of Bovine Insulin FVNQHLCGSHL SHLV LVGA VGAL ALY YLVC VCGERGF 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 g of sample is sufficient. 4.Has been automated. 5.Uses phenyl isothiocyanate. NCS

Edman Degradation peptide H 3 NCHC OR + NHNHNHNH C6H5NC6H5NC6H5NC6H5NC S + peptide C 6 H 5 NHCNHCHC OR NHNHNHNHS Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid.

Edman Degradation peptide C 6 H 5 NHCNHCHC OR NHNHNHNHSHCl peptide H3NH3NH3NH3N + + C6H5NHC6H5NHC6H5NHC6H5NH CSC N CH R O

Peptide Bond Formation Random peptide bond formation in a mixture of phenylalanine and glycine, for example, will give: Phe—PheGly—GlyPhe—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 NHCHCOH CH 2 C 6 H 5 O X H 2 NCH 2 C O Y Only Phe- Gly is formed

Amino groups are normally protected by converting them to amides. Benzyloxycarbonyl (C 6 H 5 CH 2 O—) 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

CH 2 OCCl O + H 3 NCHCO CH 2 C 6 H 5 O– + 1. NaOH, H 2 O 2. H + NHCHCOH CH 2 C 6 H 5 O CH 2 OC O (82-87%) Z-Phe

An advantage of the benzyloxycarbonyl protecting group is that it is easily removed by: a) hydrogenation (H 2 /Pd) b) cleavage with HBr in acetic acid Removing Z-Protection

The tert-Butoxycarbonyl Protecting Group NHCHCOH CH 2 C 6 H 5 O (CH 3 ) 3 COC O is abbreviated as: BocNHCHCOH CH 2 C 6 H 5 O or Boc-Phe

HBr Cleavage of Boc-Protecting Group NHCHCNHCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 O (CH 3 ) 3 COC O HBr H 3 NCHCNHCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 O CO 2 (86%) + Br – CH 2 C H3CH3CH3CH3C H3CH3CH3CH3C

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. (H 2 /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 CH 2 C 6 H 5 O+ H 2 NCH 2 COCH 2 CH 3 O DCCI, chloroform ZNHCHC CH 2 C 6 H 5 O NHCH 2 COCH 2 CH 3 O (83%)

DCCI-Promoted Coupling CH 2 C 6 H 5 O C 6 H 11 N C 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 CH 2 C 6 H 5 O C 6 H 11 N C NC 6 H 11 +

Mechanism of DCCI-Promoted Coupling H 2 NCH 2 COCH 2 CH 3 O C 6 H 11 N C C 6 H 11 NH H O + ZNHCHC CH 2 C 6 H 5 O NHCH 2 COCH 2 CH 3 O CH 2 C 6 H 5 O C 6 H 11 N C H OCCHNHZ

The Active Ester Method ZNHCHCO CH 2 C 6 H 5 O+ H 2 NCH 2 COCH 2 CH 3 O NO 2 chloroform ZNHCHC CH 2 C 6 H 5 O NHCH 2 COCH 2 CH 3 O (78%) + HOHOHOHO NO 2 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 Support CH 2 CHCHCHCH CH 2 Cl The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (S N 2).

The Merrifield Procedure CH 2 CHCHCHCH CH 2 Cl BocNHCHCO RO–

The Merrifield Procedure BocNHCHCO RO CH 2 CHCHCHCH Next, the Boc protecting group is removed with HCl.

The Merrifield Procedure H 2 NCHCO R CH 2 CHCHCHCH O DCCI-promoted coupling adds the second amino acid

The Merrifield ProcedureNHCHCOR O CH 2 CHCHCHCH BocNHCHCR' O Remove the Boc protecting group.

The Merrifield Procedure CH 2 CHCHCHCH NHCHCOR O H 2 NCHC R' O Add the next amino acid and repeat.

The Merrifield Procedure Remove the peptide from the resin with HBr in CF 3 CO 2 H CH 2 CHCHCHCH NHCHCOR ONHCHC R'OC O+ H3NH3NH3NH3N peptide

The Merrifield Procedure CH 2 CHCHCHCH CH 2 Br NHCHCO R O NHCHC R'OC O+ H3NH3NH3NH3N 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 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.