Molecular Biology Has Revolutionized The Determination Of Primary Structure

Knowledge of DNA sequences permits deduction of the primary structures of polypeptides. DNA sequencing requires only minute amounts of DNA and can readily yield the sequence of hundreds of nucleotides. To clone and sequence the DNA that encodes a partic-

Phenylisothiocyanate (Edman reagent) and a peptide

Peptide X Peptide Y Peptide Z

Carboxyl terminal portion of peptide X

Amino terminal portion of peptide Y

Figure 4-7. The overlapping peptide Z is used to deduce that peptides X and Y are present in the original protein in the order X ^ Y, not Y ^ X.

A phenylthiohydantoic acid

A phenylthiohydantoic acid

A phenylthiohydantoin and a peptide shorter by one residue

Figure 4-6. The Edman reaction. Phenylisothiocyanate derivatizes the amino-terminal residue of a peptide as a phenylthiohydantoic acid. Treatment with acid in a nonhydroxylic solvent releases a phenylthiohydantoin, which is subsequently identified by its chromatographic mobility, and a peptide one residue shorter. The process is then repeated.

A phenylthiohydantoin and a peptide shorter by one residue

Figure 4-6. The Edman reaction. Phenylisothiocyanate derivatizes the amino-terminal residue of a peptide as a phenylthiohydantoic acid. Treatment with acid in a nonhydroxylic solvent releases a phenylthiohydantoin, which is subsequently identified by its chromatographic mobility, and a peptide one residue shorter. The process is then repeated.

ular protein, some means of identifying the correct clone—eg, knowledge of a portion of its nucleotide sequence—is essential. A hybrid approach thus has emerged. Edman sequencing is used to provide a partial amino acid sequence. Oligonucleotide primers modeled on this partial sequence can then be used to identify clones or to amplify the appropriate gene by the polymerase chain reaction (PCR) (see Chapter 40). Once an authentic DNA clone is obtained, its oligonucleotide sequence can be determined and the genetic code used to infer the primary structure of the encoded polypeptide.

The hybrid approach enhances the speed and efficiency of primary structure analysis and the range of proteins that can be sequenced. It also circumvents obstacles such as the presence of an amino-terminal blocking group or the lack of a key overlap peptide. Only a few segments of primary structure must be determined by Edman analysis.

DNA sequencing reveals the order in which amino acids are added to the nascent polypeptide chain as it is synthesized on the ribosomes. However, it provides no information about posttranslational modifications such as proteolytic processing, methylation, glycosylation, phosphorylation, hydroxylation of proline and lysine, and disulfide bond formation that accompany maturation. While Edman sequencing can detect the presence of most posttranslational events, technical limitations often prevent identification of a specific modification.

Table 4-1. Methods for cleaving polypeptides.

Method

Bond Cleaved

CNBr

Met-X

Trypsin

Lys-X and Arg-X

Chymotrypsin

Hydrophobic amino acid-X

Endoproteinase Lys-C

Lys-X

Endoproteinase Arg-C

Arg-X

Endoproteinase Asp-N

X-Asp

V8 protease

Glu-X, particularly where X is hydro-phobic

Hydroxylamine

Asn-Gly

o-Iodosobenzene

Trp-X

Mild acid

Asp-Pro

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