Practical Protein Chemistry - A. Darbre 1989

Traditional strategy for protein structure determination
Introduction

G. WINTER (MRC Laboratory of Molecular Biology, Hill Road, Cambridge, CB2 2QH, U.K.)

All modern Methods for determining the Amino Acid Sequence of Peptides are based on the Edman Degradation [19, 21].

In The First stage of this reaction, phenylisothiocyanate (PITC) attaches to the α-amino group of the peptide. Then, under the action of an anhydrous acid, the N-terminal residue is cleaved from the phenylthiocarbamyl (PTC) derivative as a 5'-thiazolinone, yielding a shortened peptide whose new N-terminus is the next amino acid in the sequence (Fig. 10.1). Excess Reagents and by-products at intermediate stages are removed by extraction with organic Solvents. Repeating this process while simultaneously identifying the cleaved amino acid—released from the thiazolinone as a phenylthiohydantoin (PTH) derivative—or the N-terminal residue of the shortened peptide makes it possible to determine The amino acid sequence of the starting compound.

The length of the sequence determined in this manner is limited due to the gradual decrease in product yields during the coupling, Cleavage, and extraction stages, potential blocking of the newly formed N-terminal residue, Side Reactions of cleavage at Arginine and Histidine residues [58, 61] or other amino acid residues of the polypeptide chain [39], as well as several other factors. For instance, in the presence of trace amounts of oxygen in the reaction medium, peptide PTC-derivatives undergo desulfurization [33] and fail to undergo subsequent cleavage upon acid Treatment. Peptides with N-terminal residues of glutamine [4], Tryptophan [75], and various Cysteine derivatives [53, 68, 76] can form cyclic products. This, much like the potential rearrangement in the Asn-Gly sequence [20, 35, 72] or the O→N acyl migration in acylserine and acylthreonine residues [20, 34, 69], leads to the Termination of the cleavage process. Additional complications arise with poorly soluble hydrophobic peptides, which fail to react completely during Condensation and are prone to partitioning into the organic phase during extractions.

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FIG. 10.1. Scheme 1.

The yields in the classical ("manual") Edman Procedure are typically lower than those obtained using automated instruments, primarily due to the inability to completely exclude oxygen from the reaction environment and less efficient aqueous washes. Since the cleavage reaction with trifluoroacetic acid is reversible [21] (Fig. 10.1), the presence of thiazolinones not fully extracted in previous steps inhibits the Cleavage of the PTC-peptide. In Hartley's procedure [26], unlike the standard Edman method and its DABITC/FITC variants [9] (Ch. 14), an aliquot of the peptide is taken for Dansylation and subsequent identification of the N-terminal amino acid (Ch. 11), leading to an additional reduction in step-by-step yields. As the number of cleaved Amino Acids increases, Background "noise" levels rise, complicating the reliable identification of the amino acid. Typically, these methods allow for the determination of relatively short sequences containing up to 15 residues. Therefore, it is quite evident that cleaving Proteins into short peptides is an essential prerequisite for structural analysis using the classical approach; this is usually achieved through enzymatic or chemical reagents (the latter yielding larger fragments).