Peptide sequencing cyclic peptides

When the receptor binding domain is encoded in a small peptide sequence, the peptide hg-and can also be synthesized and conjugated chemically to the carrier protein. This approach was followed in our laboratory by Beljaars et al. for the development of carriers aimed at the hepatic stellate cell, a cell type involved in liver fibrosis [33] (see also Chapter 4). A peptide sequence derived from the receptor binding domains of collagen VI was incorporated into a cyclic peptide homing device, and subsequently conjugated to lysine residues of HSA. This carrier bound selectively to activated hepatic stellate cells and rapidly accumulated in the livers of fibrotic rats. 

L. Ngoka and M. L. Gross, Multistep tandem mass spectrometry for sequencing cyclic peptides in an ion-trap mass spectrometer, J. Am. Soc. Mass Spectrom. 

A number of polypeptide biomarkers have also been identified in the mass range below 4000,28-31 which are cyclic secondary metabolites bonded to lipids or sugars. These peptide sequences are not directly translated from DNA,32... 

Fig. 21. Peptide-based glycoclusters at the wedge of linear peptide sequences or as cyclic scaffolds.

Cyclic peptides are analyzed less frequently then the linear ones but their sequencing and nomenclature are much more complicated. There is no generally accepted nomenclature scheme but we describe here the one proposed by Ngoka and Gross [4]. It takes account of Roepstorff-Fohlmann s and Biemann s conventions but introduces amendments to address cyclic peptide fragmentation. 

The influence of backbone flexibility was seen, for example, when the stability of the linear tetrapeptide 6.70 was compared to that of a cyclic hexa-peptide derivative of the same sequence [91]. Indeed, the cyclic peptide was approximately one order of magnitude more stable than the linear peptide in the pH range of 2-7. The results at higher pH values were inconclusive since the stability of the cyclic peptide decreased dramatically due to the degradation of a disulfide bond absent in the linear peptide. 

The peptides discussed so far are defined by a common genetic pattern or architectural feature, such as their sequence or disulfide bond pattern. In this section we discuss peptides that share a common mode of action but may arise from different peptide families. Proteinase inhibitors (Pis) come in an astounding range of sizes, from the smallest gene-encoded cyclic peptide known to date, sunflower trypsin inhibitor 1 (SFTI-1), ° a 14-residue cyclic peptide with a single disulfide bond, to squash inhibitors that are approximately 30 residues in size and feature the cystine knot motif, to 53-residue Pis found in Nicotiana... 

The rates of formation of various cyclic peptides and DKPs have been documented and shown to be affected by a wide range of physicochemical and structural parameters. Goolcharran and Borchardt examined the effects of exogenous (i.e., pH, temperature, buffer species, and concentration) and endogenous (i.e., primary sequences) factors affecting the rate of cyclic dipeptide formation, using the dipeptide analogues of X-Pro-/)-nitroaniline (X-Pro-/>NA where X represents the amino acid residue of the respective cyclic dipeptide). 

By convention, the left-hand amino acid in this sequence is the one with a free amino group, the A-terminus, and the right-hand amino acid has the free carboxyl, the C-terminus. Thus, Ser-Phe-Ala is different from Ala-Phe-Ser, and represents a quite different molecule. Sometimes, the termini identities are emphasized by showing H- and -OH H- represents the amino group and -OH the carboxyl group. Some peptides are cyclic, and this convention can have no significance, so arrows are incorporated into the sequence to indicate peptide bonds in the direction CO— NH. As sequences become longer, one-letter abbreviations for amino... 

The important difference between linear and cyclic peptides is the reduced conformational flexibility imposed by the cyclization. 14,151 Cyclic peptides have, therefore, been under intense investigation with respect to their conformational preferences 30,31 and their different turn motifs, e.g. reverse turns. 32 35 To differentiate between the structural influences of the numerous sequence elements the following special concepts have been described 161 (1) Global constraints are structural characteristics that restrict the whole molecule, e.g. the macrocyclic structure of the peptide framework and (2) local constraints comprise a large number of structure-inducing building blocks, such as proline, D-amino acids, and turn mimetics. 

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