Protonation, amino acid sequence-specific

Fragmentation of Protonated Peptides. The amino acid sequence-specific fragmentation of peptides is directed by the site of protonation. For peptides containing arginine and lysine, protonation occurs primarily on these basic residues. However, in order to anticipate the entire set of possible protonated fragments that may be... 

Ovchinnikov 234 237) has shown that bovine rhodopsin, although quite different in amino acid sequence (348 residues), also forms seven transmembrane helices. This structural similarity between bacterial and mammalian light activated membrane proteins is remarkable. Since the two amino acid sequences have little in common it would appear that the necessary requirement is seven transmembrane helices to form a channel which is specific for proton migration. For example it has been suggested that a similar arrangement and function is performed by the lactose permease of E. coli237). 

The /r dependence of p causes the buildup rate to fall off very rapidly with internuclear distance, with the result that NOEs are short-range interactions that are typically not observed between protons separated by more than 5.5 A (but see below). Nevertheless, this provides extremely valuable structural information since spatially proximal protons will yield a crosspeak in NOESY spectra regardless of how distal they are in the amino acid sequence. Since several thousand NOEs will be observed for even a protein of modest size, NOEs provide the most important structural restraints for structure calculations. But first they need to be assigned to specific proton pairs, quantified, and converted into distance information. 

The heme-copper oxidase superfamily is defined hy two criteria (1) a high degree of amino acid sequence similarity within the largest suhunit (suhunit I) and (2) a unique bimetallic active site, consisting of a heme and a closely associated copper atom (see Figure 8), where dioxygen is reduced to water. There are two main branches of the superfamily, which have distinct substrate specificities the mitochondrial respiratory oxidases use cytochrome r as a substrate and, hence, are called cytochrome c oxidases (COX). Bacteria, unlike most mitochondria, contain multiple respiratory oxidases. Many of the prokaryotic respiratory oxidases use membrane-bound quinol (ubiquinol or menaquinol) as a substrate rather than cytochrome c. A number of these quinol oxidases have been shown to be members of the heme-copper oxidase superfamily and to pump protons as efficiently as COXs. " ... 

As with electrophoresis and NMR spectroscopy, mass spectrometry is also helping in medical research - to both identify and research the amino acid sequences in proteins. It can be used to analyse the whole protein molecule or the peptides left after breaking down the protein with specific enzymes. Figure 29.28 shows the mass spectrum of a pentapeptide that is made into a charged compound by the addition of a proton, hence the MH peak. 

Using DQF-COSY and TOCSY we can link all of the protons within a single spin system, which corresponds to a single amino acid residue. We can classify each spin system as a pattern of chemical shifts unique to one amino acid or as a member of a class AMX or five spin. In order to get sequence-specific assignments, however, we have to have some way to correlate protons in one residue to protons in the next residue in the sequence. For unlabeled proteins this is done by NOE interactions certain protons in one residue are constrained by the peptide bond to be close in space to certain protons in the next residue. These NOE correlations are called sequential or z, i + 1 because they correlate a proton in residue z with a proton in the next residue in the sequence, residue z + 1. Specifically, we expect to see NOE correlations between Ha of residue z and Hn of residue z + 1 (Fig. 12.15) and sometimes between the protons of residue z and the Hn of the next residue. Because the DQF-COSY and TOCSY spectra correlate protons within a residue, we can move from... 

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