Blocked N-termini

For about 50% of all proteins, the N-terminus is blocked by N-acetyl amino acids glycosylated amino acids, pyrrolidone groups, or others. This blockage prevents Edman degradation. Edman degradation is still today the basis for most sequencing technologies (see Sections 7.6.4 and 7.6.6). 

Furthermore, I imagined, glycerine would stabilize the protein structure. But to be honest even if I had known about the glycerine s protective effect at the time, I would have given the identical response. 

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A number of ribosomal proteins contain modihed amino acids at the N terminus or at other positions of the protein chain (Table IV). The N termini of three proteins (S5, S18, and L7) are acetylated, thus they cannot be subjected successfully to manual or automatic Edman degradation because of their blocked N termini. Mutants have been isolated in... 

Microsequencers permit sequence analysis on minute amounts of protein. Microsequencing can be used in conjunction with two-dimensional electrophoretic separations of proteins such as that shown in Box 3-C. The proteins in the polyacrylamide gel are electropho-retically transferred onto a porous sheet (membrane) of an inert material such as polyvinyl difluoride.249-251 After staining, a selected spot is cut out and placed into the sequencer. To avoid the problems associated with blocked N termini, the protein may be treated with proteases on the membrane and the resulting peptide fragments may then be separated on a narrow-bore HPLC column and sequenced.240... 

Many proteins, maybe even the majority in eukaryotes by some estimates, have blocked N-termini, which render them refractive to coupling with PITC. By far the most frequent blocking modification is acetylation. Contrary to frequent assumptions,... 

The introduction of retro-, retro-inverso-, and PMRI-peptides with free and blocked C-and N-termini has been successful in numerous biological systems such as neurotransmitters, inhibitors of proteases and protein kinases, sweeteners, antimicrobial peptides, hormones, adhesion molecules, antigenic epitopes, immuno-modulators, and immunological probes. Table 1 provides an exhaustive list of retro-, retro-inverso-, PMRI-, and end-group-modified re/ro-mvmo-pseudopeptides derived from bioactive peptides. 

If your protein sample has an acetylated N-terminus, it will not yield any information from Edman degradation of the intact protein. Enzymes have been described that remove N-acetyl groups from peptide chains, but, in general, these have been found to function well only on peptides and not on intact proteins. Procedures have been described for cleavage of blocked proteins into peptides followed by modihcation of all newly revealed peptide N-termini (e.g., by succiny-lation) and then incubation of the mixture with a deacetylase. The entire mixture can then be subjected to Edman degradation. Only the deacetylated peptide representing the original N-terminus will provide sequence. 

Figure 7.10. Ladder sequencing of peptides (following Chait et al.) (A) Peptide is coupled with PICT and PIC. TFA cleaves the PICT-coupled amino acid. The PIC-derivatized peptide remains unchanged. (B) Several cycles (here three) of PICT/PIC coupling and subsequent acid cleavage generate a peptide ladder with PIC N-termini, a residue peptide with free N-terminus, and the PICT-derivatized amino acids. For the analysis of the peptide ladder, you also block the residue peptide with PIC.

For the first step of the analysis of PSI biogenesis, we determined which subunits of PSI are nuclear-encoded. PSI complex was prepared from several species of Nicotiana, and each subunit was separated by high resolution LDS PAGE. Partial amino acid sequences were determined for each subunit, and compared with the amino acid sequences deduced from tobacco chloroplast DNA sequence in order to determine which subunits are chloroplast-encoded. As far as the PSI subunits having mol. wt. from 5.6 kDa to 20 kDa are concerned, it was only psaC protein that was proved to be of chloroplast origin. All the others, some of which are blocked at their N termini, are not coded for by chloroplast DNA, indicating that they are of nuclear origin. 

Gianneschi and coworkers [29] have utilized a similar approach in functionalizing ROMP copolymers with hydrophilic peptides. This work involves a block copolymer synthesis incorporating a phenyl NBE moiety as the hydrophobic block and an NHS moiety as the chemically addressable block. Subsequent conjugation of peptides via their N-termini affords amphiphilic block copolymers in which the hydrophilic shell is defined by a short peptide sequence programmed to be an enzyme substrate. For graft-to functionalization, A/ ,A/ -diisopropylethylamine was added to the activated ester diblock copolymer in DMF, followed by the addition of peptide (with respect to NHS units). 

Two major CRABP 1 molecules with different N-termini are produced by this procedure Sixty to 100% of the product, depending on the preparation, is authentic CRABP I, with a pi of 4.76. The 40% remaining in some preparations contains a major N-terminal blocked CRABP I variant with a pi of 4.79, and a minor variant with a pi of 4 83. The combination of the two vanants binds RA as well as authentic CRABP I... 

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