PubMed · 9116821
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R Wang, B T Chait. 1997. N-terminal protein ladder sequencing.. https://doi.org/10.1385/0-89603-353-8%3A175
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We report here the application of electrospray ionization tandem mass spectrometry for the characterization of protein ubiquitylation, an important posttranslational modification of cellular proteins. Trypsin digestion of ubiquitin-conjugated proteins produces diglycine branched peptides containing the modification sites. Chemical derivatization by N-terminal sulfonation was carried out on several model peptides for the formation of a characteristic fragmentation pattern in their MS/MS analysis. The fragmentation of derivatized singly charged peptides results in a product ion distribution similar to that already observed by MALDI-TOF MS/MS. Signature fragments distinguished the diglycine branched peptides from other modified and unmodified peptides, while the sequencing product ions reveal the amino acid sequence and the location of the ubiquitylation site. Doubly charged peptide derivatives fragment in a somewhat different manner, but several fragments characteristic to diglycine branched peptides were observed under low collision energy conditions. These signature peaks can also be used to identify peptides containing ubiquitylation sites. In addition, a marker ion corresponding to a glycine-modified lysine residue produced by high-energy fragmentation provides useful information for identity verification. The method is demonstrated by the analysis of three ubiquitin-conjugated proteins using LC/MS/MS.
The phosphorylation of the amylopectin fraction of starch catalyzed by the alpha-glucan, water dikinase (GWD, EC 2.7.9.4) plays a pivotal role in starch metabolism. Limited proteolysis of the potato tuber (Solanum tuberosum) GWD (StGWD, 155 kDa) by trypsin primarily produced stable fragments of 33 and 122 kDa, termed the SBD fragment and N11, respectively, as generated by trypsin cleavage at Arg-286. SBD and N11 were generated using recombinant DNA technology and purified to near homogeneity. Tandem repeat sequences, SBD-1 and SBD-2, of a region that is significantly similar in sequence to N-terminal regions of plastidial alpha-amylases are located in the N-terminus of StGWD. The SBD-1 motif is located within the sequence of the SBD fragment, and our results demonstrate that the fragment composes a new and novel carbohydrate-binding module (CBM), apparently specific for plastidial alpha-glucan degradation. By mutational analyses of conserved Trp residues located within the SBD-1 motif, W62 and W117, we show that these aromatic residues are vital for carbohydrate binding. N11 still possessed starch phosphorylating activity, but with a 2-fold higher specific activity compared to that of wild type (WT) StGWD using potato starch as the glucan substrate, whereas it had double the K(m) value for the same substrate. Furthermore, investigation of the chains phosphorylated by WT StGWD and N11 shows that N11 exhibits a higher preference for phosphorylating shorter chains of the amylopectin molecule as compared to WT. From analyses of the glucan substrate specificity, we found up to 5-fold higher specific activity for N11 using amylose as the substrate.
The field of protein-structure prediction has been revolutionized by the application of "mix-and-match" methods both in template-based homology modeling and in template-free de novo folding. Consensus analysis and recombination of fragments copied from known protein structures is currently the only approach that allows the building of models that are closer to the native structure of the target protein than the structure of its closest homologue. It is also the most successful approach in cases in which the target protein exhibits a novel three-dimensional fold. This review summarizes the recent developments in both template-based and template-free protein structure modeling and compares the available methods for protein-structure prediction by recombination of fragments. A convergence between the "protein folding" and "protein evolution" schools of thought is postulated.