Uncharacterized proteins

Obviously, not all proteins known to interact with ubiquitin or ubiquitin-like domains contain one of the professional ubiquitin-interaction domains. RptS and Rpnl, two subunits of the proteasome that bind to ubiquitin and UbLs, respectively, do not belong to any of the classes described above. Most probably, a large number of uncharacterized proteins with high affinity and specificity for ubiquitin are still waiting to be discovered. The bioinformatical tools described in the early sections of this chapter will be instrumental for this task. 

Putative functional domains within the uncharacterized protein can be identified using the CDD search tools while performing protein-protein BLAST (BLASTP) alignments to obtain insight into possible functions of the predicted protein. The predicted protein sequence is used since amino-acid sequences are more evolutionarily conserved than nucleotide sequences, and therefore a broader search across all available sequence information from a variety of organisms can be conducted to identify potential functionality. Results from the BLASTP and CDD analysis (Fig. 10) have identified... 

Once the set of superfamily members has been confirmed, the next step is to identify conserved sequence elements that can be associated with conserved functional steps among the member proteins. This will be most easily achieved for superfamilies in which one or more members have been mechanistically characterized and residues or motifs playing important chemical roles already identified. Here, inference based on analogy can be highly useful in predicting properties of uncharacterized proteins. This, generally, is one of the most common procedures in... 

Fig. 3. Sequence alignment of an uncharacterized protein to phosphinothricin N-acetyltransferase. The sequence alignment obtained in the third iteration of Position Specific Iterative Basic Local Alignment Search Tool of the query uncharacterized protein BAE56987.1 to the sbjct phosphinothricin N-acetyltransferase 1VHS is displayed. The Q/RxxGxG/A motif found in a large number of coenzyme A-binding proteins is highlighted by a box.

Highly sophisticated computational methods are invaluable assets in the search for the biological function of uncharacterized proteins. For many proteins computational sequence analysis can suggest at best a general biochemical function. Still, this information can be used to design experimentally testable hypotheses aimed at identifying... 

When performing a sequence similarity search, it is not simply the similarity statistics that determine biological insight into the uncharacterized protein. The bioinformatician uses the knowledge about the proteins already characterized in order to arrive at any insights. Thus it has been said that biology is a knowledge-based discipline [1]. 

Tlie availability of over 20 fully sequenced genomes has forced the development of new methods to find protem functions and interactions. Proteins were grouped by correlated evolution, correlated mRNA expression patterns and patterns of domain fusion to deter-mme functional relationships among the 6217 proteins of the yeast S. cerevisiae. Using these methods, over 93,000 pair-wise finks between functionally related yeast proteins were discovered. Links between characterized and uncharacterized proteins allow a general function to be assigned to more than half of the 2557 previously uncharacterized yeast proteins. 

A major requirement in the purification of peptides generated from previously uncharacterized proteins is to maximize the detection sensitivity due to limited supplies of the proteins. Separations by RPLC are now carried out with narrow-bore columns of 1-2 mm internal diameter which increases the solute concentration thereby increasing the sensitivity. Further miniaturization of RPLC systems has also been achieved with the use of capillary columns of internal diameters of 0.32-0.075 mm. 

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