Protein sequencing strategy

This strategy can be divided roughly into the following steps  

These steps are discussed in more detail in the following sections. 

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These initial systematic studies regarding the directed evolution of PAL allowed several conclusions to be made. Protein sequence space can be explored successfully by applying the following strategies [8c,33j ... 

The choice of the particular upward pathway in the kinetic resolution of rac-19, that is, the specific order of choosing the sites in ISM, appeared arbitrary. Indeed, the pathway B C D F E, without utilizing A, was the first one that was chosen, and it led to a spectacular increase in enantioselectivity (Figure 2.15). The final mutant, characterized by nine mutations, displays a selectivity factor of E=115 in the model reaction [23]. This result is all the more remarkable in that only 20000 clones were screened, which means that no attempt was made to fully cover the defined protein sequence space. Indeed, relatively small libraries were screened. The results indicate the efficiency of iterative CASTing and its superiority over other strategies such as repeating cycles of epPCR. 

A particular goal of chemical theory is to predict protein structure from the amino acid sequence—to calculate how polypeptides fold into the compact geometries of proteins. One strategy is to develop methods (often based on bioinformatics) for predicting structures approximately and then refining the structures... 

Example 3. The last example covers the biggest peptide obtained from a tryptic digestion of an unknown protein. Although there are better ways to identify such peptides than de novo sequencing, this example allows us to present some additional hints that can be used in sequencing strategies. 

This section focuses on the use of SWISS-PROT + TrEMBL for sequence similarity searches. Searches in protein sequence databases have now become a standard research tool in the life sciences. To produce valuable results, the source databases should be comprehensive, nonredundant, well annotated, and up-to-date. However, lack of a single protein sequence database that satisfies all four criteria has previously forced users to perform searches across multiple databases to avoid incomplete results. This strategy normally produces complete but redundant results owing to different versions of the same sequence report in different databases. 

Livingstone, C. D., and Barton, G.J. (1993). Protein sequence alignments a strategy for the hierarchical analysis of residue conservation. Comput. Appl. Biosci. 9, 745—756. 

Several steps were needed to determine the structure of the core particle to higher resolution (Fig. Id). The X-ray phases of the low-resolution models were insufficient to extend the structure to higher resolution, since the resolution of the early models of the NCP was severely limited by disorder in the crystals. The disorder was presumed to derive from both the random sequences of the DNA and from heterogeneity of the histone proteins caused by variability in post-translational modification of the native proteins. One strategy for developing an atomic position model of the NCP was to develop a high-resolution structure of the histone core. This structure could then be used with molecular replacement techniques to determine the histone core within the NCP and subsequently identify the DNA in difference Fourier electron density maps. 

Clearly, all of these strategies for navigating in protein sequence space are successful, but it is not obvious which ones are optimal. 

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