Protein sequence characterization

Mann, M., and Wilm, M., 1995. Electro.spray ma.ss. spectrometry for protein characterization. Trends in Biochemical Sciences 20 219-224. A review of die ba.sic application of ma.ss. spectrometric methods to the analysis of protein. sequence and. structure. 

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. 

At this time, more then thirty channel DNAs have been cloned and characterized from various sources, predominantly from Drosophila melanogaster, mouse, rat and human cDNA/genomic libraries [6-31]. Inspection of the derived primary K channel protein sequences indicates that voltage-gated channels belong to a... 

Historically the Shaker (Sh) K channel was the first K channel which was cloned and characterized [6-10]. Subsequently many more channel cDNAs and genes have been isolated and studied. Yet Sh channels remained in the forefront of channel research. The study of Sh channel mutants has provided the most thorough insight into structure-function relationships of K channels to date. I will first discuss in this chapter the primary sequences of voltage-gated channels. I will only use a few selected examples for discussion. As of this time, so many related K channel protein sequences have been published that it is not feasible to discuss all of them. Subsequently, I will describe in detail the present knowledge about functional K" " channel domains which are implicated in activation, inactivation and selectivity of the channel. 

KOYAMA, Y., OHMORI, H. Nucleotide sequence of the Escherichia coli solA gene encoding a sarcosine oxidase-like protein and characterization of its product, Gene, 1996, 181, 179-183. 

The systematic characterization of secondary protein modifications is different from sequencing a protein. For a systematic analysis of protein modifications, the complete protein sequence should be covered by the investigation. This is difficult to achieve since the enzymatic digestion of a protein is not homogeneous. The sequence coverage depends on the nature of the protein, its quantity, and the enzyme used. Typical... 

All predicted protein sequences lacking any significant sequence similarity to characterized proteins are labeled hypothetical proteins. The majority of these cases come from the genome sequencing projects. Example ... 

Although a number of assays and technologies are available to characterize and test protein molecules, such as peptide mapping, protein sequencing, carbohydrate analysis, electrophoresis, ELISA, and mass spectroscopy, they are not as definitive as the methods used for small molecule drugs. Hence, the test for similarity is not as well defined in the case of proteins. However, as... 

Subtilisins are a class of related serine endo proteases produced by members of the Bacillus genus. The B.amvloliauefaciens subtilisin (BPN ) is well-characterized with regard to its DNA sequence 4 protein sequence (5), X-ray crystal structure (6) and kinetic properties (7). With this wealth of information available, BPN was chosen as the model enzyme for our recombinant approach. 

The structure and function of enzymes is determined by both the amino acid sequence and the surrounding solvent. The overall stability of proteins is characterized by a subtle balance of into- and inter-molecular interactions. The basic principle of the structure (and of the stability) of the proteins is related to the nature of its normal enviromnent for (water) soluble globular proteins this is the minimization of the hydrophobic surface area, whereas the contrary is the case for membrane proteins (Jaenicke, 1991). 

NMR analysis allows characterization of proteins to an atomic level. The most frequently used nuclei on protein NMR are 41, 2H, 13C, 15N, and 170 with proton NMR (Jefson, 1988). The use of NMR methods for protein sequence and conformational studies was limited to the small proteins or peptides because high magnetic fields were required but not widely available to study larger molecules and it was very time consuming with the capability of instruments in the past. 

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