Protein sequencing, methods

Fig, 10.25 The six environment categories used by the 3D profiles method. (Figure adapted from Bowie j U, R Liith. and D Eisenberg 1991. A Method to Identify Protein Sequences That Fold into a Known Three-Dunensinnal Structure. Science 253 164-170.)... 

Cuff IA and G J Barton 1999. Evaluation and Improvement of Multiple Sequence Methods for P Secondary Structure Prediction. Proteins Structure, Function and Genetics 34 508-519. 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

Although the techniques described have resulted in the determination of many protein stmctures, the number is only a small fraction of the available protein sequences. Theoretical methods aimed at predicting the 3-D stmcture of a protein from its sequence therefore form a very active area of research. This is important both to understanding proteins and to the practical appHcations in biotechnology and the pharmaceutical industries. 

W. Machleidt, Proc. Int. Conf. So/idPhase Methods in Protein Sequence Anal. 17 (1975). 

WR Pearson. Comparison of methods for searching protein sequence databases. Protein Sci 4 1145-1160, 1995. 

JU Bowie, R Liithy, D Eisenberg. A method to identify protein sequences that fold into a known three-dimensional structure. Science 253 164-170, 1991. 

WR Taylor. Multiple protein sequence alignment Algorithms and gap insertion. Methods Enzymol 266 343-367, 1996. 

G Vriend, C Sander, PFW Stouten. A novel search method for protein sequence-structure relations using property profiles. Protein Eng 7 23-29, 1994. 

To gain the most predictive utility as well as conceptual understanding from the sequence and structure data available, careful statistical analysis will be required. The statistical methods needed must be robust to the variation in amounts and quality of data in different protein families and for structural features. They must be updatable as new data become available. And they should help us generate as much understanding of the determinants of protein sequence, structure, dynamics, and functional relationships as possible. 

K Sjdlander, K Karplus, M Brown, R Hughey, A Krogh, IS Mian, D Haussler. Dirichlet mixtures A method for improved detection of weak but significant protein sequence homology. Comput Appl Biosci 12 327-345, 1996. 

Combinatorial methods are often referred to as in vitro or directed evolution techniques. In nature, the random DNA mutations that lead to changes in protein sequences occur rarely and so evolution is usually a slow... 

Natural selection works through the complementary processes of mutation and genetic reassortment by recombination. The oligonucleotide-directed mutagenesis methods used in the foregoing examples do not allow for recombination instead, mutations are combined manually to optimize a protein sequence. Willem Stemmer at Maxygen invented a method of directed evolution that uses both mutation and recombination. This method, called... 

Heilman, EJ., Wiksell, E., and Karlsson, B.-M. (1990). A new approach to micropreparative desalting exemplified by desalting a reduction/alkylation mixture, presenred ar Eighr International Conference on Methods in Protein Sequence Analysis, Kiruna, Sweden, July 1-6. 

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. 

Moreover, molecular modeling is one key method of a wide range of computer-assisted methods to analyze and predict relationships between protein sequence, 3D-molecular structure, and biological function (sequence-structure-function relationships). In molecular pharmacology these methods focus predominantly on analysis of interactions between different proteins, and between ligands (hormones, drugs) and proteins as well gaining information at the amino acid and even to atomic level. 

Besides sensitive methods for the analysis of proteins, bioinformatics is one of the key components of proteome research. This includes software to monitor and quantify the separation of complex samples, e.g., to analyze 2DE images. Web-based database search engines are available to compare experimentally measured peptide masses or sequence ions of protein digests with theoretical values of peptides derived from protein sequences. Websites for database searching with mass spectrometric data may be found at http //www.expasy.ch/tools, http //prospector.ucsf. edu/ and http //www.matrixscience.com. 

Specific sequencing methods (eg, for proteins and nucieic acids)... 

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