PROTEIN EXPERIMENTS

Protein Structure Experiments

Protein Structure Experiments

The Structure of Proteins

Before proceeding to a discussion of proteomics, it is necessary to elaborate on the major issues related to proteins that we have already started to discuss in Chapter 1. Proteins consist of amino acids. The side chains (radicals), amino acids are hydrocarbon chains, which sometimes forms a cyclic structure. Amino acids contain an amino group (-NH2) and carboxyl (-COOH). Amino acids form polymers, connecting with each other through a specific connection, called peptide bonds. The peptide bond is formed with the participation of the amino group of one amino acid and the carboxyl group of another amino acid (Brendan & Tooze, 1999, pp. 231).

Determination of the primary structure of proteins is reduced to finding the order of the amino acids in the polypeptide chain. This problem is solved using the method of sequencing. Properly sequenced at its current level to determine the amino acid sequence and polypeptides whose size does not exceed a few tens of amino acid residues. At the same time studied polypeptide fragments much shorter than those of natural proteins, which have to deal with. Therefore, you must pre-cut source of the polypeptide into short fragments. After sequencing of fragments obtained by their need to re-make of the original sequence.

Thus the definition of the primary sequence of the protein is reduced to the following basic steps:

The splitting of the protein into several fragments in length, available for sequencing.

Sequencing of each of the resulting fragments.

Build a complete protein structure from the established structures of its fragments.

For the specific cleavage of proteins in certain points apply to both enzymatic and chemical methods. Of the enzymes that catalyze the hydrolysis of proteins in certain points, the most widely used trypsin and chymotrypsin. Trypsin catalyzes the hydrolysis of peptide bonds located after lysine and arginine residues. Chymotrypsin predominantly cleaves proteins after aromatic amino acid residues - phenylalanine, tyrosine and tryptophan. If necessary, the specificity of trypsin can be improved or changed. For example, treatment of citraconic anhydride studied protein leads to the acylation of lysine residues. In this modified protein cleavage will take place only on the balance of arginine. Along with enzymatic methods using chemical methods and the breakdown of proteins. For this purpose, is often used cyanogen bromide, proteolytic at methionine residues.

Sequencing Experiment

Sequencing is carried out by a method known as the method of Edman. Sequential processing of a polypeptide having a free terminal -Amino group, any alkyl or arilizotiotsianatom in weakly alkaline medium leads to the formation of the corresponding thiourea, which is in a moderately acidic environment leads (for values of acidity, not damaging the peptide bond) is cleaved to form the corresponding tiogidantoina. The original procedure is based on the Edman phenyl isothiocyanate, and thus on the formation of feniltiogidantoinov.

The result is a feniltiogidantoin containing the amino acid residue side R 1, which can be identified by measurement of any physical or physico-chemical characteristics, which allows to distinguish between hydantoins corresponding to the different composition of proteins within ...