Proteins primary

Fi.sieh, Y. L., et al., 1996. Automated analytical. sy.stem for the examination of protein primary. structure. Analytical Chemistry 68 455-462. An analyti-... 

Complex peptide mixmres can now be analyzed without prior purification by tandem mass spectrometry, which employs the equivalent of two mass spectrometers linked in series. The first spectrometer separates individual peptides based upon their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses determined. As the sensitivity and versatility of mass spectrometry continue to increase, it is displacing Edman sequencers for the direct analysis of protein primary strucmre. 

Figure 3. Hydropathy Profile and mature protein primary sequence of the deduced p-Subunit Precursor Protein.

Upon its generation, sequence information is normally submitted to various databases. The major databases in which protein primary sequence data are available are listed in Table 2.4. Also included in this table are the major nucleic acid sequence databases, as amino acid sequence information can potentially be derived from these. 

Empirical statistical methods, which are based upon data generated from studying proteins of known three-dimensional structure and correlation of such proteins primary amino acid sequences with structural features. 

Protein primary structure databases include the following ExPASy Molecular Biology Server (Swiss-Prot) expasy.ch/ Protein Information resources (PIR) pir.georgetown.edu Protein Research Foundation (PRF) prf.or.jp/en/os.html. 

MOLECULAR SHAPE OE PROTEINS PRIMARY, SECONDARY AND TERTIARY STRUCTURES... 

Molecular shape of proteins primary, secondary and tertiary structures... 

While these stages I-IV represent generic processes applying to all food proteins, it is significant to note that even small differences in the protein primary structure can sometimes cause major changes in functionality (Wilde, 2000). For example, the genetic variants A and B of P-... 

In the absence of crystallographic or NMR data, predictive techniques based on protein primary sequences can be used to elaborate crude 3D models. Such models will suggest that certain amino acid residues are involved in forming the active (receptor) site. The assignment of structural or functional roles to particular residues can be tested by site-directed mutagenesis, and the model can be further refined by consideration of SAR among ligands. 

The differences in primary structure can be especially informative. Each protein has a distinctive number and sequence of amino acid residues. As we shall see in Chapter 4, the primary structure of a protein determines how it folds up into a unique three-dimensional structure, and this in turn determines the function of the protein. Primary structure is the focus of the remainder of this chapter. We first consider empirical clues that amino acid sequence and protein function are closely linked, then describe how amino acid sequence is determined finally, we outline the many uses to which this information can be put. 

Various procedures are used to analyze protein primary structure. Several protocols are available to label and identify the amino-terminal amino acid residue (Fig. 3-25a). Sanger developed the reagent l-fluoro-2,4-dinitrobenzene (FDNB) for this purpose other reagents used to label the amino-terminal residue, dansyl chloride and dabsyl chloride, yield derivatives that are more easily detectable than the dinitrophenyl derivatives. After the amino-terminal residue is labeled with one of these reagents, the polypeptide is hydrolyzed to its constituent amino acids and the labeled amino acid is identified. Because the hydrolysis stage destroys the polypeptide, this procedure cannot be used to sequence a polypeptide beyond its amino-terminal residue. However, it can help determine the number of chemically distinct polypeptides in a protein, provided each has a different amino-terminal residue. For example, two residues—Phe and Gly—would be labeled if insulin (Fig. 3-24) were subjected to this procedure. 

The primary structure of a peptide or protein is defined by the sequence of amino acids. In this experiment the procedures that are in common use to determine protein primary structure are applied to an unknown dipeptide. Amino acid composition of the peptide will be determined by acid hydrolysis followed by HPLC, CE, or paper chromatography. The identity of the NH2-terminal amino acid will be achieved by the dansyl method followed by thin-layer chromatography. 

R Garrett and C Grisham, Biochemistry, 2nd ed (1999), W B Saunders (Orlando, FL), pp 107-152 Protein primary structure... 

A major problem, until recently, was the determination of the protein primary structure, but with the advent of modern analysis of DNA this has become comparatively easy. One of the first structures to be described was that of insulin which contains 60 amino-acids and has a molecular weight of 12,000. Once the primary structure is known, it is possible to predict the secondary and tertiary structures using additional information obtained through X-ray crystallography of the crystallised protein. 

Fig. 5.A2. Protein primary structure. The amino-acid sequence of ox insulin...

Protein primary hydration Native conformation g H20/g protein Denaturated conformation g H20/g protein... 

Similar examples of the dependence of intramolecular transfer rates on protein primary gtructure are found in reactions in the cytc/cytc peroxidase system. 

The polypeptide chain folds up to form a specific shape (conformation) in the protein. This conformation is the three-dimensional arrangement of atoms in the structure and is determined by the amino acid sequence. There are four levels of structure in proteins primary, secondary, tertiary and, sometimes but not always, quaternary. 

Application of Mass Spectrometry to Protein Primary Sequence Determination... 

National Biomedical Research Foundation specializes in providing a database for protein primary structure. This database contains all the information from the Atlas of Protein Sequence and Structure edited by M.O. Dayhoff. In this database proteins are categorized according to their super family grouping. In addition to the primary structure information, detailed descriptions of proteins, including active site, prosthetic group, etc., are included. 

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