Protein! s primary

Although a protein s primary structure is determined by covalent bonds along the peptide backbone, the secondary and higher levels of structure depend in large part... 

As we saw in Section 13.4, the shape of a protein molecule is determined by the protein s primary structure, which is the sequence of amino acids. So what, then, controls a protein s amino acid sequence The answer to this question, as we are now ready to explore, is the gene. 

Once the protein s primary sequence has been determined, the location of disulfide bonds in the intact protein can be established by repeating a specific enzymatic cleavage on another sample of the same protein in which the disulfide bonds have not previously been cleaved. Separation of the resulting peptides shows the appearance of one new peptide and the disappearance of two other peptides, when compared with the enzymatic digestion product of the material whose disulfide bonds have first been chemically cleaved. In fact, these difference techniques are generally useful in the detection of sites of mutations in protein mole-... 

A protein s primary structure is its amino acid sequence. Its secondary structure is the orientation of segments of the protein chain into a regular pattern, such as an a-helix or a P-pleated sheet. Its tertiary structure is the three-dimensional shape into which the entire protein molecule is coiled. 

Two of the most common secondary structures found in proteins are helical and pleated-sheet conformations, shown in the diagram above. One might compare the helical structure of a protein, for example, with the spiral-shaped cord found on many home telephones. These structures form when atoms, ions, or other chemical species in one part of the protein s primary structure are attracted to other atoms, ions, or chemical species with opposite electrical charges in another part of the structure. 

As well as predicting protein structure, it is also of interest to try to predict solvent accessibility from the protein s primary sequence of amino acid residues. Solvent accessibility concerns the area of a protein s surface that is exposed to the surrounding solvent. The importance of this concept is that these accessible regions have the potential to interact with other entities, including endogenous proteins and drugs. Similarly, if the protein of interest is an enzyme, only residues with solvent accessibility could be part of the enzyme s active site. This means that an interaction site of interest, one involved in signal transduction (see Krauss, 2003), requires spatial accessibility to the solvent (Ofiran and Rost, 2005). 

While still far from perfect, in silico methods of predicting secondary structure and solvent accessibility using only a protein s primary sequence have matured considerably in recent years (Ofran and Rost, 2005). Ability to predict tertiary structure is currently less well developed but improving. 

This technique gives information about the protein s primary structure, which may include its amino and/or carboxyl terminal groups (Edman, 1950). For recombinant DNA-derived proteins, this analysis serves to confirm the amino acid sequence predicted by the DNA sequence. The analysis can also be useful to determine the protein s homogeneity. 

Peptide mapping is a method that enables the determination of protein identity when compared to a standard. When compared to previous lots of the same product, it serves to determine the stability of the protein s primary sequence, which in turn reflects the genetic stability of the producer cells. This method is capable of detecting small differences between proteins in one or more amino acids. The detection will be dependent upon an amino acid alteration affecting the observed peptide profile (Figure 13.1 see color section). 

Chemists have long appreciated that a protein s primary amino acid sequence determines its three-dimensional structure. It has also been known for some time that proteins are able to carry out their diversified functions only when they have folded up into compact three-dimensional structures. The protein-folding problem first gained prominence in the 1950s and 1960s, when Christian Anfinsen demonstrated that ribonuclease could be denatured (unfolded) and renatured reversibly. 

There exists a general concensus that the primary structure of a protein eventually determines its tertiary structure. Therefore it is extremely important to be able to study a protein s primary structure. 

Determining a protein s primary structure is similar to solving a complex puzzle. Several steps are involved in solving the amino acid sequence of any protein. 

Reversible unfolding of proteins has been known for some time. The folding of several proteins occurs spontaneously showing that the required information for folding is present in the protein s primary structure [1]. 

A protein s primary structure, as its name su ests, is of fundamental importance. For the protein to carry out its particular function, the primary structure must be correct. We shall see later that when the primary structure is correct, the protein s polyamide chain folds in particular ways to g ve it the shape it needs for its particular task. 

The order or sequence of amino acids in the protein chain is called the protein s primary structure. Differences in primary structure are what enable proteins to be tailored for different and very specific functions. 

Once a peptide map is characterized using online MS, the chromatographic profile alone can serve as a routine analytical tool to monitor the protein s primary structure and covalent modifications, and is often used for batch release or stability testing of biopharmaceuticals. However, whenever in-depth characterization of a protein is needed, such as that required for comparability studies or reference material characterization, the peptide map should be coupled with MS to ensure a thorough examination of all peptides in the map. 

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