Protein structural alteration

Haemoglobin is one of the most well studied and best understood proteins thanks largely to the early work of Max Perutz, John Kendrew and colleagues. Haemoglobin is now often used as a model allosteric protein and to illustrate the impact of protein structural alterations in disease. 

During this period I was frustrated by my inability to obtain tryptophan synthetase in pure form from Neurospora. Therefore, I searched for a more suitable enzyme for studies on protein structure alterations resulting from mutations. I characterized d- and L-serine deaminases from Neurospora - with this objective in mind but was unable to develop a convenient isolation procedure for mutants lacking these enzymes and so stopped these investigations. 

Noncovalent Forces Stabilizing Protein Structure. Much of protein engineering concerns attempts to alter the stmcture or function of a protein in a predefined way. An understanding of the underlying physicochemical forces that participate in protein folding and stmctural stabilization is thus important. 

WA Lim, A Hodel, RT Sauer, FM Richards. The crystal structure of a mutant protein with altered but improved hydrophobic core packing. Proc Natl Acad Sci USA 91 423-427, 1994. PB Harbury, B Tidor, PS Kim. Repacking proteins cores with backbone freedom Structure prediction for coiled coils. Pi oc Natl Acad Sci USA 92 8408-8412, 1995. 

If cellular redox state, determined by the glutathione status of the heart, plays a role in the modulation of ion transporter activity in cardiac tissue, it is important to identify possible mechanisms by which these effects are mediated. Protein S-,thiolation is a process that was originally used to describe the formation of adducts of proteins with low molecular thiols such as glutathione (Miller etal., 1990). In view of the significant alterations of cardiac glutathione status (GSH and GSSG) and ion-transporter activity during oxidant stress, the process of S-thiolation may be responsible for modifications of protein structure and function. 

Figure 4.14 Diagrammatic representation of (a) oxy-radical>mediated S-thioiation and (b) thiol/disulphide-initiated S-thiolation of protein suiphydryl groups. Under both circumstances mixed disuiphides are formed between glutathione and protein thiois iocated on the ion-translocator protein resulting in an alteration of protein structure and function. Both of these mechanisms are completely reversible by the addition of a suitabie reducing agent, such as reduced glutathione, returning the protein to its native form.

N6. Neubauer, B. A., Pekrun, A., Eber, S. W Lakomek, M and Schroter, W., Relation between genetic defect, altered protein structure, and enzyme function in triose-phosphate isomerase (TPI) deficiency. Eur. J. Pediatr. 151,232a (1992). 

Thus, aside from the covalently polymerized a-chain itself, the majority of protein structure is determined by weaker, noncovalent interactions that potentially can be disturbed by environmental changes. It is for this reason that protein structure can be easily disrupted or denatured by fluctuations in pH, temperature, or by substances that can alter the structure of water, such as detergents or chaotropes. 

The modification of amino acids in proteins and peptides by oxidative processes plays a major role in the development of disease and in aging (Halliwell and Gutteridge, 1989, 1990 Kim et al., 1985 Tabor and Richardson, 1987 Stadtman, 1992). Tissue damage through free radical oxidation is known to cause various cancers, neurological degenerative conditions, pulmonary problems, inflammation, cardiovascular disease, and a host of other problems. Oxidation of protein structures can alter activity, inhibit normal protein interactions, modify amino acid side chains, cleave peptide bonds, and even cause crosslinks to form between proteins. 

The main contributions to AadsG for a globular protein are from electrostatic, dispersion, and hydrophobic forces and from changes in the structure of the protein molecule. Although in this section these contributions are discussed individually, strict separation of the influence of these forces on the overall adsorption process of a protein is not possible. For instance, adsorption-induced alteration of the protein structure affects the electrostatic and hydrophobic interaction between the protein and the surface. When the sorbent surface is not smooth but is covered with (polymeric)... 

Many of the initial biopharmaceuticals approved were simple replacement proteins (e.g. blood factors and human insulin). The ability to alter the amino acid sequence of a protein logically coupled to an increased understanding of the relationship between protein structure and function (Chapters 2 and 3) has facilitated the more recent introduction of several engineered therapeutic proteins (Table 1.3). Thus far, the vast majority of approved recombinant proteins have been produced in the bacterium E. coli, the yeast S. cerevisiae or in animal cell lines (most notably Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. These production systems are discussed in Chapter 5. 

A number of different molecular mechanisms can underpin the loss of biological activity of any protein. These include both covalent and non-covalent modification of the protein molecule, as summarized in Table 6.5. Protein denaturation, for example, entails a partial or complete alteration of the protein s three-dimensional shape. This is underlined by the disruption of the intramolecular forces that stabilize a protein s native conformation, namely hydrogen bonding, ionic attractions and hydrophobic interactions (Chapter 2). Covalent modifications of protein structure that can adversely affect its biological activity are summarized below. 

The monoclonal antibody techniques provide a means of producing a specific antibody for binding antigen. This technique is useful for studying protein structure relations (or alterations) and has been used for devising specific RIAs. 

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

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