Self-association of proteins

In the pH range close to the protein s lEP an interesting phenomenon of non-uniform redistribution of protein molecules among polysaccharide chains occurs (Tolstoguzov et al. 1985). The reason is that in the vicinity of the protein lEP the hydrophobic protein-protein and electrostatic protein-polysaccharide interactions can be energetically comparable with each other. Protein-protein association on the anionic polysaccharide matrix (or self-association of proteins), which is mainly due to hydrophobic interactions, is usually enhanced when the pH approaches the protein lEP. Accordingly, under conditions of a relatively weak protein-polysaccharide interaction, each free site situated near the site on the polysaccharide chain already occupied by a protein molecule becomes thermodynamically preferable for further binding of protein molecules. This leads to cooperative protein adsorption on an anionic polysaccharide. Some parts of polysaccharide chains tend to be completely covered by protein molecules (as in a virus) while other parts are completely free of protein. 

Our analysis indicates no self-association of protein molecules for BSA solutions [112] at pH = 5.4 and 7.3 (Fig. 11). The fraction of dimers giving good agreement with experiment in this case is zero this holds true for both the one-component [41] and the multicomponent model. The results obtained by the two theoretical models for pH = 5.4, where experimental results are denoted by (x), practically coincide. Experimental data for pH=7.3, are denoted by (+) and one-component model results by the dashed line. In this case no IET results since the multicomponent model could be obtained for concentrations above 330 g/dm-3 and therefore only one-component calculations are shown. 

The purpose of this chapter is to review the principles of large zone size exclusion chromatography (large zone SEC) as applied to the study of self-association of macromolecules, specifically proteins. This selfassociation is not only of fundamental scientific interest, but also has practical consequences with respect to protein purification and to maintenance of biological function. The recent development of recombinant DNA technology has made available substantial amounts of sosie proteins, that were formerly available in very limited amounts if at all. These recombinant proteins must be obtained in a state that is fully biologically active. In some cases proteins are active as monomers, but in others the active protein exists as a dimer or an oligomer. Clearly it is of practical interest to understand the self-association of proteins under a variety of conditions. 

The application of large zone SEC to the study of the self-association of proteins has been discussed in several comprehensive reviews during the past 10-15 years (ref. 1-3). However, a basic presentation for the practicing protein chemist is needed, and that is the goal of this chapter. 

Self-Association of Proteins The idea that many proteins are non-covalent complexes of two or more identical subunits can be traced back to the early work of Svedberg with the analytical ultracentrifuge (ref. 4). Such protein assembly is referred to as quaternary structure, a topic reviewed by Klotz et al. in 1970 (ref. 5). 

Investigations into the self-association of proteins have focused primarily on (1) stoichiometry, (2) energetics, and (3) the influence of substrates or other effector molecules. In addition, much effort has gone into the understanding of the geometry of these interactions, but this subject will not be considered in this chapter. 

This chapter has been an attenpt to illustrate the utility of the large zone SEC approach to the study of the self-association of proteins. Perhaps the greatest drawback to the application of this technique is the propensity of certain proteins to adsorb to the coluam packings being used. This possibility must be carefully assessed for every protein studied and for every colusm packing and buffer system used. Failure to exclude the possibility of protein-gel interactions can lead to an incorrect interpretation of the experimental results. In spite of this limitation this approach offers the advantages of (1) experimental simplicity,... 

In spite of the overwhelming evidence suggesting that recombinant resilin is amorphous, there are some results that suggest that a level of defined stmcmre cannot be completely ruled out. In particular, the fact that the protein solution coacervates when cooled (Figure 9.7) suggests that there is a degree of self-association between protein molecules. 

Other clues to the self-association of recombinant resilin in solution, and thus a degree of defined stmcture, include the propensity of the monomer proteins to covalently cross-link very rapidly through dityrosine side chains using a mthenium-based photochemical method [29]. Proteins which do not naturally self-associate do not form biomaterials when exposed to the Ru(ll)-based photochemical procedure (Elvin, C.E. and Brownlee, A.G., personal communication). Furthermore, Kodadek and colleagues showed that only intimately associated proteins are cross-linked via this zero-A photochemistry procedure [45]. 

Fig. 2b. The appearance of two crystal forms shows that the protein in the membrane exists in equilibrium between the protomeric aj8 unit and oligomeric (aj8>2 forms. The high rate of crystal formation of the protein in vanadate solution shows that transition to the E2 form reduces the difference in free energy required for self association of the protein. This vanadate-method for crystallization has been very reproducible [34-36] and it also leads to crystalline arrays of Ca-ATPase in sarcoplasmic reticulum [37] and H,K-ATPase from stomach mucosa [38]. 

Adams, C.A., Kar, S.R., Hopper, J.E., and Fried, M.G. (2004) Self-association of the amino-terminal domain of the yeast TATA-binding protein./. Biol. Chem. 279, 1376-1382. 

CaM), of fatty acid carboxylate to intestinal fatty acid binding protein (IFABP), and of peptides (e.g, melittin) to Ca +-saturated calmodulin (holo CaM)]. We also extended PLIMSTEX to protein-protein interactions involving self associations of various insulins [33]. These are widely studied systems, and their individual K values range from to 10 M h... 

Self-association of Insulin A Protein-Protein Interaction... 

Cytochromes serve as electron donors and electron acceptors in biological electron transfer chains, and with >75,000 members (53) they provide the bulk of natural heme proteins in biology. Cytochromes may be fixed into place within an extended electron transfer chain, such as the membrane-bound 6l and 6h of the cytochrome bci complex, or may be soluble and act as mobile electron carriers between proteins, for example, cytochrome c (54). In either role, the cytochrome may be classified by the peripheral architecture of the porphyrin macrocycle. Figure 1 shows the dominant heme types in biological systems, which are hemes a, b, c, and d, with cytochomes b and c being most prevalent. The self-association of a protein with heme via two axial ligands is a... 

For instance, denaturation and partial hydrolysis of proteins oppositely influence their incompatibility with other biopolymers (Tolstoguzov 1991). Most biopolymers are polyelectrolytes. Factors such as pH and salt concentration affect their interactions with one another, with the solvent and their compatibility. For instance, when the pH is shifted to their isoelectric point (lEP), the thermodynamic incompatibility of proteins is usually enhanced by self-association of the protein molecules. Generally, protein-neutral polysaccharide mixtures separate into two phases when the salt concentration exceeds 0.15 M. 

The actual mechanism of self-association of monomeric protein into amyloid is complex and three mechanisms of stmcture conversion have been proposed (32) In templated assembly, a monomeric native state peptide binds to an existing nucleus. Upon binding, there is a change in the secondary stmcture of the monomer as it is added to the growing chain. Monomer directed conversion involves the presence of a misfolded monomer that templates the stmcture conversion of a native monomer, followed by disassociation and chain formation. The third model is nucleated polymerization, which is the most widely accepted model for the fibril growth. 

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