Unfolding of oligomeric proteins

There have been several studies of the stability of homodimeric or other oligomeric proteins (30-35). These proteins are interesting as models for understand- 

The latter situation is particularly interesting because the intermediate state, M, may or may not have a structured conformation similar to that of the monomeric units of the dimer. 

Tlic following paragraphs give a detailed experimental description of the unfolding study with Cro. 

Since urea slowly decomposes to form cyanate, stock solutions should be prepared fresh every day or two Access to a CD instrument, such as an Aviv 62DS spectropolarimeter, equipped with a thermoelectric (or thermojacketed, with a circulating water bath) cell holder to maintain constant temperature Quartz cuvettes 

A Stock solution of the protein dialysed against (or, if lyophilized, dissolved in) the same buffer. The concentration will depend upon the optical techniques being used. For CD studies, a 50-200 j,m protein stock solution should allow a minimal volume to be added to the denaturant solution to give a working concentration in the 5-20 (jlm range Pipettes 

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For studying the thermodynamic stability of oligomeric proteins, several aspects have to be taken into account (i) the reversibility of unfolding, (ii) the type of the equilibrium between the native and the completely unfolded protein, (iii) the choice of the detection and denaturation method, and (iv) the specific contribution of the association between the monomers to the overall stability. 

Figure 7. Numerical integration of a Cp-curve to determine tlu population sizes using equations 38 and 39. Note that for obtaining the population 011(1 has to integrate twice, while the enthalpy is obtaiiu d after the first integration. Therefore it cannot be expected that the two (quantities vary in the same manner with temperature. This discrepancy between ai) T) and H T) — H T) is actually seen in the denaturation transition of the dimeric ROP protein. It should occur generally in transitions of oligomeric proteins. The Cp-curve of ROP protein was taken from Steif et al. [54]. The insert shows a comparison of the variation with temperature of the population ai) T) of unfolded proteins and of the relative enthalpy change H — calculated using equations 38 and 39.

The generic tendency of proteins to aggregate into nonfunctional, and sometimes cytotoxic, structures poses a universal problem for all types of cells. This problem is exacerbated by the high total concentrations of macromolecules found within most intracellular compartments, but it is solved by the actions of certain proteins that function as molecular chaperones. Different chaperones act by distinct mechanisms on both the folding of polypeptide chains and their subsequent assembly into oligomeric structures. Many chaperones, but not all, are also stress (or heat shock) proteins because the need for a chaperone function increases under stress conditions that cause proteins to unfold. 

Native state (N) is formed from the unfolded state through intermediate(s) (I). The intermediate(s) acts as a template and propagates to form ordered oligomeric intermediates or protofibrils (PF), which builds up to form stable, insoluble fibrils (F) as shown in Fig. 2. The reaction mechanism involved in this sequence is similar to initiation, propagation, and termination steps in a polymerization reaction. Amorphous aggregates, formed are in equilibrium with the intermediates. In other words, this process is reversible. However, amyloid fibrils are the byproducts of irreversible protein aggregation. 

Fig. 33. Schematic representation of the effects of pressure on oligomeric proteins a) native dimeric protein with cavities/voids b) dissociation of the oligomer, hydration with electrostriction of polar/ionic groups, hydrophobic hydration of unpolar groups (-CR), release of void volume c) weakening of hydrophobic interactions provides pathways for water to penetrate into the interior of the protein, swelling of the core - molten-globule like state d) unfolding of subunits, disruption of the secondary/tertiary structure (hydration of residues not plotted here), loss of cavity volume within protein (adopted from ref. 139).

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