Dissociative mechanism for irreversible thermal denaturation of oligomeric proteins

Abstract

Protein stability is a fundamental characteristic essential for understanding conformational transformations of the proteins in the cell. When using protein preparations in biotechnology and biomedicine, the problem of protein stability is of great importance. The kinetics of denaturation of oligomeric proteins may have characteristic properties determined by the quaternary structure. The kinetic schemes of denaturation can include the multiple stages of conformational transitions in the protein oligomer and stages of reversible dissociation of the oligomer. In this case, the shape of the kinetic curve of denaturation or the shape of the melting curve registered by differential scanning calorimetry can vary with varying the protein concentration. The experimental data illustrating dissociative mechanism for irreversible thermal denaturation of oligomeric proteins have been summarized in the present review. The use of test systems based on thermal aggregation of oligomeric proteins for screening of agents possessing anti-aggregation activity is discussed.

Keywords: Conformational lock; Dissociative mechanism; Oligomeric proteins; Protein denaturation.

Fig. 1

Kinetics of thermal inactivation of Phb from rabbit skeletal muscles at 53 °C (0.08 M Hepes–NaOH, pH 6.8). The relative enzymatic activity A/A ₀ versus time plot was constructed on the basis of the data represented in the work by Kurganov et al. (2000). Enzyme concentration: 1 1.6 and 2 5.2 mg/mL

Fig. 2

The kinetics of thermoinactivation of Phb. The dependence of the relative enzymatic activity A/A ₀ on time is plotted for the data represented in Eronina et al. (2009). Conditions: 0.08 M Hepes buffer, pH 6.8, containing 0.1 M NaCl; 48 °C

Fig. 3

Contact regions in the dimeric molecule of Phb. a Ribbon diagram of the crystal structure of Phb (PDB 9GPB), consisting of two identical subunits, viewed perpendicular to the molecular dyad axis (depicted as a double-ended arrow). Two contact regions are encircled. The amino acid residues belonging to different subunits (subunits A and A′, respectively) are shown in red and blue. b–d The expanded view of the contact regions I (b) and II (c, d) along the dyad axis of the dimer (b, c) and perpendicular to it (d). Residues involved in the formation of hydrogen bonds (depicted by dots) are shown by toggle wire representation and identified by the one-letter code. Residues and structural elements of the subunit A′ are designated by primes. The figure is generated by using the MolBrowser 3.8-4a software (MolSoft)

Fig. 4

Temperature dependences of excess heat capacity (C _p^ex) of GAPDH (10 mM sodium phosphate buffer, pH 7.5) at various concentrations of the protein: 1 0.5, 2 1.5 and 3 3.0 mg/mL. C _p^ex was calculated per GAPDH tetramer. The heating rate was 1 °C/min. Adapted from Markossian et al. (2006)

Scheme 1

The general scheme of thermal aggregation of oligomeric proteins proceeding under crowded conditions. Oligomer* is an oligomer with weak conformational lock [intermediate E₂ ^(m) in Eq. (6)]