A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

Eronina, Tatiana B.; Borzova, Vera A.; Maloletkina, Olga I.; Kleymenov, Sergey Y.; Asryants, R.A.; Markossian, Kira A.; Bi, Kurganov

doi:10.1371/journal.pone.0022154

Cited by 15 publications

(15 citation statements)

References 58 publications

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“…This process is often seen by the appearance of an exothermic heat effect immediately following the endothermic denaturation peak. The exothermic heat effect is most likely due to precipitation since it can be minimized by modifying the geometry of the cell and reducing the length of the precipitation path . Even if the exothermic heat effect is suppressed, denaturation under these conditions is not coupled to a significant exposure of nonpolar groups to the solvent (due to aggregation) and no positive change in heat capacity is observed.…”

Section: Resultsmentioning

confidence: 99%

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

et al. 2017

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The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (~100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day−1. Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.

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Section: Resultsmentioning

confidence: 99%

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

et al. 2017

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“…Each experiment was repeated in triplicate. A reduction in intensity at high temperatures indicates precipitation of large-sized aggregates70.…”

Section: Figurementioning

confidence: 99%

Structural implications of Ca2+-dependent actin-bundling function of human EFhd2/Swiprosin-1

Park

Kwon

et al. 2016

Sci Rep

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EFhd2/Swiprosin-1 is a cytoskeletal Ca2+-binding protein implicated in Ca2+-dependent cell spreading and migration in epithelial cells. EFhd2 domain architecture includes an N-terminal disordered region, a PxxP motif, two EF-hands, a ligand mimic helix and a C-terminal coiled-coil domain. We reported previously that EFhd2 displays F-actin bundling activity in the presence of Ca2+ and this activity depends on the coiled-coil domain and direct interaction of the EFhd2 core region. However, the molecular mechanism for the regulation of F-actin binding and bundling by EFhd2 is unknown. Here, the Ca2+-bound crystal structure of the EFhd2 core region is presented and structures of mutants defective for Ca2+-binding are also described. These structures and biochemical analyses reveal that the F-actin bundling activity of EFhd2 depends on the structural rigidity of F-actin binding sites conferred by binding of the EF-hands to Ca2+. In the absence of Ca2+, the EFhd2 core region exhibits local conformational flexibility around the EF-hand domain and C-terminal linker, which retains F-actin binding activity but loses the ability to bundle F-actin. In addition, we establish that dimerisation of EFhd2 via the C-terminal coiled-coil domain, which is necessary for F-actin bundling, occurs through the parallel coiled-coil interaction.

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“…(3) can be used for the description of the initial parts of the kinetic curves of aggregation in the experiments where temperature was elevated with a constant rate [91]:where T 0 is the initial temperature of aggregation, i.e., the temperature at which the light scattering intensity begins to increase, and k agg is a parameter which characterizes the rate of aggregation. Parameters T 0 and k agg can be used for quantitative characterization of the ability of various agents to suppress protein aggregation.…”

Section: Theory Quantification Of the Chaperone-like Activitymentioning

confidence: 99%

“…According to theoretical views developed by Kurganov and co-workers [68], [91]–[93], the point in time t = t 0 or point in temperature T = T 0 corresponds to the appearance of start aggregates. A start aggregate contains hundreds of denatured protein molecules.…”

Section: Theory Quantification Of the Chaperone-like Activitymentioning

confidence: 99%

Quantification of Anti-Aggregation Activity of Chaperones: A Test-System Based on Dithiothreitol-Induced Aggregation of Bovine Serum Albumin

et al. 2013

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The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin allowed us to make a conclusion that a non-linear character of the dependence of v agg on concentration of intact α-crystallin was due to the dynamic mobility of the quaternary structure of α-crystallin and polydispersity of the α-crystallin–target protein complexes. To characterize the anti-aggregation activity of the chemical chaperones (arginine, arginine ethyl ester, arginine amide and proline), the semi-saturation concentration [L]0.5 was used. Among the chemical chaperones studied, arginine ethyl ester and arginine amide reveal the highest anti-aggregation activity ([L]0.5 = 53 and 58 mM, respectively).

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A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

Cited by 15 publications

References 58 publications

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

Structural implications of Ca2+-dependent actin-bundling function of human EFhd2/Swiprosin-1

Quantification of Anti-Aggregation Activity of Chaperones: A Test-System Based on Dithiothreitol-Induced Aggregation of Bovine Serum Albumin

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