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

et al. (2011) A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins. PLoS ONE 6(7): e22154. doi:10.1371/journal.pone.0022154 A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins Tatyana Eronina 0 Vera Borzova 0 Olga Maloletkina 0 Sergey Kleymenov 0 Regina Asryants 0 Kira Markossian 0 Boris Kurganov 0 Darren R. Flower, Aston University, United Kingdom 0 1 Bach Institute of Biochemistry, Russian Academy of Sciences , Moscow , Russia , 2 Kol'tsov Institute of Developmental Biology, Russian Academy of Sciences , Moscow , Russia , 3 Belozersky Institute of Physico-Chemical Biology, Moscow State University , Moscow , Russia To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = Kagg(T2T0)2, where Kagg is the parameter characterizing the initial rate of aggregation and T0 is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T0 corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets. - Funding: This study was funded by the Russian Foundation for Basic Research (grants 11-04-00932-?, 11-04-01271-a and 11-04-01350-?), the Program Molecular and Cell Biology of the Presidium of the Russian Academy of Sciences and Dmitry Zimin Dynasty Foundation, the Federal Target Program Scientific and Scientific-Pedagogical Personnel of Innovative Russia in 20092013 (the state contract No. P1356). The funders had no role in study design,data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Senisterra and coworkers [1,2] elaborated a high-throughput light-scattering-based method for screening of ligands specifically interacting with protein targets. Thermal protein denaturation is used to characterize the binding of ligands to their target protein. This method is based on the assumption that the proteins under study irreversibly denaturate and form aggregates during thermal denaturation. Light scattering as a measure of protein aggregation is a very sensitive technique. Protein aggregation is studied in the regime of heating with a constant rate. The dependence of the light scattering intensity on temperature has a sigmoid shape. At rather high temperatures the light scattering intensity (I) reaches a limiting value (Ilim). To characterize thermostability of a protein, Senisterra and coworkers used the temperature (Tagg) corresponding to the middle point of the transition, i.e., a temperature at which I = Ilim/2. Parameters Ilim and Tagg are determined by fitting of the experimental dependence of I on temperature with the following empiric equation, analogous to the Boltzmann equation: where B is a constant. According to the idea advanced by Senisterra and coworkers a change in the Tagg value in the presence of a ligand characterizes the effect of the latter on protein thermostability. To substantiate this conclusion, the authors additionally used differential scanning calorimetry (DSC), which is a source of direct information on the protein resistance to high-temperature exposure. The authors also constructed a plot demonstrating the existence of a correlation between the Tagg value and the position of the maximum on the DSC profiles (Tm). It is evident that the accuracy of determining parameter Tagg is connected with the reliability of the estimation of parameter Ilim. When trying to estimate parameter Ilim we should take into account that the true limiting level of the light scattering intensity may not be reached because of precipitation of the largesized aggregates formed at high temperatures. Such a precipitation results in the decrease in the light scattering intensity, and the real experimental dependence of I on temperature looks like a curve passing through a maximum. The maximum value of the light scattering intensity may be lower than the Ilim value calculated from Eq. (1). Besides, the correlation between the increment of the light scattering intensity and the degree of protein denaturation should be controlled not only by checking the correlation between parameters Tagg and Tm, but by stricter analysis of the relationship between turbidimetric data and calorimetric data, supplying direct information on the degree of protein denaturation. To avoid the uncertainty in the estimation of parameter Ilim, in the present work we proposed new parameters, which characterize the rate of aggregation. To determine these parameters, there is no need for the full dependences of the light scattering intensity on temperature, since the proposed parameters (the initial temperature of aggregation and the parameter characterizing the rate of change in the light scattering intensity with temperature) are calculated from the initial parts of the dependences of I on temperature. The use of these parameters allows us to obtain the quantitative characteristics of the effect of the agents to be tested on the rate of aggregation. Since the initial stage of thermal aggregation of the proteins is the stage of denaturation, the ligands under study involve agents affecting both the stage of denaturation and the stage of aggregation. To demonstrate the applicability of the proposed approach, thermal aggregation of glycogen phosphorylase b (Phb; EC 2.4.1.1) from rabbit skeletal muscles and bovine liver glutamate dehydrogenase (GDH; EC 1.4.1.3) were used as examples. Mathematical description of the initial parts of the dependences of the light scattering intensity on temperature As a preliminary step we discuss the kinetics of thermal aggregation of the proteins registered at a fixed temperature. To analyze the initial parts of the dependences of the light scattering intensity (I) on time, the following empiric equation was proposed [3]: In this equation Kagg is a constant with the dimension of (counts/ s)?min22 and t0 is the duration of the lag period (t0 is a point in time at which the light scattering intensity begins to increase). The applicability of Eq. (2) was demonstrated for thermal aggregation of Ph (...truncated)