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

Tatyana Eronina,Boris Kurganov,Sergey Kleymenov,Olga Maloletkina,Regina Asryants,Kira Markossian,Vera Borzova,Darren R Flower

doi:10.1371/journal.pone.0022154

Abstract

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 = K agg(T−T 0)2, where K agg is the parameter characterizing the initial rate of aggregation and T 0 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 T 0 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.

Highlights

Senisterra and coworkers [1,2] elaborated a high-throughput light-scattering-based method for screening of ligands interacting with protein targets
When studying the kinetics of thermal aggregation of some proteins by dynamic light scattering (DLS) we have shown that the hydrodynamic radius (Rh) of the initially registered aggregates amounts to tens of nanometers [23]
Turbidimetric Registration of phosphorylase b (Phb) Aggregation Thermal aggregation of Phb was studied in the regime wherein temperature was elevated with a constant rate (1uC/min)

Summary

Introduction

Senisterra and coworkers [1,2] elaborated a high-throughput light-scattering-based method for screening of ligands 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. 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: I~

Methods

Results

Conclusion