Kinetic data analysis of chaperone-like activity of Wt, R69C and D109H αB-crystallins

The α-Crystallin (α-Cry) functions as a molecular chaperone, preventing the formation of stress-induced protein aggregation which is important for maintenance of lens transparency. The kinetic data of Wt, R69C and D109H αB-Crys chaperone-like activity were obtained by UV–Vis spectroscopy in both thermal- and chemical-induced aggregation methods. The data were analyzed using physical parameters describing the aggregation process including t* (the characteristic of the stage of nucleation), and t0.5 (the characteristic of the stage of aggregate growth) and Ilim (the limiting value of the light scattering intensity). Parameter t* is duration of the lag phase and the lower t* value is associated with the higher rate of the nucleation stage. Also, the lower values of t0.5 indicated the higher rate of aggregate growth stage. The change in parameter Ilim in the presence of chaperones can be connected with the change in the size of protein aggregates. These data are related to the research article entitled “Structural and functional characterization of D109H and R69C mutant versions of human αB-crystallin: the biochemical pathomechanism underlying cataract and myopathy development” [1].

© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).

Kinetic data analysis of chaperone-like activity of different aB-Crys
The aggregation process, obeying the mechanism of nucleation-dependent aggregation, involves the stage of nucleation and the stage of aggregate growth. When studying the aggregation kinetics by registration of increment of the light scattering intensity, the following equation is often applicable for description of the dependence of the light scattering intensity on time [2e4]: I ¼ I lim f1 À exp½ À k I ðt À t* Þg; ðt > t*Þ (1) where k I is the rate constant of the first order, I, I 0 and I lim are the current, initial (at t ¼ 0) and limiting (at t / ∞) values of the light scattering intensity and t* is a point in time corresponding to crossing of the theoretical curve, which calculated with this equation, with the horizontal line I ¼ 0 calculated with this equation. Parameter t* is duration of the lag phase and may be considered as a characteristic of the rate of the nucleation stage. The lower the t* value, the higher is the rate of the nucleation stage. Eq. (1) can be transformed as follows: Specifications

Value of the Data
The data provide a further mechanistic insight into anti-aggregation ability of human aB-Cry and its mutant forms (R69C and D109H). The data might be used for modulating chaperone activity of the mutant proteins using chemical chaperones. These data also show the effect of each chaperone on the important parameters shaping chaperoning activity. These data clearly display the client protein-specific chaperone activity of the mutant proteins.
The physical sense of parameter t 0.5 is the following. At t ¼ (t* þ t 0.5 ) the value of I is equal to I lim /2. Parameter t 0.5 may be considered as a characteristic of the rate of the stage of aggregate growth. The lower the t 0.5 value, the higher is the rate of the stage of aggregate growth. The change in parameter I lim in the presence of chaperones can be connected with the change in the size of protein aggregates. The diminishing of the I lim value in the presence of chaperones can be due to the decrease in the size of protein aggregates.  Table S1 in supplementary materials [1]. As can be seen from this Figure  this peculiarity of the shape of the kinetic curve, the following equation can be proposed for description of the dependence of the light scattering intensity on time: where B is constant. This equation was used to describe the kinetic curves of insulin aggregation in the absence of any additives (Fig. 1A, B ¼ 0.00834 ± 0.00006 min À1 ) and in the presence of Wt aB-Cry ( Fig. 1B; B ¼ 0.00220 ± 0.00002 min À1 ) and in the presence of R69C mutant form of aB-Cry ( Fig. 1C; When studying the effect of D109H mutant form of aB-Cry on insulin aggregation, Eq. (2) was used for description of the kinetic curve (B ¼ 0). Parameters I lim , t* and t 0.5 for insulin aggregation calculated using theoretical equations (2) and (3) are given in Table 1. Fig. 2A shows the kinetics of aggregation of catalase at 60 C. The initial kinetic data are represented in Table S2 in supplementary materials. To analyze the shape of the kinetic curve, we have constructed the dependence of derivative dI/dt on I (Fig. 2B). The dependence of dI/dt on I can be described by equation [3]:

Aggregation of catalase at 60 C
where D is constant. Parameter m was found to be equal to 3.4 ± 0.2. Integration of Eq. (4) gives the following expression: It should be noted, if m ¼ 1, the dependence of the light scattering intensity on time follows Eq. (2). Parameters I lim , t*, t 0.5 and m calculated for the kinetic curves using Eq. (5) are given in Table 1.

Aggregation of lysozyme in the presence of 20 mM DTT (42 C)
Kinetics of DTT-induced aggregation of lysozyme at 42 C in the absence and in the presence of Wt, R69C and D109H aB-Crys (Fig. 4) was analyzed using Eq. (5). The initial kinetic data are represented in Table S3 in supplementary materials. Parameters I lim , t*, t 0.5 and m for lysozyme aggregation are given in Table 1.

1.1.4.
Aggregation of g-crystallin at 60 C Fig. 5 shows the kinetics of aggregation of g-crystallin (g-Cry) at 60 C in the absence and in the presence of Wt, R69C and D109H aB-Crys. The initial kinetic data are represented in Table S4 in supplementary materials. Parameters I lim , t*, t 0.5 and m for lysozyme aggregation calculated using Eq. (5) are given in Table 1.

Chaperone-like activity assessment of R69C and D109H mutant aB-Crys
The chaperone-like activity of mutant aB-Crys was measured using different client proteins including insulin, lysozyme, catalase and g-Cry [5]. Aggregation of bovine pancreatic insulin (0.3 mg mL À1 ) and chicken egg white lysozyme (0.2 mg mL À1 ) was induced with dithiothreitol (DTT; 20 mM) in buffer A at 40 C. The heat-induced aggregation of g-Cry and bovine liver catalase was performed at 60 C. The molar ratio of chaperone/g-Cry was set at 1:2. The aggregation of catalase (0.3 mg mL À1 ) was induced in the presence of different chaperones. The light scattering of the client proteins was measured while the concentration of the chaperone was fixed at 0.1 mg mL À1 . The aggregation of g-Cry was obtained in the presence of 0.08 mg mL À1 of Wt and mutant aB-Cry chaperones.
The aggregation progress of the client proteins was monitored by measuring light scattering at 360 nm as a function of time, using a T90 þ UVeVis spectrophotometer (PG Instrument Ltd., UK) equipped with a Peltier temperature controller. Moreover, all of the measurements were done in the absence of shaking/stirring condition. Origin Pro 8.0 SR0 software was used for the calculations. To characterize the degree of agreement between experimental data and calculated values, we used the coefficient of determination R 2 (see Ref. [6]).