The study of amorphous aggregation of tobacco mosaic virus coat protein by dynamic light scattering

https://doi.org/10.1016/j.bpc.2006.11.006Get rights and content

Abstract

The kinetics of heat-induced and cetyltrimethylammonium bromide induced amorphous aggregation of tobacco mosaic virus coat protein in Na+/Na+ phosphate buffer, pH 8.0, have been studied using dynamic light scattering. In the case of thermal aggregation (52 °C) the character of the dependence of the hydrodynamic radius (Rh) on time indicates that at certain instant the population of aggregates is split into two components. The size of the aggregates of one kind remains practically constant in time, whereas the size of aggregates of other kind increases monotonously in time reaching the values characteristic of aggregates prone to precipitation (Rh = 900–1500 nm). The construction of the light scattering intensity versus Rh plots shows that the large aggregates (the start aggregates) exist in the system at the instant the initial increase in the light scattering intensity is observed. For thermal aggregation the Rh value for the start aggregates is independent of the protein concentration and equal to 21.6 nm. In the case of the surfactant-induced aggregation (at 25 °C) no splitting of the aggregates into two components is observed and the size of the start aggregates turns out to be much larger (107 nm) than on the thermal aggregation. The dependence of Rh on time for both heat-induced aggregation and surfactant-induced aggregation after a lapse of time follows the power law indicating that the aggregation process proceeds in the kinetic regime of diffusion-limited cluster–cluster aggregation. Fractal dimension is close to 1.8. The molecular chaperone α-crystallin does not affect the kinetics of tobacco mosaic virus coat protein thermal aggregation.

Introduction

Amorphous (unordered) aggregates of different proteins have been recently implicated in pathogenesis of many important human diseases [1], [2], [3], [4], [5]. However, structural studies of such aggregates are greatly hampered by their large size, transient character, and heterogeneity. Probably, because of this, up to now, as far as we know, only one detailed model of mechanism of amorphous protein aggregation has been proposed. This is the Goldberg–Wetzel model [6], [7] according to which unordered aggregates are formed by intermolecular interactions of those domains of partly disordered protein molecules, which in the native state were involved in intramolecular interactions between the same domains.

Tobacco mosaic virus (TMV) coat protein (CP) is well known for its ability to produce ordered assemblies [8]. It is well known that at room temperature, pH values in the range from 7.5 to 9.0, and ionic strength in the range from 10 to 100 mM, the TMV CP exists in the form of so called 4S-protein (a dynamic mixture of pentamers and trimers, with a minor amount of monomers). At pH of about 7.0 and ionic strength of about 100 mM TMV CP with high efficiency specifically assembles in vitro with TMV RNA with formation of completely native virions. At pH  6.0 in the absence of RNA, the protein produces long virus-like helical aggregates called repolymerized protein [8].

However, TMV CP also turned out to be a good model for studies of thermal amorphous aggregation. In contrast to most of other unspecific protein aggregation system [7], [9], the process of amorphous TMV CP aggregation is highly reproducible and its rate can be easily manipulated by changing solution ionic strength, protein concentration, and temperature [10], [11], [12], [13]. Recently we have observed that the TMV CP amorphous aggregation can be also induced in neutral phosphate buffer (PB) at room temperature (25 °C) by low micromolar concentrations of cationic surfactant cetyltrimethylammonium bromide (CTAB) [14].

And finally, possible existence of amyloidogenic potential in TMV CP have been suggested by R. Diaz-Avalos and D. Caspar from the results of their studies of the TMV CP “off-pathway” aggregate (stacked disks) structure [15].

Dynamic light scattering (DLS) gives valuable information on the size of protein aggregates and is widely used for the study of the kinetics of protein aggregation. DLS, as applied to analysis of protein aggregates, has some evident merits. Modern variants of DLS technique allow registering the initial stages of protein aggregation, where the fraction of the aggregated protein constitutes some tenth of a percent of the total amount of the protein in the system. Besides, this method allows quantitative determination of the individual components in heterogeneous populations of aggregates [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26].

Recently we have studied with the help of DLS the process of aggregation of βL-crystallin in the presence of α-crystallin. We have found that three different types of amorphous aggregates (named “start aggregates”, “basic aggregates” and “superaggregates”) are formed [27]. The construction of the light scattering intensity versus hydrodynamic radius plot allows estimating the size of the start aggregates [27]. The formation of the start aggregates (the primary clusters) has been also observed on thermal aggregation of several other proteins [27], [28], [29].

The goal of the present work was to carry out the comparative study of the kinetics of irreversible heat-induced and reversible CTAB-induced amorphous aggregation of the TMV CP by DLS. It has been shown that in both cases aggregation proceeds by way of the formation of the start aggregates.

Section snippets

TMV purification and proteins preparation

Wild-type (strain U1) TMV was obtained as described elsewhere [30] and its coat protein was isolated by the acetic acid method [31]. The CP preparations were stored at concentrations of 4.5 to 6 mg/ml (259–345 μM) in 5 mM Na+/Na+ PB, pH 8.0, at + 4 or − 20 °C. The TMV CP concentrations were measured by UV spectroscopy using the extinction coefficient A2800.1% of 1.30 [32].

α-Crystallin from bovine eye lenses was purchased from Sigma (USA).

DLS measurements

DLS is widely used in biochemistry to measure the size of

The kinetics of thermal aggregation of TMV CP

Dynamic light scattering allows the changes in the size of aggregates in the course of protein aggregation to be registered. Fig. 1 shows the typical autocorrelation functions measured at various times of incubation of 5.75 μM (0.1 mg/ml) TMV CP in 50 mM PB at 52 °C. Using the DynaLS software we calculated the size distribution of particles formed in the course of aggregation. At sufficiently short times of incubation the distribution function was unimodal (Fig. 2A, B). The mean value of the

Discussion

With the help of DLS it was found that two types of aggregates (basic aggregates and superaggregates) are formed in the course of the TMV CP thermal aggregation (Fig. 2, Fig. 3). The splitting of aggregate population into two components is not a general phenomenon, but it was registered by DLS for thermal aggregation of bovine serum albumin at 58 °C [26], aggregation of dithiothreitol-denatured α-lactalbumin [43], and thermal aggregation of βL-crystallin in the presence of α-crystallin [27].

Conclusions

The results of the present and previous [27], [28], [29] studies testify that in many cases amorphous protein aggregation begins with the formation of rather large start aggregates (with the Rh value from 20 to 100 nm) and further occurs according to the diffusion-limited cluster–cluster aggregation mechanism [35], [36], [37], [38]. Nevertheless, important differences in the process of aggregation are observed for individual systems. In the course of thermal aggregation of mixtures of βL- and

Acknowledgements

This work was supported by the Russian Foundation for Basic Research (grants 05-04-49503 and 05-04-48691), the Program for Fundamental Research “Molecular and Cell Biology” of the Presidium of Russian Academy of Sciences, and INTAS (grant 03-51-4813).

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