The use of oil in a counter-diffusive system allows to control nucleation and coarsening during protein crystallization

Abstract

A method is described to control nucleation and coarsening in the process of protein crystallization. The control of these events is achieved by combining a biphasic oil/water system with a multi-compartment crystallization device. The addition of oil resulted in a significant decrease of the diffusion rate of a precipitant agent solution. When compared with crystal growth in the absence of oil, significantly smaller amounts of initially precipitated protein were observed. Thus, this method shifts crystal growth from a nucleation-coarsening (Ostwald ripening) equilibrium to a new equilibrium where Ostwald ripening is drastically diminished. Moreover, larger, rod-shape, high quality single crystals can also be obtained as a result of this equilibrium shift.

Introduction

The formation of high quality protein crystals remains a challenge for high-throughput protein crystallography and structural genomics. This is mainly due to the complex multi-factorial nature of crystallization. Even though crystal screens can reduce the number of assays, it is still usually necessary to evaluate crystallization conditions by trial and error [1]. Moreover, once initial crystallization conditions have been identified, it is a common practice to further evaluate a subset of new conditions in order to improve crystal size and quality.

Protein crystallization in capillary tubes provides a simple procedure for controlling the growth process [2], [3], [4]. A multi-compartment device using this procedure was developed to evaluate simultaneously some of the parameters that determine crystal growth [5]. More recently, a single-compartment device named the Granada Box has been commercialized [6]. With both single cell and multi-compartment devices, it is possible to obtain large, high quality, rod-shaped protein crystals when gels and capillary tubes are used together to reduce convective transport and sedimentation.

Protein crystallization combining the use of gels and capillary tubes was first described more than three decades ago [7]. High-quality crystals of E. coli thioredoxin, for example, could be grown in quartz capillary tubes using agarose gel as a neutral network to control diffusion; these crystals diffracted up to 1.9 Å resolution [8]. More recently, different approaches to control diffusion in order to obtain protein single crystals have been proposed, including the control of water evaporation by quick release of vapour diffusion pressure [9], [10], as well as the addition of paraffin oil [11]. The addition of certain oils to crystallization trials is particularly attractive since oils can help to improve the size and quality of protein crystals [12], [13].

In a previous report discussing the advantages of the combination of oils and gels to grow crystals inside capillary tubes [13], we noted the difficulty of forming a homogeneous layer of oil using dimethylphenylmethoxylane, a high-density oil. Here, an improved method to control coarsening and diffusion in order to crystallize proteins into capillary tubes is presented and discussed. This method affords a tighter control of the diffusion of a precipitating agent solution. The results obtained indicate that coarsening (Ostwald ripening) can be drastically diminished under the conditions described here. Besides, the multi-compartment device designed to carry out all the experiments is also suitable for simultaneously evaluating oil/water systems of different composition as well as for obtaining large, high-quality protein single crystals for high-resolution, X-ray diffraction analysis.

Coarsening, which is also referred as Ostwald ripening, is a term originally used to explain the process by which droplets of a minority phase in a binary mixture evolve during the late stages of phase separation, giving place to condensed, larger, low-curvature droplets [14], [15]. It has more recently been recognized that coarsening is relevant to understanding phase transitions in a wide variety of systems, such as block-copolymers [16], electrically charged colloidal suspensions [17], ferrofluids and ferromagnetic films at constant magnetization [18], [19], proteins in Langmuir monolayers [20], [21] as well as protein crystallization [22], [23]. In the last case, coarsening (Ostwald ripening) is characterized by the diffusion of single protein molecules to form small size aggregates following a first-order phase transition. Eventually, the small size aggregates form larger ones (clusters) of such a size that protein precipitation can be observed. These aggregation events take place at early stages of crystal growth (i.e., during the last stages of liquid phase separation) and depending on the kinetics of cluster growth, they can eventually give rise to the formation of new crystals.