Determination of the phase diagram for soluble and membrane proteins

Abstract

Methods to efficiently determine the phase behavior of novel proteins have the potential to significantly benefit structural biology efforts. Here, we present protocols to determine both the solubility boundary and the supersolubility boundary for protein/precipitant systems using an evaporation-based crystallization platform. This strategy takes advantage of the well-defined rates of evaporation that occur in this platform to determine the state of the droplet at any point in time without relying on an equilibrium-based end point. The dynamic nature of this method efficiently traverses phase space along a known path, such that a solubility diagram can be mapped out for both soluble and membrane proteins while using a smaller amount of protein than what is typically used in optimization screens. Furthermore, a variation on this method can be used to decouple crystal nucleation and growth events, so fewer and larger crystals can be obtained within a given droplet. The latter protocol can be used to rescue a crystallization trial where showers of tiny crystals were observed. We validated both of the protocols to determine the phase behavior and the protocol to optimize crystal quality using the soluble proteins lysozyme and ribonuclease A as well as the membrane protein bacteriorhodopsin.

Figure 1

(a) Photograph of the automated data acquisition setup. (b) Three 16-compartment evaporation-based crystallization platforms on the X−Y stage. The top inset shows a single crystallization compartment with a 2 μL droplet. The bottom inset shows a schematic depiction of a crystallization compartment with the cross-sectional area (A) and evaporation channel length (L) indicated.

Figure 2

(a) Schematic depiction of the process to determine the solubility boundary of a protein/precipitant system by evaporation (step A) followed by an iterative reverse vapor diffusion process (steps B−D). (b) Depiction of the process from part a in a generalized protein/precipitant phase diagram. From the initial state (point 1), evaporation causes the droplet to increase in concentration along the straight line connecting point 1 to the origin (constant C_P/C_S). Crystals nucleate and are observed at point 2 after the droplet has traversed the metastable zone and crossed the supersolubility boundary into the labile zone. Evaporation is arrested, and the reservoir is filled with a precipitant solution of a lower concentration than point 2. As the droplet equilibrates with this reservoir, the concentration of protein in solution will decrease as the crystal grows until the solubility boundary is reached (point 3). Subsequent iterations of this process cause the droplet to move along the solubility boundary, gradually dissolving the protein crystal (point 4). The point at which the protein crystal has dissolved completely (point 5) represents the point where the initial path of the droplet first crossed the solubility boundary. (c) Repeating the process with droplets of different initial protein/precipitant concentration ratio (C_P/C_S) allows for determination of additional points on the supersolubility and solubility curves.

Figure 3

(a) Solubility and critical supersolubility boundaries for lysozyme vs NaCl as obtained using an evaporation-based crystallization protocol. The solid curves have been fit through our experimental data to provide a guide for the eye. The data shows good agreement with solubility data from the literature.^, (b) Nucleation time as a function of initial protein concentration for lysozyme/NaCl solutions at different C_P/C_S ratios used to obtain the critical supersolubility boundary. The convergence of the various lines at a single y-intercept corresponds to the drying time for the drop based on droplet size and evaporation rate. All data obtained at 23 °C.

Figure 4

(a) Solubility and critical supersolubility boundaries for RNase A vs NaCl as obtained using an evaporation-based crystallization protocol. The solid curves have been fit through our experimental data to provide a guide for the eye. (b) Nucleation time as a function of initial protein concentration for RNase A/NaCl solutions at different C_P/C_S ratios used to obtain the critical supersolubility boundary. The convergence of the various lines at a single y-intercept corresponds to the drying time for the drop based on droplet size and evaporation rate. All data obtained at 23 °C.

Figure 5

Solubility boundary for the membrane protein bacteriorhodopsin vs NaH₂PO₄ obtained using the same protocol as before but with a common vapor diffusion platform. The solid curve was fit through our experimental data to provide a guide for the eye. Data obtained at 23 °C.

Figure 6

(a) Schematic depiction of the method to decouple nucleation and growth events during evaporation-based protein crystallization (step A) followed by reverse vapor diffusion to dissolve some nuclei (step B), and then a second evaporation step to drive crystal growth of the remaining nuclei (step C). (b) Graphical depiction of the process from part a shown in reference to a generalized protein/precipitant phase diagram. From the initial state (point 1), evaporation causes the droplet to increase in concentration along the straight line connecting point 1 to the origin. Crystals nucleate and are observed at point 2 after the droplet has crossed the supersolubility boundary to enter the labile zone. Evaporation is arrested, and the reservoir is filled with a precipitant solution of a concentration between point 2 and where the droplet originally crossed the solubility boundary. As the droplet equilibrates with this reservoir, the number of nuclei present in the droplet will decrease until the solubility boundary is reached (point 3). The well is then opened to evaporation again to allow the few remaining crystal nuclei to grow.

Figure 7

Optical micrographs comparing the results from evaporation-based crystallization experiments (a1, b1, c1) to those using the dilution protocol to decouple nucleation and growth (a2, b2, c2) for three different protein/precipitant systems: (a) lysozyme/NaCl starting from C_P0 = 16 mg/mL, C_S0 = 0.32 M; (b) RNase A/NaCl starting from C_P0 = 60 mg/mL, C_S0 = 2 M; and (c) bacteriorhodopsin/NaH₂PO₄ starting from C_P0 = 17.2 mg/mL, C_S0 = 0.025 M.