Understanding Water Equilibration Fundamentals as a Step for Rational Protein Crystallization

Abstract

BackgroundVapor diffusion is the most widely used technique for protein crystallization and the rate of water evaporation plays a key role on the quality of the crystals. Attempts have been made in the past to solve the mass transfer problem governing the evaporation process, either analytically or by employing numerical methods. Despite these efforts, the methods used for protein crystallization remain based on trial and error techniques rather than on fundamental principles.Methodology/Principal FindingsHere we present a new theoretical model which describes the hanging drop method as a function of the different variables that are known to influence the evaporation process. The model is extensively tested against experimental data published by other authors and considering different crystallizing conditions. Aspects responsible for the discrepancies between the existing theories and the measured evaporation kinetics are especially discussed; they include the characterization of vapor-liquid equilibrium, the role of mass transfer within the evaporating droplet, and the influence of the droplet-reservoir distance.Conclusions/SignificanceThe validation tests show that the proposed model can be used to predict the water evaporation rates under a wide range of experimental conditions used in the hanging drop vapor-diffusion method, with no parameter fitting or computational requirements. This model combined with protein solubility data is expected to become a useful tool for a priori screening of crystallization conditions.

Protein Crystallization As a Process Step in a Novel Meso Oscillatory Flow Reactor: Study of Lysozyme Phase Behavior

“…The solubility 39,40 and the metastability 41 curves were drawn based on the data available in the literature at quiescent conditions. Clear solutions above the solubility curve were indicative of the metastable region, once in spite of being supersaturated these solutions…”

“…Based on lysozyme phase behavior (Figure 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 and metastability curves reported in the literature for microbatch experiments 39,40 were linearized (see Figure 6) according to Martins et al 40 equation:…”

“…The values of the constants A and B that best fit the experimental results are given in Table 1. According to Figure 6 and Table 1, metastability data can be described by the exponential relationship proposed by Martins et al 40 , used for the description of lysozyme solubility curve with sodium chloride as the precipitant agent. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Under agitation, collision between protein molecules is increased, increasing thus nucleation probability.…”

Interaction between Plate Make and Protein in Protein Crystallisation Screening

“…Recently, a new model for the equilibration processes was reported for the hanging drop crystallisation format [6]. This model extends previously proposed models designed to describe the rate of equilibration of hanging drop crystallisation experiments, and the authors consider the effect of well geometry, drop to reservoir distance, drop volume and precipitant type on the rate of equilibration.…”

The effect of diluting crystallization droplets on protein crystallization in vapor diffusion method

“…The vapor diffusion crystallization technique (hanging or sitting drop method) is the most widely used protein crystallization method [7,8]. The protein crystallization droplet and the reservoir solution are put at different locations in a sealed plate well, which is aimed to concentrate the protein crystallization droplet to supersaturation to form crystals.…”