A strategy for selecting the pH of protein solutions to enhance crystallization

Abstract

The pH of a solution is an important parameter in crystallization that needs to be controlled in order to ensure success. The actual pH of the crystallization droplet is determined by the combined contribution of the buffers in the screening and protein solutions, although the contribution of the latter to the pH is often ignored. In this study, the effects of the buffer and protein solution pH values on the results of screening are systematically investigated. It was found that these parameters significantly affected the results and thus the following strategy for the selection of appropriate pH values is proposed: (i) when screening with only one protein solution, the pH should be as low, as high or as divergent from the pI as possible for a basic, acidic or neutral protein, respectively, within its stable pH range; (ii) when screening with two protein solutions, the pH values should be well separated from one another; and (iii) when multiple pH values are utilized, an even distribution of pH values is the best approach to increase the success rate of crystallization.

Using isoelectric point to determine the pH for initial protein crystallization trials

“…, 2006 ). However, such a link has also been disputed ( Huber and Kobe, 2004 ) and it has also been suggested that ‘the pI value of a protein should be avoided when choosing the pH for a protein solution’ ( Zhang et al. , 2013 ).…”

“…In-house proteins were also buffered at near neutral pH (either pH 6.5 or pH 7.5). In order to determine the conditions for crystallization, each protein was initially screened using sitting-drop vapour diffusion with a bespoke 96 condition sparse matrix screen buffered at 6 different pHs using the multi-component buffer PCTP ( Newman, 2004 ; Zhang et al. , 2013 ).…”

“…The isoelectric point for each sequence in the SGC dataset was determined in the same manner using an Excel spreadsheet with visual basic for applications (Microsoft VBA). For the AZ dataset, the pI was either obtained from Zhang et al. (2013) or calculated as above and was confirmed using isoelectric focusing.…”

“…It should therefore follow that a solution with a pH matching the isoelectric point would be ideal for crystallization although this has never been confirmed. One possible reason for this is that the recorded pH is that of the buffer in the crystallization solution rather than the final pH of the crystallization cocktail ( Zhang et al. 2013 ).…”

A comparative study on the quality of protein crystals obtained using the cross-diffusion microbatch and sitting-drop vapor diffusion methods

“…Because higher-quality protein crystals can provide more detailed information regarding their threedimensional structure, all structural biologists are eager to improve crystals to the required quality. Moreover, other chemical optimization approaches, such as pH value optimization, 8 which affects crystal nucleation and growth processes, 9,10 precipitant concentration change, which causes a transition of the crystal growth mechanism from twodimensional nucleation to dislocation growth, 11 and protein engineering, 12,13 which uses mutagenesis or recombinant techniques to modify the protein in order to enhance its crystallizability, are widely used in structural biology. 1,2 Hence, the development of novel methods for improving crystal quality has been pursued.…”