Effects of Ionic Strength and Liquid-Liquid Phase Separation on the Nucleation of Protein Crystals

The growth of protein crystals that are suitable for X-ray crystallography is the most difficult and time-consuming step in the determination of the three dimensional structures of the protein molecules due to the lack of fundamental understanding of the protein crystallization. In this respect, a detailed understanding of the effects of solution parameters, such as protein concentration and ionic strength, on the protein is advantageous. In this study, the effects of ionic strength and liquid-liquid separation on the nucleation of protein crystals have been investigated systematically. An optical microscope with a heating/cooling stage has been utilized to investigate liquid-liquid phase separation through measuring cloud-point temperature for supersaturated lysozyme solution. The effects of ionic strength on the cloud-point temperature are studied in detail, which provides the effects of ionic strength on the net attractive interactions among lysozyme molecules. In addition, the phase diagram of the lysozyme crystallization solution can be divided into two parts: the solid-liquid coexistence region and the liquid-liquid co-existence region by measuring the cloud-point temperature. The crystal nucleation and growth processes that happen in both of these two regions were observed by the same experimental set up. Depending on the solution conditions two different nucleation and crystal growth processes were observed and recorded. When nucleation happens in the solid-liquid co-exstience region, the crystals nucleate from the solution without the happening of liquid-liquid phase separation. When the nucleation happens in the liquid-liquid co-existence region, the nucleation and growth mechanism is different. The crystals nucleate after the happening of liquid-liquid separation. The growth and the following nucleation compete with the liquid-liquid separation. Furthermore, micro-batch crystallization experiments of a model protein, lysozyme, were carried out at a series of given lysozyme and sodium chloride concentrations with a fix pH value and temperature. The number of crystals per well were recorded and correlated with solution conditions. It was found that the number of crystals did not increase monotonically with the ionic strength. The nucleation was found to be promoted at the liquid-liquid co-existence region by combining the number of the crystals per well with the measured cloud-point temperature at the solution conditions studied. The increase of the ionic strength in the solid-liquid region increases the net attractive interaction among protein molecules until the liquid-liquid coexistence boundary, where the free energy barrier is reduced significantly. Conversely, the increase of the ionic strength may increase the repulsive force among the protein molecules, which would result in the decrease of the number of the crystals. The experimental results and theoretical analysis in this work may work as a further support to those previously reported work. And the effects of the liquid-liquid phase separation on the protein crystal nucleation may provide opportunities for further tuning of the nucleation.