Elucidation of protein-precipitant phase diagrams and their link to crystal quality.

摘要

Proteins are critical components of many fundamental biological processes in human body. Our understanding of such fundamental biological processes at a molecular level is limited due to the lack of knowledge of the high-resolution structure of the proteins which depends on obtaining X-ray quality crystals of proteins. High throughput screening of a wide range of different conditions is typically required to obtain such high quality crystals of proteins, and unfortunately, these screens are often not successful. In this work we use an evaporation-based crystallization platform in which droplets containing protein and precipitant are gradually concentrated through evaporation of solvent until dryness. Gradual desiccation of protein-containing droplets ensures a phase transition, and the nature of the resulting solid phase(s) observed during and at the end of the experiment is used to guide the identification of crystallization conditions. The outcomes of individual experiments - the formation of gels, precipitates, micro crystals, or crystals - guide the search for and optimization of conditions resulting in diffraction quality crystals. The number, quality, and size of the resulting protein crystals is shown to depend significantly on the rate of increase in protein concentration, a key variable in the method described.;In order to gain a deeper understanding of the phase behavior of solutions used for protein crystallization, an experimental protocol is introduced to determine the solubility boundary and metastable zone width for protein/precipitant systems using the evaporation-based crystallization platform. Recently, generalized phase diagrams for small molecules and nanoparticles have been introduced in which the pair interaction of the long-time self diffusivity, D2, is used as a measure of the strength of particle interactions governing the solubility of a particular compound. The solubility data obtained using the methods developed in this work is mapped onto the generalized phase diagram where D2 is determined by measuring the concentration dependence of protein diffusivities using Pulsed-Field Gradient Nuclear Magnetic Resonance (PFG NMR). We demonstrate that from the knowledge of the metastable zone width we are able to separate the nucleation and growth of crystals to produce larger, good quality crystals.;Understanding of protein crystal nucleation and growth mechanisms is limited by the difficulty of measuring rates of crystal formation and growth. Here we develop a kinetic model capable of predicting changes in the number and size of protein crystals as a function of time under continuous evaporation. The determination of kinetic parameters is greatly simplified if the protocol of decoupling nucleation and growth is applied to collect experimental growth data. Moreover, this model successfully predicts the initial condition of drops that will result in gel formation. We use this model with experimental crystal growth data of hen egg white lysozyme and are able to determine the crystal nucleation and growth rate parameters.;Recently, bicelles (disk shaped "particles") have been introduced as a promising medium for the crystallization of membrane proteins but not much progress has been made due to the lack of understanding of the mechanism underlying the crystallization process. To this end a systematic characterization of the interaction between these bicelles using PFG NMR under different experimental parameters (such as concentrations of ions and other solutes in the solution, temperature and the q ratio of the lipids used to make bicelles) is reported.;In sum, we report new evaporation- and dilution-based protocols that will enable structural biologist to rapidly determine the phase diagram (e.g. solubility boundary, metastable zone width) of proteins of unknown structure using a very small sample volume. The knowledge of phase diagram of a protein/precipitant system thus obtained will be useful in obtaining high quality crystals for X-ray diffraction studies. Moreover, we use theory to compare different protein molecules on a generalized phase diagram using the solubility data obtained from our experiments. The comparison of the solubility boundary and the metastable boundary on the same footing will provide a reasonable estimate of the metastable zone width, which will aid crystallographers in screening conditions conducive for protein crystallization. We also develop a kinetic model that describes the competition between the rates of supersaturation, crystal nucleation and crystal growth occurring in the regulated-evaporation crystallization process. The knowledge of these rates coupled with the knowledge of the phase diagram of a protein/precipitant system will enable crystallographers to predict a priori the outcome of an experiment performed in an evaporation-based crystallization platform.