The effect of relative humidity (rH) on protein crystal structures, an area that has attracted high scientific interest during the past decade, is investigated in this study on hen egg‐white lysozyme (HEWL) polycrystalline precipitates via in situ laboratory X‐ray powder diffraction (XRPD) measurements. For this purpose, HEWL was crystallized at room temperature and pH 4.5, leading to a novel monoclinic HEWL phase which, to our knowledge, has not been reported before. Analysis of XRPD data collected upon rH variation revealed several structural modifications. These observations, on a well‐studied molecule like HEWL, underline not only the high impact of humidity levels on biological crystal structures, but also the significance of in‐house XRPD as an analytical tool in industrial drug development and its potential to provide information for enhancing manufacturing of pharmaceuticals.
Variation of relative humidity (rH) greatly affects the internal order of solventbased protein crystals, and the rearrangement of molecules can be efficiently recorded in distinct diffraction patterns. This study focuses on this topic, reporting the effect of rH variation experiments on hen egg white lysozyme (HEWL) polycrystalline precipitates of tetragonal symmetry using X-ray powder diffraction (XRPD). In situ XRPD data were collected on HEWL specimens during dehydration and rehydration processes using laboratory instrumentation. A known polymorph [space group P4 3 2 1 2, a = 79.07181 (1), c = 38.0776 (1) Å ] was identified during gradual dehydration from 95 to 63% rH and vice versa. Pawley analysis of collected data sets and accurate extraction of unitcell parameters indicated a characteristic evolution of the tetragonal axes with rH. In addition, there is a low humidity level below which samples do not retain their crystallinity. This work illustrates the accuracy of laboratory XRPD as a probe for time-resolved studies of proteins and in situ investigations of gradual structural modifications upon rH variation. These experiments provide essential information for improving production and post-production practices of microcrystalline protein-based pharmaceuticals. research papers J. Appl. Cryst. (2019). 52, 816-827 S. Logotheti et al. Effects of relative humidity on HEWL 817
Obtaining well diffracting crystals of membrane proteins is often challenging, but chances can be improved by crystallizing them in lipidic conditions that mimic their natural membrane environments. One approach is the high lipid–detergent (HiLiDe) method, which works by mixing the target protein with high concentrations of lipid and detergent prior to crystallization. Although this approach is convenient and flexible, understanding the effects of systematically varying lipid/detergent ratios and a characterization of the lipid phases that form during crystallization would be useful. Here, a HiLiDe phase diagram is reported for the model membrane protein MhsT, which tracks the precipitation and crystallization zones as a function of lipid and detergent concentrations, and is augmented with data on crystal sizes and diffraction properties. Additionally, the crystallization of SERCA1a solubilized directly with native lipids is characterized as a function of detergent concentration. Finally, HiLiDe crystallization drops are analysed with transmission electron microscopy, which among other features reveals liposomes, stacked lamellae that may represent crystal precursors, and mature crystals with clearly discernible packing arrangements. The results emphasize the significance of optimizing lipid/detergent ratios over broad ranges and provide insights into the mechanism of HiLiDe crystallization.
The simplicity of XRPD data collection and the uniqueness of the diffraction pattern that each polymorph shows, marks powder diffraction as the most suitable technique for quick and accurate characterization of microcrystalline suspensions. However, for protein samples, the intense synchrotron beam for ultrahigh-resolution XRPD data collection, typically causes radiation-damage effects. The damage rapidly appears during the measurement, affecting both angular (FWHM) and dspacing resolution. As radiation damage can be a serious obstacle for collecting high quality data, measurements on a laboratory system have a lot to offer. Here, we present our analysis results of well known tetragonal and of a new monoclinic HEWL polymoph on a laboratory X-ray powder diffractometer including in situ measurements under variable relative humidity conditions for both polymorphs. Proteins often crystallize in microcrystalline precipitates. The protein molecules are then surrounded by solvent and their packing arrangement is retained by limited intermolecular contacts. A change in the crystal environment first affects the bulk solvent that fills the intermolecular space, with resulting changes in the crystal structure. In literature it is reported that protein crystals in controlled humidity environments show a large change in unit-cell parameters when the humidity is decreased [1][2]. When a protein crystal is carefully dehydrated, it is in a metastable state in which the crystal initially still retains the original packing structure [2]. Further dehydration may cause the collapse of the crystal lattice: the crystal no longer maintains its packing structure because of the loss of a large amount of bulk solvent. However in some crystals, the dehydration induces a molecular arrangement change resulting in a new crystal structure. This has been already reported for hen egg-white (HEW) lysozyme [3]. While dehydration can induce structural changes, this is also likely to happen upon hydration of the same crystals. The observed gradual structural changes during our experiments as well as phase transitions upon dehydration and hydration of HEWL are analyzed in the relative humidity range 50% -95%. Dehydration and hydration processes are reversible in humidity cycles in the range of 95% rH to 75% rH. Without stabilizing PEG the lower limit for dehydration of tetragonal HEWL is around 75% rH. With PEG the tetragonal HEWL samples remain crystalline below 75% rH, but show phase transitions and larger variations of the cell parameters. Below 75% rH another new tetragonal polymorph was discovered.
The bacterial amino acid transporter MhsT of the SLC6 family was crystallized in complex with different substrates in order to understand the determinants of substrate specificity of the transporter. Surprisingly, crystals of the different MhsT-substrate complexes showed interrelated, but different crystal packing arrangements. Space group assignment and structure determination of these different crystal forms presented challenging combinations of pseudosymmetry, twinning and tNCS
The bacterial amino-acid transporter MhsT from the SLC6A family has been crystallized in complex with different substrates in order to understand the determinants of the substrate specificity of the transporter. Surprisingly, crystals of the different MhsT–substrate complexes showed interrelated but different crystal-packing arrangements. Space-group assignment and structure determination of these different crystal forms present challenging combinations of pseudosymmetry, twinning and translational noncrystallographic symmetry.
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