In situdetection of a novel lysozyme monoclinic crystal form upon controlled relative humidity variation

Abstract

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.

Revisiting the structure of a synthetic somatostatin analogue for peptide drug design

“…Significant methodological advances over the past decades transformed macromolecular XPD from an impossible task to a respectable method, circumventing the need for a suitable single crystal of the molecule of interest (Margiolaki & Wright, 2008;Karavassilia & Margiolaki, 2016). This approach has already been effectively employed for phase identification (Basso et al, 2005;Norrman et al, 2006;Collings et al, 2010;Papageorgiou et al, 2010;Karavassili et al, 2012;Valmas et al, 2015;Fili et al, 2015Fili et al, , 2016Trampari et al, 2018) as well as structure determination (Margiolaki et al, 2005(Margiolaki et al, , 2007(Margiolaki et al, , 2013Valmas et al, 2017) of a wide range of proteins and peptides (Tedesco et al, 2000(Tedesco et al, , 2001Cheung et al, 2002;Inouye & Kirschner, 2006;Fujii et al, 2011;Das et al, 2015) of pharmacological interest.…”

Applications of X-ray Powder Diffraction in Protein Crystallography and Drug Screening

“…However, after long exposure, collapse of the crystal matrix was irreversible. These experiments indicate that the lowest rH at which crystals preserve their structure is between 75% and 80% for those of monoclinic symmetry, and between 71% and 75% for those of tetragonal symmetry, while they underlined the need of long enough waiting time for the crystalline samples to reach their equilibrium [28,29]. This is the first study establishing a preliminary protocol for quick and accurate extraction of structural information from protein polycrystalline precipitates upon humidity variation using X-ray powder diffraction and laboratory instrumentation.…”

“…The amount of water is closely related to relative humidity or temperature levels around the sample. Even small changes in the sample's environment may cause subsequent alterations in solvent channels, driving protein molecules not to occupy exactly equivalent positions within or between unit cells, frequently leading to insufficient resolution of their diffraction patterns [28,29,162,163].…”

“…The main goals of such experiments are either the improvement of the diffraction patterns obtained, or, from a biological point of view, the structural characterization of a molecule in a very specific condition or the inspection of its behavior upon rH variation. Recently, the effect of relative humidity on protein crystal structures, was investigated in two studies of hen egg-white lysozyme (HEWL) polycrystalline precipitates, via in situ laboratory XRPD measurements [28,29]. Two different crystallization protocols were employed in which microcrystals were grown using the salting-out approach [177] in batch by mixing equal amounts of protein solution and crystallization buffer [28,29].…”

“…Although the use of polycrystalline specimens has some disadvantages, as this approach may provide low or medium resolution diffraction data (3-10Å), these samples are produced more easily and quicker than a single crystal. XRPD methods allow for the examination of low-quality crystals as an ensemble, protein polymorph screening becomes a common procedure, while time-resolved studies are also possible [23,26,28,29]. Preliminary structural features extracted from these experiments can provide crucial information toward their complete structural characterization.…”