Hydration-Induced Structural Changes in the Solid State of Protein: A SAXS/WAXS Study on Lysozyme

Abstract

The stability of biologically produced pharmaceuticals is the limiting factor to various applications, which can be improved by formulation in solid-state forms, mostly via lyophilization. Knowledge about the protein structure at the molecular level in the solid state and its transition upon rehydration is however scarce, and yet it most likely affects the physical and chemical stability of the biological drug. In this work, synchrotron small- and wide-angle X-ray scattering (SWAXS) are used to characterize the structure of a model protein, lysozyme, in the solid state and its structural transition upon rehydration to the liquid state. The results show that the protein undergoes distortion upon drying to adopt structures that can continuously fill the space to remove the protein–air interface that may be formed upon dehydration. Above a hydration threshold of 35 wt %, the native structure of the protein is recovered. The evolution of SWAXS peaks as a function of water content in a broad range of concentrations is discussed in relation to the structural changes in the protein. The findings presented here can be used for the design and optimization of solid-state formulations of proteins with improved stability.

Wide-angle X-ray scattering and molecular dynamics simulations of supercooled protein hydration water

“…Such void effects can also influence the experimental data, although they mainly affect the lower momentum transfer region Q o 0.1 Å À1 due to the density difference at the protein-air interface of lysozyme powders. 36 However we are able to reproduce the peak related to interatomic distances, which contains contributions from both water and protein molecules and according to the experiment exhibits the more significant temperature dependence.…”

“…The scattering intensity exhibits two main contributions: the low-Q peaks are attributed to length scales associated with the protein packing arrangements and secondary structures, whereas the high-Q peak is attributed to interatomic distances. 36 We mainly observed temperature-dependent changes in the relative amplitude and peak position of the latter. The MD simulations reproduce well the scattering peak associated with interatomic distances and allows to decompose the protein and water contribution to the scattering intensity.…”

“…2A shows the temperature dependence of the angularly integrated scattering intensity of hydrated lysozyme with variable hydration levels, ranging from h = 0.05 to h = 0.47. The peak at lower momentum transfer exhibits two sub-peaks at Q = 0.50 Å À1 and Q = 0.65 Å À1 , labeled as i and ii, which arise due to the protein packing arrangements and secondary structures 36 and are enhanced with increasing hydration level. Moreover, the peak at Q = 1.54 Å À1 , labeled iii, relates to the interatomic distances and contains contributions from both protein and water molecules.…”

“…Moreover, the peak at Q = 1.54 Å À1 , labeled iii, relates to the interatomic distances and contains contributions from both protein and water molecules. 36 The scattering intensity of bulk water, shown for comparison, exhibits a peak centered near Q = 1.95 Å À1 , marked by a vertical line. At this momentum transfer, the scattering intensity of peak iii of hydrated lysozyme is shown in the inset as a function of the hydration level.…”

Multiscale X-ray study of Bacillus subtilis biofilms reveals interlinked structural hierarchy and elemental heterogeneity

“…S6). In addition to the contribution at q ~ 14 nm -1 , natively hydrated biofilms had additional contributions around q = 19 nm -1 and q = 26 nm -1 , corresponding to scattering form water (40,41).…”