# In Situ XRPD Investigation of Relative Humidity-Induced Lattice Responses in Tetragonal Hen Egg-White Lysozyme

## TL;DR

This study uses X-ray diffraction to show how humidity changes affect the structure of a protein crystal, revealing its stability and resilience.

## Contribution

The study introduces in situ XRPD to investigate humidity-induced structural changes in tetragonal HEWL protein crystals.

## Key findings

- Tetragonal HEWL crystals maintain structural integrity under humidity changes with limited lattice variation.
- Dehydration causes a-axis contraction and c-axis expansion, while rehydration reverses this trend.
- Prolonged low humidity leads to partial crystallinity degradation but not structural collapse.

## Abstract

Protein crystals are intrinsically hydrated systems, and their structural integrity is strongly influenced by environmental humidity. Understanding the effects of relative humidity (RH) variation on crystal stability is therefore essential for both fundamental research and applied studies. In this work, the structural response of tetragonal hen egg-white lysozyme (HEWL) to controlled RH variation was investigated using in situ X-ray powder diffraction (XRPD). Polycrystalline HEWL samples were subjected to systematic gradual dehydration and rehydration cycles, as well as to non-gradual RH variation protocols. Pawley analysis of the XRPD data enabled monitoring of the evolution of unit cell parameters and unit cell volume as a function of RH. Under all experimental conditions, the tetragonal polymorph (space group P43212; a = 79.105 (4) Å, c = 38.231 (2) Å) was preserved. RH variation induced smooth, continuous and anisotropic lattice changes, characterized by a decrease in the a (=b)-axis and a concomitant increase in the c-axis upon dehydration, while rehydration resulted in the opposite behavior. The overall magnitude of lattice variation remained limited (within ±2%), indicating a high degree of structural stability. Partial degradation of crystallinity was observed only after prolonged exposure to low RH levels. These findings demonstrate the remarkable structural resilience of tetragonal HEWL and highlight the effectiveness of in situ XRPD as a powerful tool for probing hydration-driven lattice responses in protein crystals under realistic environmental conditions.