Application of differential scanning fluorimetry to X-ray protein crystallography

X-ray crystallography remains a powerful technique for determining high-resolution protein structures; however, obtaining high-quality crystals is a significant bottleneck. This study presents a detailed experimental workflow that employs differential scanning fluorimetry (DSF) to optimize protein crystallization. DSF, which measures protein thermal stability, was used to refine both protein buffer composition and crystallization conditions. The method was applied to two distinct proteins: CreD, a nitrosuccinate lyase, and HIRA, a histone chaperone. For CreD, DSF-based optimization of the protein buffer enhanced the crystal quality, increasing the resolution from 3.32 to 2.18 Å. For HIRA(644-1017), DSF-guided optimization of the protein buffer significantly improved the protein solubility from 0.1 to 19.1 mg ml^-1, facilitating the growth of initial crystals. Further optimization of the crystallization conditions using DSF, combined with microseeding, improved the crystal quality, leading to structure determination at 2.45 Å resolution. This study demonstrates that DSF is a valuable tool for efficiently optimizing protein crystallization. The workflow presented here, involving initial DSF-based optimization of protein buffers followed by DSF-guided optimization of crystallization conditions, offers a rational approach to enhancing protein crystal quality, thereby facilitating structure determination by X-ray crystallography.