INTEGRATING COMPUTATIONAL PREDICTIONS AND BIOPHYSICAL CHARACTERIZATION TO GUIDE GRH1 CONSTRUCT SELECTION FOR STRUCTURAL STUDIES

Golgi Reassembly and Stacking Protein Homologue 1 (Grh1) is the Saccharomyces cerevisiae member of the GRASP family, involved in Golgi organization and unconventional protein secretion. Like other GRASPs, Grh1 comprises a conserved N-terminal GRASP domain containing two tandem PDZ domains and a highly disordered C-terminal serine/proline-rich (SPR) region. Phylogenetic analysis of representative GRASP homologs highlighted the evolutionary conservation of the GRASP domain, supporting its use as the structural core for construct design. Nevertheless, the intrinsic disorder of the SPR region is expected to increase conformational heterogeneity, hindering high-resolution structural studies. Here, we employed an integrated computational and experimental approach to rationally design and evaluate truncated Grh1 constructs for structural biology applications. Sequence and structure-based computational analyses included phylogenetic reconstruction, intrinsic disorder prediction, structural prediction, crystallization propensity assessment and stereochemical validation, followed by preliminary biophysical characterization of the selected construct. Intrinsic disorder prediction (IUPred3) identified the C-terminal SPR region as the major source of structural disorder, providing the rationale for generating three truncated constructs (D1, D2 and D3). AlphaFold3 predictions consistently indicated improved structural confidence for the truncated constructs, accompanied by lower predicted aligned error and reduced fractions of disordered residues. XtalPred-RF further supported construct selection by predicting shorter intrinsically disordered regions and lower instability indices while maintaining favorable crystallization scores for all constructs. Consistently, stereochemical validation using MolProbity revealed high overall model quality, with most Ramachandran outliers in the full-length model located within the predicted disordered C-terminal region and absent from the truncated constructs. Preliminary biophysical characterization of the selected construct demonstrated efficient soluble expression and purification, preservation of secondary structure, favorable solution behavior and the formation of initial crystallization hits. Together, these results demonstrate that rational construct design supported by complementary computational analyses and preliminary biophysical characterization provides a robust strategy for selecting Grh1 constructs with improved structural properties for high-resolution studies using X-ray crystallography and NMR spectroscopy.