Alana Gudinas

All Publications

• Ion-selective structural transitions of a disordered repeats-in-toxin protein domain Gudinas, A. P., Shambharkar, G. M., Chang, M. P., Fernandez, D., Matsui, T., Mai, D. J. CELL PRESS. 2026: 373a

  View details for Web of Science ID 001717743900082

• Repetitive proteins that undergo large conformational changes evade structural prediction algorithms. The Journal of chemical physics Chang, M. P., Jin, T., Gudinas, A. P., Fernandez, D., Alexander-Katz, A., Matsui, T., Mai, D. J. 2025; 163 (22)

  Abstract

  Protein structure prediction algorithms, such as AlphaFold, have accelerated protein design and advanced the understanding of the relationship between amino acid sequence and protein structure. However, these algorithms are limited in their ability to predict the structures of conformationally dynamic, intrinsically disordered, and stimuli-responsive proteins. To evaluate sequence-to-structure predictions of such challenging proteins, we explored a class of conformationally dynamic, repeats-in-toxin (RTX) proteins. RTX proteins adopt intrinsically disordered conformations in the absence of calcium and undergo reversible folding into β-roll structures upon binding to calcium. RTX proteins are characterized by tandem repeats of the sequence GGXGXDXUX, in which X can be any amino acid and U is an aliphatic amino acid. We designed RTX sequence variants with global substitutions of nonconserved amino acids, tandem repeats of consensus sequences GGAGXDTLY, and tandem repeats of scrambled sequences GGAGXDTYL. AlphaFold2 and AlphaFold3 predicted that all of these RTX variants adopt β-roll structures, characteristic of wild-type RTX bound to calcium. However, modeling the predicted structures with molecular dynamics simulations and characterizing the protein variants with circular dichroism spectroscopy, small-angle x-ray scattering, and x-ray crystallography revealed that variants adopt diverse, sequence-dependent structures in the absence and presence of calcium. To better design proteins for applications in biotechnology and sustainability, it is critical to build predictive tools that consider intrinsically disordered protein states and validate these tools with multi-mode, multi-scale experimental data.

  View details for DOI 10.1063/5.0304777

  View details for PubMedID 41379425

• Ion-selective conformational stabilization of a disordered repeats-in-toxin protein domain. Biophysical journal Gudinas, A. P., Shambharkar, G. M., Chang, M. P., Fernández, D., Matsui, T., Mai, D. J. 2025

  Abstract

  Ion-binding intrinsically disordered proteins (IDPs) recruit and bind to specific metal ions to perform critical biological functions. In proteins where ion binding and structural transitions are coupled, interactions with off-target toxic metals can dramatically disrupt protein structure and function, exemplified by lead and mercury poisoning. Understanding the complex mechanisms underlying how IDPs exclude or allow binding to different ionic species is crucial for addressing the origins of metal toxicity in biological systems. Here, we elucidate mechanisms of ion selectivity in an IDP that adopts a structure upon Ca2+ binding. We probed ion-induced conformational changes of a repeats-in-toxin (RTX) protein domain in the presence of different ion ligands-Mg2+, Ca2+, Sr2+, and Ba2+-with chemical similarities but drastically different ionic radii. RTX adopts ion-selective conformations measured by X-ray crystallography, small-angle X-ray scattering (SAXS), and circular dichroism (CD). High resolution X-ray structures reveal that Sr2+ induces a nearly identical RTX structure as natively binding Ca2+, enabled by the intrinsic flexibility and disorder of the protein. SAXS and CD indicate that smaller Mg2+ does not induce a significant conformational change in RTX, whereas larger Ba2+ induces a partially folded structure. These results highlight the importance of geometric constraints imposed by protein structure in determining metal ion selectivity, yielding insights into how off-target ion binding may result in protein misfolding and malfunction.

  View details for DOI 10.1016/j.bpj.2025.10.014

  View details for PubMedID 41088755

• Polymeric protagonists for biological processes. Nature chemistry Gudinas, A. P., Mai, D. J. 2023

  View details for DOI 10.1038/s41557-023-01219-9

  View details for PubMedID 37248342