Characterizing the conformational landscape of arf1 and mutant altering equilibria
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Authors
Peters, Edgar, Vance
Issue Date
2025-08
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Chemistry
Alternative Title
Abstract
The adenosine diphosphate (ADP) ribosylation factor (Arf) family of small guanosine triphosphatases (GTPases) function as molecular switches regulating membrane dynamics and intracellular trafficking in eukaryotic organisms. Among this family, Arf1 serves as the founding member, offering a structurally and functionally conserved framework for investigation into their regulatory mechanisms. Central Arf1’s switch-like behavior is the regulated exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP), a process that requires an extensive reorganization within its central -sheet and switch regions. To elucidate the structural and energetic basis of this switch mechanism, we combined pressure perturbation with nuclear magnetic resonance spectroscopy (NMR), Fourier Transform infrared spectroscopy (FTIR), and small-angle X-ray scattering, and computational modeling. Application of high hydrostatic pressure revealed that Arf1 populates a molten globule ensemble of partially unfolded conformers characterized by retained secondary structure but increased conformational flexibility. Using Arf1 as a model, we characterized energetic contributions of its autoinhibitory N-terminal helix. Truncation of the first 17 residues destabilizes the GDP-bound state promoting pressure-stabilized conformations that enhance nucleotide exchange, indicating a global stabilizing role for the helix. To further probe determinants of conformational activation, we introduced a corollary mutation from Arf6, the most distantly related Arf isoform, substitution isoleucine 42 with serine (I42S). In both context, this substitution modulated the energetic landscape and shifted the distribution of accessible conformational state within the Arf1 native state ensemble in a pressure and pH- dependent manner. Together these findings establish Arf1 as a reference system for investigating the wide functional divergence that exists within the Arf family of small GTPases.
Description
August2025
School of Science
School of Science
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY