About

Gail E. Fanucci is a Professor in the Department of Chemistry at the University of Florida. Her research focuses on using spin‑labeling magnetic resonance (SLMR) to characterize macromolecular motions linked to function and disease. Her work spans biological macromolecules, polymer assemblies, and glycolipids, with current projects probing membrane‑binding proteins and membrane biophysics, conformational changes in HIV‑1 protease and large RNAs, and natively unstructured proteins. She complements electron paramagnetic resonance (EPR) with solution NMR relaxation and Overhauser DNP to relate conformational sampling, backbone dynamics, and changes in surface water hydration. Fanucci has a distinguished academic background, including postdoctoral fellowships at the University of Virginia and the University of Pennsylvania, and a Ph.D. in Chemistry from the University of Florida. She has received several awards, such as the UF Term Professorship Award in 2021, the UF Research Foundation Professorship Award in 2018, and the NSF CAREER Award from 2008 to 2013. She has also served on various professional committees and editorial boards, contributing significantly to her field.

Research topics

• Materials science
• Chemistry
• Organic chemistry
• Biology
• Nanotechnology
• Cell biology
• Computational biology
• Biochemistry
• Polymer chemistry

Selected publications

• Evolutionary tuning of an intrinsically disordered inhibitor: conserved IA3 binding helices and divergent C-terminal linkers control conformational ensembles

  SSRN Electronic Journal · 2026-01-01

  preprintOpen access1st authorCorresponding
  PublisherDOI

• The overlooked C‐terminal domain of <scp>IA3</scp> encodes a disordered concentration and salt sensor for inhibitory‐helix dynamics

  Protein Science · 2026-05-22

  articleSenior authorCorresponding

  Intrinsically disordered proteins (IDPs) often use charge-patterned polyampholyte regions to couple concentration and ionic strength to conformational changes, but the mechanisms by which such regions redistribute secondary-structure elements and modulate local dynamics remain incompletely understood. Here, we use the yeast vacuolar aspartyl protease inhibitor IA3 as a minimal model to dissect how a C-terminal polyampholyte controls the conformational ensemble and dynamics of a helix-forming N-terminal inhibitory segment. IA3 comprises a conserved N-terminal binding helix (residues 1-32), a mixed-charge linker (residues 33-49), and a near-neutral C-terminal polyampholyte (residues 50-68). We show by far-UV circular dichroism (CD) spectroscopy and BeStSel deconvolution that, at very low ionic strength, increasing IA3 concentration from 10 to 100 μM drives a reversible redistribution from helix/turn-enriched to antiparallel/coil-enriched ensembles, without visible phase separation. Raising salt to moderate levels recapitulates these high-concentration spectra at low micromolar IA3, indicating that electrostatic screening effectively reduces the interaction-driven "effective concentration" of chains. Single-site charge substitutions in the C-terminal region (D46K, E68N) reproduce the high-concentration ensemble at 10 μM, whereas other mutations (N52E, K61D, L60K, K24L) preserve the low-concentration, helix/turn-rich state. Site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) at position 9 within the inhibitory helix reveals that conditions favoring antiparallel/other-rich ensembles increase local mobility, whereas helix/turn-enriched states exhibit more restricted motion. These results establish IA3 as a minimal system in which a C-terminal polyampholyte functions as a tunable electrostatic sensor, coupling charge patterning, concentration, and ionic strength to secondary-structure redistribution and N-terminal helix dynamics. The findings define sequence-encoded electrostatic rules that may generalize to other polyampholyte-containing IDPs and suggest how IA3 couples its inhibitory activity to cellular ionic conditions.

  PublisherDOI

• Hydrophobic Clusters Regulate Surface Hydration Dynamics of <i>Bacillus subtilis</i> Lipase A

  The Journal of Physical Chemistry B · 2024-04-17 · 2 citations

  articleSenior authorCorresponding

  Lipase A (BSLA) has been characterized by low-field Overhauser dynamic nuclear polarization (ODNP) relaxometry using a series of spin-labeled constructs. Sites for spin-label incorporation were previously designed via an atomistic computational approach that screened for surface exposure, reflective of the surface hydration comparable to other proteins studied by this method, as well as minimal impact on protein function, dynamics, and structure of BSLA by excluding any surface site that participated in greater than 30% occupancy of a hydrogen bonding network within BSLA. Experimental ODNP relaxometry coupling factor results verify the overall surface hydration behavior for these BSLA spin-labeled sites similar to other globular proteins. Here, by plotting the ODNP parameters of relative diffusive water versus the relative bound water, we introduce an effective "phase-space" analysis, which provides a facile visual comparison of the ODNP parameters of various biomolecular systems studied to date. We find notable differences when comparing BSLA to other systems, as well as when comparing different clusters on the surface of BSLA. Specifically, we find a grouping of sites that correspond to the spin-label surface location within the two main hydrophobic core clusters of the branched aliphatic amino acids isoleucine, leucine, and valine cores observed in the BSLA crystal structure. The results imply that hydrophobic clustering may dictate local surface hydration properties, perhaps through modulation of protein conformations and samplings of the unfolded states, providing insights into how the dynamics of the hydration shell is coupled to protein motion and fluctuations.

  PublisherDOI

• Designing Surface Exposed Sites on Bacillus Subtilis Lipase a for Spin-Labeling and Hydration Studies

  SSRN Electronic Journal · 2024-01-01

  preprintOpen access1st authorCorresponding
  PublisherDOI

• Natural Polymorphisms D60E and I62V Stabilize a Closed Conformation in HIV-1 Protease in the Absence of an Inhibitor or Substrate

  Viruses · 2024-02-02 · 1 citations

  articleOpen accessSenior authorCorresponding

  HIV infection remains a global health issue plagued by drug resistance and virological failure. Natural polymorphisms (NPs) contained within several African and Brazilian protease (PR) variants have been shown to induce a conformational landscape of more closed conformations compared to the sequence of subtype B prevalent in North America and Western Europe. Here we demonstrate through experimental pulsed EPR distance measurements and molecular dynamic (MD) simulations that the two common NPs D60E and I62V found within subtypes F and H can induce a closed conformation when introduced into HIV-1PR subtype B. Specifically, D60E alters the conformation in subtype B through the formation of a salt bridge with residue K43 contained within the nexus between the flap and hinge region of the HIV-1 PR fold. On the other hand, I62V modulates the packing of the hydrophobic cluster of the cantilever and fulcrum, also resulting in a more closed conformation.

  PublisherOA PDFDOI

• Designing surface exposed sites on Bacillus subtilis lipase A for spin-labeling and hydration studies

  Biophysical Chemistry · 2024-02-16 · 3 citations

  articleSenior authorCorresponding
  PublisherDOI

• Different Biophysical Properties of Cell Surface α2,3- and α2,6-Sialoglycans Revealed by Electron Paramagnetic Resonance Spectroscopic Studies

  The Journal of Physical Chemistry B · 2023-02-21 · 8 citations

  articleOpen accessCorresponding

  Sialoglycans on HeLa cells were labeled with a nitroxide spin radical through enzymatic glycoengineering (EGE)-mediated installation of azide-modified sialic acid (Neu5Ac9N3) and then click reaction-based attachment of a nitroxide spin radical. α2,6-Sialyltransferase (ST) Pd2,6ST and α2,3-ST CSTII were used for EGE to install α2,6- and α2,3-linked Neu5Ac9N3, respectively. The spin-labeled cells were analyzed by X-band continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy to gain insights into the dynamics and organizations of cell surface α2,6- and α2,3-sialoglycans. Simulations of the EPR spectra revealed average fast- and intermediate-motion components for the spin radicals in both sialoglycans. However, α2,6- and α2,3-sialoglycans in HeLa cells possess different distributions of the two components, e.g., a higher average population of the intermediate-motion component for α2,6-sialoglycans (78%) than that for α2,3-sialoglycans (53%). Thus, the average mobility of spin radicals in α2,3-sialoglycans was higher than that in α2,6-sialoglycans. Given the fact that a spin-labeled sialic acid residue attached to the 6-O-position of galactose/N-acetyl-galactosamine would experience less steric hindrance and show more flexibility than that attached to the 3-O-position, these results may reflect the differences in local crowding/packing that restrict the spin-label and sialic acid motion for α2,6-linked sialoglycans. The studies further suggest that Pd2,6ST and CSTII may have different preferences for glycan substrates in the complex environment of the extracellular matrix. The discoveries of this work are biologically important as they are useful for interpreting the different functions of α2,6- and α2,3-sialoglycans and indicate the possibility of using Pd2,6ST and CSTII to target different glycoconjugates on cells.

  PublisherOA PDFDOI

• Spin-Labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells

  Applied Magnetic Resonance · 2023-10-09 · 1 citations

  articleOpen accessSenior author
  PublisherOA PDFDOI

• Charge Distribution Patterns of IA₃ Impact Conformational Expansion and Hydration Diffusivity of the Disordered Ensemble

  The Journal of Physical Chemistry B · 2023-11-08 · 5 citations

  articleSenior authorCorresponding

  IA3 is a 68 amino acid natural peptide/protein inhibitor of yeast aspartic proteinase A (YPRA) that is intrinsically disordered in solution with induced N-terminal helicity when in the protein complex with YPRA. Based on the intrinsically disordered protein (IDP) parameters of fractional net charge (FNC), net charge density per residue (NCPR), and charge patterning (κ), the two domains of IA3 are defined to occupy different domains within conformationally based subclasses of IDPs, thus making IA3 a bimodal domain IDP. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and low-field Overhauser dynamic nuclear polarization (ODNP) spectroscopy results show that these two domains possess different degrees of compaction and hydration diffusivity behavior. This work suggests that SDSL EPR line shapes, analyzed in terms of their local tumbling volume (VL), provide insights into the compaction of the unstructured IDP ensemble in solution and that protein sequence and net charge distribution patterns within a conformational subclass can impact bound water hydration dynamics, thus possibly offering an alternative thermodynamic property that can encode conformational binding and behavior of IDPs and liquid–liquid phase separations.

  PublisherDOI

• Spin-labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells

  Research Square · 2023-06-13

  preprintOpen accessSenior authorCorresponding

  As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction. Continuous wave X-band electron paramagnetic resonance spectroscopy is employed to characterize how the chronological sequence of chemical fixation and spin labeling impacts the local mobility and accessibility of the nitroxide-labeled glycans in the glycocalyx of HeLa cells. Results demonstrate that chemical fixation with paraformaldehyde can alter local glycan mobility and care should be taken in the analysis of data in any study where chemical fixation and cellular labeling occur.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R01GM105409

  NIH · $1.1M · 2018

• Thermodynamics and Chain Dynamics in Spin-labeled Peptide Block Copolymer Assemblies

  NSF · $600k · 2020–2025

• Spin Labeling Studies of Biomolecular Flexibility and Hydration

  NSF · $822k · 2017–2023

• CAREER: Site-Directed Spin Labeling EPR Applications in Intrinsically Unstructured Proteins

  NSF · $604k · 2008–2014

• NIH Grant R01GM077232

  NIH · $1.2M · 2012

Frequent coauthors

• Daniel R. Talham

  University of Florida

  40 shared

• Alexander Angerhofer

  University of Florida

  38 shared

• Karen A. Hyde

  Université Claude Bernard Lyon 1

  36 shared

• Malcom Forbes

  Bridge University

  36 shared

• Michael Bowman

  Iowa State University

  36 shared

• Gary J. Gerfen

  Albert Einstein College of Medicine

  36 shared

• Kurt W. Zilm

  Yale University

  36 shared

• Martin L. Kirk

  The Ohio State University

  36 shared

Awards & honors

• UF Term Professorship Award (2021)
• UF Research Foundation Professorship Award (2018)
• Wessell's Excellence Award, UF College Outstanding Assistant…
• HHMI Distinguished Mentor Award (2008)
• NSF CAREER Award (2008–2013)