Claire Stewart

All Publications

• Development and characterization of LipoCatch: a bacterial lipoprotein-based biomaterial that self-assembles into nanostructures. Nanoscale advances Starvaggi, F. A., Stewart, C. J., Arslanian, M. A., Treviño, M. A., Weston, S. A., Sharaf, N. G. 2026

  Abstract

  Genetically encoded nanomaterials enable the control of molecular composition and function, yet the use of the biosynthetic bacterial lipidation pathway to build hybrid protein/lipid nanostructures has not been reported. Here, we designed a versatile bacterial lipoprotein, named LipoCatch, for modular nanostructure formation. Lipidation was achieved by appending a signal peptide to the protein-encoding gene of SpyCatcher from the SpyCatcher/SpyTag protein/peptide binding pair. This protein was biosynthetically produced in E. coli and purified in the presence of detergent. LC-MS, SEC, DLS, and TEM confirmed site-specific lipidation and formation of nanoparticles with an average diameter of 13 nm. We show that LipoCatch supports two orthogonal functionalization strategies: (1) covalent modification through the SpyCatcher/SpyTag system and (2) the non-covalent incorporation of phospholipids that permits the tuning of particle size and compositions. Finally, stability studies show LipoCatch and hybrid LipoCatch/phospholipid nanostructures are tolerant to lyophilization, in contrast to phospholipid-only liposomes. Together, this proof-of-principle study establishes an engineered bacterial lipoprotein, LipoCatch, as a genetically encoded platform for building customizable bacterial lipoprotein-based biomaterials.

  View details for DOI 10.1039/d6na00554c

  View details for PubMedID 42494381

  View details for PubMedCentralID PMC13394007

• Structural basis for selective thymidine binding by the Borrelia burgdorferi substrate-binding protein BmpA. The Journal of biological chemistry Liu, Q., Nun Ez, V. A., Fernandez, D., Stewart, C. J., Sharaf, N. G. 2026: 113206

  Abstract

  BmpA is a putative substrate-binding protein from Borrelia burgdorferi, the causative agent of Lyme disease, an organism with limited metabolic capacity that relies on salvage pathways rather than de novo nucleotide biosynthesis. Here, we determine the crystal structure of BmpA to a resolution of 2.6 Å, revealing a conserved substrate-binding protein fold with a deeply buried nucleoside-binding pocket. Using microscale thermophoresis, we show that BmpA binds thymidine with high affinity followed by cytidine and adenosine, whereas binding to ribose, guanosine, inosine, and uridine was not detected. Structure-guided mutagenesis further demonstrates that two conserved aromatic residues (Phe27 and Phe176) are essential for thymidine recognition, as alanine substitution at either position abolishes detectable binding. Additionally, a Foldseek-based structural homology search identified related proteins across diverse bacterial and archaeal species that share a conserved overall fold and binding-site architecture despite low sequence similarity, consistent with an evolutionarily conserved scaffold that can accommodate distinct nucleoside ligands. Together, our work illustrates how conserved binding protein architectures enable selective nucleoside acquisition and provides a foundation for understanding nutrient uptake strategies in organisms with reduced genomes.

  View details for DOI 10.1016/j.jbc.2026.113206

  View details for PubMedID 42208901

• Crowding beyond excluded volume: A tale of two dimers PROTEIN SCIENCE Olgenblum, G. I., Stewart, C. J., Redvanly, T. W., Young, O. M., Lauzier, F., Hazlett, S., Wang, S., Rockcliffe, D. A., Parnham, S., Pielak, G. J., Harries, D. 2025; 34 (4): e70062

  Abstract

  Protein-protein interactions are modulated by their environment. High macromolecular solute concentrations crowd proteins and shift equilibria between protein monomers and their assemblies. We aim to understand the mechanism of crowding by elucidating the molecular-level interactions that determine dimer stability. Using 19F-NMR spectroscopy, we studied the effects of various polyethylene glycols (PEGs) on the equilibrium thermodynamics of two protein complexes: a side-by-side and a domain-swap dimer. Analysis using our mean-field crowding model shows that, contrary to classic crowding theories, PEGs destabilize both dimers through enthalpic interactions between PEG and the monomers. The enthalpic destabilization becomes more dominant with increasing PEG concentration because the reduction in PEG mesh size with concentration diminishes the stabilizing effect of excluded volume interactions. Additionally, the partially folded domain-swap monomers fold in the presence of PEG, contributing to dimer stabilization at low PEG concentrations. Our results reveal that polymers crowd protein complexes through multiple conjoined mechanisms, impacting both their stability and oligomeric state.

  View details for DOI 10.1002/pro.70062

  View details for Web of Science ID 001445948400001

  View details for PubMedID 40095390

  View details for PubMedCentralID PMC11912439

• Expression, purification, and characterization of diacylated Lipo-YcjN from Escherichia coli. The Journal of biological chemistry Trevino, M. A., Amankwah, K. A., Fernandez, D., Weston, S. A., Stewart, C. J., Gallardo, J. M., Shahgholi, M., Sharaf, N. G. 2024: 107853

  Abstract

  YcjN is a putative substrate binding protein expressed from a cluster of genes involved in carbohydrate import and metabolism in Escherichia coli. Here, we determine the crystal structure of YcjN to a resolution of 1.95 A, revealing that its three-dimensional structure is similar to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein (MBP). Furthermore, we found that recombinant overexpression of YcjN results in the formation of a lipidated form of YcjN that is posttranslationally diacylated at cysteine 21. Comparisons of size-exclusion chromatography profiles and dynamic light scattering measurements of lipidated and non-lipidated YcjN proteins suggest that lipidated YcjN aggregates in solution via its lipid moiety. Additionally, bioinformatic analysis indicates that YcjN-like proteins may exist in both Bacteria and Archaea, potentially in both lipidated and non-lipidated forms. Together, our results provide a better understanding of the aggregation properties of recombinantly expressed bacterial lipoproteins in solution and establish a foundation for future studies that aim to elucidate the role of these proteins in bacterial physiology.

  View details for DOI 10.1016/j.jbc.2024.107853

  View details for PubMedID 39362470

• Resolving the enthalpy of protein stabilization by macromolecular crowding PROTEIN SCIENCE Stewart, C. J., Olgenblum, G. I., Propst, A., Harries, D., Pielak, G. J. 2023; 32 (3): e4573

  Abstract

  Proteins in the cellular milieu reside in environments crowded by macromolecules and other solutes. Although crowding can significantly impact the protein folded state stability, most experiments are conducted in dilute buffered solutions. To resolve the effect of crowding on protein stability, we use 19 F nuclear magnetic resonance spectroscopy to follow the reversible, two-state unfolding thermodynamics of the N-terminal Src homology 3 domain of the Drosophila signal transduction protein drk in the presence of polyethylene glycols (PEGs) of various molecular weights and concentrations. Contrary to most current theories of crowding that emphasize steric protein-crowder interactions as the main driving force for entropically favored stabilization, our experiments show that PEG stabilization is accompanied by significant heat release, and entropy disfavors folding. Using our newly developed model, we find that stabilization by ethylene glycol and small PEGs is driven by favorable binding to the folded state. In contrast, for larger PEGs, chemical or soft PEG-protein interactions do not play a significant role. Instead, folding is favored by excluded volume PEG-protein interactions and an exothermic nonideal mixing contribution from release of confined PEG and water upon folding. Our results indicate that crowding acts through molecular interactions subtler than previously assumed and that interactions between solution components with both the folded and unfolded states must be carefully considered.

  View details for DOI 10.1002/pro.4573

  View details for Web of Science ID 000935872900001

  View details for PubMedID 36691735

  View details for PubMedCentralID PMC9942490

• A Difference between <i>In Vitro</i> and In-Cell Protein Dimer Formation BIOCHEMISTRY Chu, I., Stewart, C. J., Speer, S. L., Pielak, G. J. 2022; 61 (6): 409-412

  Abstract

  The high concentration of macromolecules in cells affects the stability of proteins and protein complexes via hard repulsions and chemical interactions, yet few studies have focused on chemical interactions. We characterized the domain-swapped dimer of the B1 domain of protein G in buffer and Escherichia coli cells by using heteronuclear, multidimensional nuclear magnetic resonance spectroscopy. In buffer, the monomer is a partially folded molten globule, but that species is not observed in cells. Experiments using urea suggest that the monomer is unfolded in cells, but again, the molten-globule form of the monomer is absent. The data suggest that attractive chemical interactions in the cytoplasm unfold the molten globule. We conclude that the intracellular environment not only modulates the stability of protein complexes but also can change the species present, reinforcing the idea that chemical interactions are more important than hard repulsions in cells.

  View details for DOI 10.1021/acs.biochem.1c00780

  View details for Web of Science ID 000771920200001

  View details for PubMedID 35188746

• Macromolecular Crowding Is More than Hard-Core Repulsions ANNUAL REVIEW OF BIOPHYSICS Speer, S. L., Stewart, C. J., Sapir, L., Harries, D., Pielak, G. J. 2022; 51: 267-300

  Abstract

  Cells are crowded, but proteins are almost always studied in dilute aqueous buffer. We review the experimental evidence that crowding affects the equilibrium thermodynamics of protein stability and protein association and discuss the theories employed to explain these observations. In doing so, we highlight differences between synthetic polymers and biologically relevant crowders. Theories based on hard-core interactions predict only crowding-induced entropic stabilization. However, experiment-based efforts conducted under physiologically relevant conditions show that crowding can destabilize proteins and their complexes. Furthermore, quantification of the temperature dependence of crowding effects produced by both large and small cosolutes, including osmolytes, sugars, synthetic polymers, and proteins, reveals enthalpic effects that stabilize or destabilize proteins.Crowding-induced destabilization and the enthalpic component point to the role of chemical interactions between and among the macromolecules, cosolutes, and water. We conclude with suggestions for future studies.

  View details for DOI 10.1146/annurev-biophys-091321-071829

  View details for Web of Science ID 000799203300014

  View details for PubMedID 35239418