More Like this

1. Distinct conformational states enable transglutaminase 2 to promote cancer cell survival versus cell death

   https://doi.org/10.1038/s42003-024-06672-x  

   Aplin, Cody; Zielinski, Kara_A; Pabit, Suzette; Ogunribido, Deborah; Katt, William_P; Pollack, Lois; Cerione, Richard_A; Milano, Shawn_K (August 2024, Communications Biology)

   Abstract Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state. 

   more » « less
2. Anisotropic Gold Nanomaterial Synthesis Using Peptide Facet Specificity and Timed Intervention

   https://doi.org/10.1021/acs.langmuir.3c01577  

   Lachowski, Kacper J.; Chiang, Huat Thart; Torkelson, Kaylyn; Zhou, Wenhao; Zhang, Shuai; Pfaendtner, Jim; Pozzo, Lilo D. (November 2023, Langmuir)

   Thin metal particles with two-dimensional symmetry are attractive for multiple ap- plications, but are difficult to synthesize in a reproducible manner. Although molecules that selectively adsorb to facets have been used to control nanoparticle shape, there is still limited research into the temporal control of growth processes to control these structural outcomes. Moreover, much of the current research into the growth of thin two-dimensional particles lacks mechanistic details. In this work, we study why the substitution of isoleucine for methionine in a gold binding peptide (Z2, RMRMKMK) results in an increase in gold nanoparticle anisotropy. Nanoplatelet growth in the pres- ence of Z2M246I (RIRIKIK) is characterized using in situ small-angle X-ray scattering (SAXS) and UV-Vis spectroscopy. Fitting time-resolved SAXS profiles reveals that 10 nm thick particles with two-dimensional symmetry are formed within the first few min- utes of the reaction. Next, through a combination of electron diffraction and molecular dynamics simulations, we show that substitution of methionine for isoluecine increases the (111) facet selectivity in Z2M246I, and conclude that this is key to the growth of nanoplatelets. However, the potential application of nanoplatelets formed using Z2M246I is limited due to their uncontrolled lateral growth, aggregation, and rapid sedimentation. Therefore, we use a liquid handling robot to perform temporally con- trolled synthesis and dynamic intervention through the addition of Z2 to nanoplatelets growing in the presence of Z2M246I at different times. UV-Vis spectroscopy dynamic light scattering, and electron microscopy show that dynamic intervention results in control over the mean-size and stability of plate-like particles. Finally, we use in situ UV-Vis spectroscopy to study plate-like particle growth at different times of interven- tion. Our results demonstrate that both the selectivity and magnitude of binding free energy towards lattices is important for controlling nanoparticle growth pathways. 

   more » « less
3. Modifying the internal substituents of self-assembled cages controls their molecular recognition and optical properties

   https://doi.org/10.1039/D2DT01451C  

   Woods, Connor Z.; Wu, Hoi-Ting; Ngai, Courtney; da Camara, Bryce; Julian, Ryan R.; Hooley, Richard J. (July 2022, Dalton Transactions)

   Self-assembled Fe4 L 6 cage complexes with variable internal functions can be synthesized from a 2,7-dibromocarbazole ligand scaffold, which orients six functional groups to the cage interior. Both ethylthiomethylether and ethyldimethylamino groups can be incorporated. The cages show strong ligand-centered fluorescence emission and a broad range of guest binding properties. Coencapsulation of neutral organic guests is favored in the larger, unfunctionalized cage cavity, whereas the thioether cage has a more sterically hindered cavity that favors 1 : 1 guest binding. Binding affinities up to 10 6 M −1 in CH3 CN are seen. The dimethylamino cage is more complex, as the internal amines display partial protonation and can be deprotonated by amine bases. This amine cage displays affinity for a broad range of neutral organic substrates, with affinities and stoichiometries comparable to that of the similarly sized thioether cage. These species show that simple variations in ligand backbone allow variations in the number and type of functions that can be displayed towards the cavity of self-assembled hosts, which will have applications in biomimetic sensing, catalysis and molecular recognition. 

   more » « less
4. Structural Assessment of Polymer-Enzyme Complex Nanoparticle Stability

   Murthy, N. Sanjeeva; Upadhya, Rahul; Kosuri, Shashank; Tamasi, Matthew; Gormley, Adam J. (April 2022, Transactions of the Society for Biomaterials)

   Statement of Purpose Hybrid nanoparticles in which a polymer is used to stabilize the secondary structure of enzyme provide a means to preserve its activity in non-native environments. This approach is illustrated here with horseradish peroxidase (HRP), an important heme enzyme used in medical diagnostic, biosensing, and biotechnological applications. Polymer chaperones in these polymer-enzyme complex (PEC) nanoparticles can enhance the utility of enzymes in unfavorable environments. Structural analysis of the PECs is a crucial link in the machine-learning driven iterative optimization cycle of polymer synthesis and testing. Here, we discuss the utility of small-angle X-ray scattering (SAXS) and quartz crystal microbalance with dissipation (QCMD) for evaluating PECs. Materials and Methods Six polymers were synthesized by automated photoinduced electron/energy transfer-reversible addition-fragmentation chain-transfer (PET-RAFT) polymerization directly in 96-well plates.1 Multiple molar ratios of enzyme:polymer (1:1, 1:5, 1:10, and 1:50) were characterized. HRP was mixed with the polymer and heated to 65 °C for 1 hr to form PECs. Enzyme assay and circular dichroism measurements were performed along with SAXS and QCMD to understand polymer-protein interactions. SAXS data were obtained at NSLS-II beamline 16-ID. Results and Discussion SAXS data were analyzed to determine the radius of gyration (Rg), Porod exponent and pair distance distribution functions (P(r)) (Figure 1). Rg, which corresponds to the size of the PEC nanoparticles, is sensitive to the polydispersity of the solution and does not change significantly in the presence of the polymer GEP1. Notably, the maximal dimension does not change as significantly upon heating to denaturation in the case of the PEC as it does with HRP alone. The effect of denaturation induced by heating seems to depend on the molar ratio of the polymer to enzyme. The Porod exponent, which is related to roughness, decreased from about 4 to 3 upon complexation indicating polymer binding to the enzyme’s surface. These were confirmed by modeling the structures of the HRP, the polymer and the PEC were modeled using DAMMIF/DAMMIN and MONSA (ATSAS software). The changes observed in the structure could be correlated to the measured enzymatic activity. Figure 2 shows the evolution of the PEC when the polymer is deposited onto the enzyme immobilized on Figure 1. P(r) plots for PEC vs. HRP before and after heating, illustrating the increased enzymatic stability due to polymer additives. gold-coated QCM sensors. The plots show the changes in frequency (f) and dissipation (D) with time as HRP is first deposited and is followed by the adsorption of the polymer. Large f and D show that the polymer forms a complex with HRP. Such changes were not observed with negative controls, Pluronics and poly(ethylene glycol). Comparison of the data from free particles in solution with QCM data from immobilized enzymes, shows that the conformation of the complexes in solution and surface-bound HRP could be different. This way, we were able to explore the various states of complex formation under different conditions with different polymers. Figure 2. QCMD data showing the interaction between the immobilized HRP and the polymer. 3rd and 5th harmonics are plotted (blue -f; red-D). Conclusion SAXS and QCMD data show that stabilization of the enzyme activity by inhibiting the unraveling of the secondary structure as seen in size, surface roughness, pair distribution function and percent helicity. Acknowledgment This work was supported by NSF grant 2009942. References [1] Tamasi, M, et al. Adv Intell Syst 2020, 2(2): 1900126. 

   more » « less
5. Understanding Synthesis and Structural Variation of Nanomaterials Through In Situ/Operando XAS and SAXS

   https://doi.org/10.1002/smll.202106017  

   Fang, Lingzhe; Seifert, Soenke; Winans, Randall E.; Li, Tao (February 2022, Small)

   Abstract Nanostructured materials with high surface area and low coordinated atoms present distinct intrinsic properties from their bulk counterparts. However, nanomaterials’ nucleation/growth mechanism during the synthesis process and the changes of the nanomaterials in the working state are still not thoroughly studied. As two indispensable methods, X‐ray absorption spectroscopy (XAS) provides nanomaterials’ electronic structure and coordination environment, while small‐angle X‐ray scattering (SAXS) offers structural properties and morphology information. A combination of in situ/operando XAS and SAXS provides high temporal and spatial resolution to monitor the evolution of nanomaterials. This review gives a brief introduction to in situ/operando SAXS/XAS cells. In addition, the application of in situ/operando XAS and SAXS in preparing nanomaterials and studying changes of working nanomaterials are summarized. 

   more » « less