Elizaveta A. Dronova

Ferritins are a widespread family of proteins involved in iron homeostasis. While classic ferritins consist of four α-helices and form 24-meric nanocages, related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity. Here, by analyzing the available genomic data, we identify a family of double ferritin-like proteins (DFLPs) composed of two four-helical domains, which arose by duplication of a ferritin fold protein. We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens , TmDFLP and CaDFLP, and show that they form homodimers and bind heme. We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase activity. Furthermore, we show that some DFLPs, including TmDFLP and CaDFLP, are highly likely to be targeted into encapsulin shells. Our work expands the range of known iron metabolism systems and highlights the power of genome mining for discovery of new proteins. Graphical Abstract for Table of Contents: A family of double ferritin-like proteins (DFLPs) composed of two four-helical domains is described and investigated. DFLPs are shown to form homodimers, bind heme, possess diiron sites and display ferroxidase activity. Some DFLPs are shown to be targeted to encapsulin shells as core or secondary cargo, thus representing a new type of iron metabolism systems.

We present the results of the study of the structural distinctions between the purple membrane (PM) of Halobacterium salinarum and the claret membrane (CM) of Haloquadratum walsbyi using small-angle and wide-angle X-ray scattering. PM and CM are rhodopsin-rich membrane regions. They are crucial for light-driven energy processes in extreme halophilic archaea. While PM structure has been extensively characterized, the structure of CM remains less understood. According to our data, PM exhibits a well-defined hexagonal crystalline lattice with unit cell parameters of a = b ≈ 62 Å, which is consistent with published data. Conversely, CM showed unexpected diffraction patterns, best fitted by rhombohedral lattice with unit cell dimensions a = b = 27.9 Å; γ = 82.9°. But the presence of several unindexed peaks indicates the complexity of the CM sample. These structural differences are attributed to variations in native lipid and pigment compositions, specifically the presence of bacterioruberin in CM, as confirmed by UV-visible spectroscopy. Bacterioruberin, a hallmark of CM, contributes to its red-shifted absorption spectrum, indicative of its organized state within the membrane. However, denaturing 12% polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy reveal potential contamination in CM samples, suggesting the need for improved purification protocols. The findings highlight significant structural disparities between PM and CM, with implications for understanding membrane organization. This research underscores the critical role of lipids and pigments in shaping the supramolecular architecture of archaeal membranes and provides a foundation for future studies into their functional adaptations.

Rhodopsins are photosensitive transmembrane proteins found in bacteria, archaea, and eukaryotes. Upon photon absorption, they undergo conformational changes critical for their physiological functions, such as ion transport and signal transduction. Microbial rhodopsins are key tools in optogenetics, with applications in biomedical research and clinical treatments. To advance optogenetic techniques, it is crucial to understand the molecular mechanisms of the rhodopsin photocycle. The key approach for studying these mechanisms, cryotrapping, has a setback associated with possible deterioration in resolution due to the unexpectedly large light-induced structural changes. To estimate the scale of these changes, small-angle X-ray scattering (SAXS) can be used. In this study, we applied SAXS to the investigation of light-induced structural changes in purple membranes (PMs) as their two-dimensional crystal organization makes them a suitable model object for studying light-induced changes in 3D rhodopsin crystals. SAXS data from illuminated and non-illuminated PM samples revealed detectable changes, including variations in membrane thickness and planar unit cell dimensions. Particularly, we observed a statistically significant increase in the radius of gyration for flat particles (Rₜ) of ~0.2 Å for the PMs with mutant HsBR_E204Q, while no significant change was detected for wild-type HsBR. However, wild-type HsBR data showed a statistically significant shift in the (1, 1) Bragg peak position (Δ q ~ –10^–4 Å^–1; ~3% of the peak width) upon illumination. For HsBR_E204Q data, the shift was two times greater; however, in this case due to the lower sample concentration ε_Δq was ~50%, making it difficult to compare the wild-type and mutant cases. Future experiments should aim for better signal-to-noise ratios using synchrotron radiation, higher sample concentrations, and longer exposure times.

Here we explore capabilities of size exclusion chromatography small-angle X-ray scattering method in case of horse spleen apoferritin solution as an example of a system with high oligomerization heterogeneity. For interpretation of the experimental data, we considered models of polydisperse solution containing monomers, dimers, and trimers of apoferritin globules. Dimers were defined only by intercenter distance L, while trimers were characterized by both L and apical corner value φ. We showed that a reasonable description of the major part of the chromatogram could be reached using the model containing monomers, dimers and one type of trimers with φ about 110°–120°. In the case of two types of trimers in the model, optimal φ values were 110°–140° and 90°–110°; however, the approximation results were considered as ambiguous. Validation of the preferred model (L = 12.5 nm, φ = 120°) showed insufficient description of the experimental data for the left part of the chromatogram (elution volume <12.2 mL) indicating the presence of aggregates in these fractions. Application of the method provided the opportunity to calculate concentrations for oligomers consisting of less than four apoferritin globules, and select averaging range for different fractions properly.

F-type ATP synthases play a key role in oxidative and photophosphorylation processes generating adenosine triphosphate (ATP) for most biochemical reactions in living organisms. In contrast to the mitochondrial F_OF₁-ATP synthases, those of chloroplasts are known to be mostly monomers with approx. 15% fraction of oligomers interacting presumably non-specifically in a thylakoid membrane. To shed light on the nature of this difference we studied interactions of the chloroplast ATP synthases using small-angle X-ray scattering (SAXS) method. Here, we report evidence of I-shaped dimerization of solubilized F_OF₁-ATP synthases from spinach chloroplasts at different ionic strengths. The structural data were obtained by SAXS and demonstrated dimerization in response to ionic strength. The best model describing SAXS data was two ATP-synthases connected through F₁/F₁′ parts, presumably via their δ-subunits, forming “I” shape dimers. Such I-shaped dimers might possibly connect the neighboring lamellae in thylakoid stacks assuming that the F_OF₁ monomers comprising such dimers are embedded in parallel opposing stacked thylakoid membrane areas. If this type of dimerization exists in nature, it might be one of the pathways of inhibition of chloroplast F_OF₁-ATP synthase for preventing ATP hydrolysis in the dark, when ionic strength in plant chloroplasts is rising. Together with a redox switch inserted into a γ-subunit of chloroplast F_OF₁ and lateral oligomerization, an I-shaped dimerization might comprise a subtle regulatory process of ATP synthesis and stabilize the structure of thylakoid stacks in chloroplasts.