Sudarev, Vsevolod V

Gette M.S., Laptenkova E.V., Sudarev V.V., Zagryadskaya Y.A., Okhrimenko I.S., Bazhenov S.V., Manukhov I.V., Ryzhykau Y.L., Vlasov A.V.

Sudarev V.V., Gette M.S., Bazhenov S.V., Tilinova O.M., Zinovev E.V., Manukhov I.V., Kuklin A.I., Ryzhykau Y., Vlasov A.V.

Ferritin is a universal protein complex responsible for iron perception in almost all living organisms and has applications from fundamental biophysics to drug delivery and structure-based immunogen design. Different platforms based on ferritin share similar technological challenges limiting their development – control of self-assembling processes of ferritin itself as well as ferritin-based chimeric recombinant protein complexes. In our research, we studied self-assembly processes of ferritin-based protein complexes under different expression conditions. We fused a ferritin subunit with a SMT3 protein tag, a homolog of human Small Ubiquitin-like Modifier (SUMO-tag), which was taken to destabilize ferritin 3-fold channel contacts and increase ferritin-SUMO subunits solubility. We first obtained the octameric protein complex of ferritin-SUMO (8xFer-SUMO) and studied its structural organization by small-angle X-ray scattering (SAXS). Obtained SAXS data correspond well with the high-resolution models predicted by AlphaFold and CORAL software of an octameric assembly around the 4-fold channel of ferritin without formation of 3-fold channels. Interestingly, three copies of 8xFer-SUMO do not assemble into 24-meric globules. Thus, we first obtained and structurally characterized ferritin-based self-assembling oligomers in a deadlock state. Deadlock oligomeric states of ferritin extend the known scheme of its self-assembly process, being new potential tools for a number of applications. Finally, our results might open new directions for various biotechnological platforms utilizing ferritin-based tools.

Osipov S.D., Ryzhykau Y.L., Zinovev E.V., Minaeva A.V., Ivashchenko S.D., Verteletskiy D.P., Sudarev V.V., Kuklina D.D., Nikolaev M.Y., Semenov Y.S., Zagryadskaya Y.A., Okhrimenko I.S., Gette M.S., Dronova E.A., Shishkin A.Y., et. al.

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 FOF1-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 FOF1-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 FOF1 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 FOF1-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 FOF1 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.

Sudarev V.V., Dolotova S.M., Bukhalovich S.M., Bazhenov S.V., Ryzhykau Y.L., Uversky V.N., Bondarev N.A., Osipov S.D., Mikhailov A.E., Kuklina D.D., Murugova T.N., Manukhov I.V., Rogachev A.V., Gordeliy V.I., Gushchin I.Y., et. al.

Ferritin is a vital protein complex responsible for storing iron in almost all living organisms. It plays a crucial role in various metabolic pathways, inflammation processes, stress response, and pathogenesis of cancer and neurodegenerative diseases. In this review we discuss the role of ferritin in diseases, cellular iron regulation, its structural features, and its role in biotechnology. We also show that molecular mechanisms of ferritin self-assembly are key for a number of biotechnological and pharmaceutical applications. The assembly pathways strongly depend on the interface context of ferritin monomers and the stability of its different intermediate oligomers. To date, several schemes of self-assembly kinetics have been proposed. Here, we compare different self-assembly mechanisms and discuss the possibility of self-assembly control by switching between deadlock intermediate states.

Vlasov A.V., Maliar N.L., Bazhenov S.V., Nikelshparg E.I., Brazhe N.A., Vlasova A.D., Osipov S.D., Sudarev V.V., Ryzhykau Y.L., Bogorodskiy A.O., Zinovev E.V., Rogachev A.V., Manukhov I.V., Borshchevskiy V.I., Kuklin A.I., et. al.

This is a review of relevant Raman spectroscopy (RS) techniques and their use in structural biology, biophysics, cells, and tissues imaging towards development of various medical diagnostic tools, drug design, and other medical applications. Classical and contemporary structural studies of different water-soluble and membrane proteins, DNA, RNA, and their interactions and behavior in different systems were analyzed in terms of applicability of RS techniques and their complementarity to other corresponding methods. We show that RS is a powerful method that links the fundamental structural biology and its medical applications in cancer, cardiovascular, neurodegenerative, atherosclerotic, and other diseases. In particular, the key roles of RS in modern technologies of structure-based drug design are the detection and imaging of membrane protein microcrystals with the help of coherent anti-Stokes Raman scattering (CARS), which would help to further the development of protein structural crystallography and would result in a number of novel high-resolution structures of membrane proteins—drug targets; and, structural studies of photoactive membrane proteins (rhodopsins, photoreceptors, etc.) for the development of new optogenetic tools. Physical background and biomedical applications of spontaneous, stimulated, resonant, and surface- and tip-enhanced RS are also discussed. All of these techniques have been extensively developed during recent several decades. A number of interesting applications of CARS, resonant, and surface-enhanced Raman spectroscopy methods are also discussed.

Sudaraka Tennakoon M.S., Lee K., Shin H.

Yang R., Chen R., Li R., Liang L., Zhang Y.

Jiskani S.A.

Xie J., Luo D., Xing P., Ding W.

Liao Y., Zeng T., Guo X., Li X.

Liu Y., Chen S., Xiong X., Wen Z., Zhao L., Xu B., Guo Q., Xia J., Pei J.

Gette M.S., Sudarev V.V., Osipov S.D., Laptenkova E.V., Bazhenov S.V., Zagryadskaya Y.A., Tilinova O.M., Dronova E.A., Kuklina D.D., Al Ebrahim R., Fedorov D.M., Kurkin T.S., Semenov Y.S., Bondarev N.A., Skoi V.V., et. al.

Xu W., Wen X., Fu Y., Yang J., Cui H., Fan R.

Nuclear receptor coactive 4 (NCOA4) is a specific receptor for ferritinophagy, transporting ferritin to lysosomal degradation, releasing free iron, and excessive iron levels may lead to cellular redox imbalance, contributing to cell death, predominantly ferroptosis. NCOA4 is regulated by a variety of transcriptional, post-transcriptional, translational, and post-translational modifications. Targeted modulation of NCOA4-mediated ferritinophagy has been successfully used as a therapeutic strategy in several disease models. Recent evidences have elucidated that ferritinophagy and ferroptosis played a major role in heavy metals toxicity. In this review, we explored the regulatory mechanism of NCOA4 as the sole receptor for ferritinophagy from multiple perspectives based on previous studies. The significant role of ferritinophagy-mediated ferroptosis in heavy metals toxicity was discussed in detail, emphasizing the great potential of NCOA4 as a target for heavy metals toxicity.

Anagnostou G., Ferragina A., Crofton E.C., Frias Celayeta J.M., Hamill R.M.

The sensory quality of meat, encompassing the traits of appearance, texture, and flavour, is essential to consumer acceptance. Conventional quality assessment techniques, such as instrumental methods and trained sensory panels, often face limitations due to their destructive and time-consuming nature. In recent years, optical sensing techniques have emerged as a fast, non-invasive, and non-destructive technique for the prediction of quality attributes in meat and meat products, achieving prediction accuracies of over 90%. This review critically examines the potential of optical sensing techniques, such as near-infrared spectroscopy (NIRS), Raman spectroscopy, and hyperspectral imaging (HSI), to inform about the sensory attributes of red meat, aligning with industrial demands for early information on the predicted sensory performance of inventory to support meeting consumer requirements. Recent trends and the remaining challenges associated with these techniques will be described. While technical issues related to spectral data acquisition and data processing are important challenges when considering industrial implementation, overall, optical sensing techniques, in tandem with recent developments in digitalisation and data analytics, provide potential for the online prediction of meat sensory quality in the meat processing industries. Establishing technologies for enhanced information on the product and improved possibilities for quality control will help the industry to meet consumer demands for a consistent quality of product.

Zhang B., Fan K.

Amrita, Chakraborti S., Dey S.

Xie Z., Peng B., Shi M., Yang G., Song Z.

In plants, Ferritin is the earliest discovered regulator of iron (Fe) metabolism and plays a critical role in maintaining Fe storage and sequestration, which contributes to cellular Fe homeostasis and tolerance to abiotic stresses. However, biological functions of Ferritin proteins in perennial fruit crops are largely rare. In this study, VvFerritin1 was isolated from ‘Irsay Oliver’ table grape, and it was mainly expressed in roots and induced under Fe toxicity, H2O2 stress, and abscisic acid (ABA) treatment. Complementation of VvFerritin2 in yeast mutant DEY1453 directly restored the mutant growth, and VvFerritin1 can transport Fe2+ in yeast. The heterologous expression of VvFerritin1 in fer1-2 mutant effectively rescued the dwarfed growth of Arabidopsis fer1-2 mutant, under the control condition, Fe toxicity, or H2O2 stress, embodied in enhanced fresh weight (126%, 81%, or 48%), total root length (140%, 98%, or 64%), total root surface (70%, 84%, or 120%), and total leaf chlorophyll (56%, 51%, or 53%), respectively. In particular, tissue Fe concentration and activities of nitrite reductase (NiR), aconitase (ACO), and succinate dehydrogenase (SDH) were significantly enhanced in fer1-2/35S::Ferritin1 lines, respectively, compared to that of fer1-2 mutant. This work contributes to the study of molecular mechanisms of Fe storage and homeostasis in ‘Irsay Oliver’ table grape.

Matveeva V.O., Grebennikova A.D., Sakharov D.I., Fomin V.V., Manukhov I.V., Bazhenov S.V.

AbstractFerritin FtnA is the main scavenger of Fe2+and storage of Fe3+in bacterial cells, together with Dps and Bfr preventing the Fenton reaction and thus protecting the cell from iron-induced oxidative stress. However, until now, it was not known how its expression is regulated under conditions of oxidative stress, and the available evidence was contradictory. To study the regulation ofE. coli ftnAexpression in response to oxidative stress, PftnA-luxCDABEtranscriptional fusion in different strains was used. It has been shown that PftnAis induced after the addition of oxidative stress inducers. The maximum amplitude of this activation did not depend on the presence of functional genesoxyRandsoxRin the cell, but completely disappeared in the absence offur. The response is amplified in theftnAmutant and is diminished in the FtnA-overproducing strain, which indicates that iron sequestration blocks the response. Exposure of a cell to H2O2initially inactivates Fur and a number of iron-utilizing proteins, and derepresses iron uptake. This results in an increase in the cellular iron content with the consequent Fur reactivation, which leads to the induction offtnAexpression. Thus, oxidative stress leads to PftnAactivation, which is mediated by Fur and time-delayed in comparison with OxyR-response.

Tang Y., Zhang Q., Chen H., Chen G., Li Z., Chen G., Lin L., Yu Z., Su M., Zhang B.

Li Y., Liu R., Zhao Z.

Brain diseases pose significant treatment challenges due to the restrictive nature of the blood–brain barrier (BBB). Recent advances in targeting macromolecules offer promising avenues for overcoming these obstacles through receptor-mediated transcytosis (RMT). We summarize the current progress in targeting brain drug delivery with macromolecules for brain diseases. This exploration details the transport mechanisms across the BBB, focusing on RMT and its use of natural ligands for drug delivery. Furthermore, the review examines macromolecular ligands such as antibodies, peptides, and aptamers that leverage RMT for effective BBB traversal. Advancements in macromolecules-based delivery systems for brain diseases are summarized, emphasizing their therapeutic potential and limitations. Finally, emerging RMT strategies, including viral vectors, exosomes, and boron neutron capture therapy, are discussed for their precision in brain-targeted treatments. This comprehensive overview underscores the potential of RMT-based approaches to revolutionize brain disease therapy.

Bou‐Abdallah F., Fish J., Terashi G., Zhang Y., Kihara D., Arosio P.

AbstractDespite ferritin's critical role in regulating cellular and systemic iron levels, our understanding of the structure and assembly mechanism of isoferritins, discovered over eight decades ago, remains limited. Unveiling how the composition and molecular architecture of hetero‐oligomeric ferritins confer distinct functionality to isoferritins is essential to understanding how the structural intricacies of H and L subunits influence their interactions with cellular machinery. In this study, ferritin heteropolymers with specific H to L subunit ratios were synthesized using a uniquely engineered plasmid design, followed by high‐resolution cryo‐electron microscopy analysis and deep learning‐based amino acid modeling. Our structural examination revealed unique architectural features during the self‐assembly mechanism of heteropolymer ferritins and demonstrated a significant preference for H‐L heterodimer formation over H‐H or L‐L homodimers. Unexpectedly, while dimers seem essential building blocks in the protein self‐assembly process, the overall mechanism of ferritin self‐assembly is observed to proceed randomly through diverse pathways. The physiological significance of these findings is discussed including how ferritin microheterogeneity could represent a tissue‐specific adaptation process that imparts distinctive tissue‐specific functions to isoferritins.

Abramson J., Adler J., Dunger J., Evans R., Green T., Pritzel A., Ronneberger O., Willmore L., Ballard A.J., Bambrick J., Bodenstein S.W., Evans D.A., Hung C., O’Neill M., Reiman D., et. al.

AbstractThe introduction of AlphaFold 21 has spurred a revolution in modelling the structure of proteins and their interactions, enabling a huge range of applications in protein modelling and design2–6. Here we describe our AlphaFold 3 model with a substantially updated diffusion-based architecture that is capable of predicting the joint structure of complexes including proteins, nucleic acids, small molecules, ions and modified residues. The new AlphaFold model demonstrates substantially improved accuracy over many previous specialized tools: far greater accuracy for protein–ligand interactions compared with state-of-the-art docking tools, much higher accuracy for protein–nucleic acid interactions compared with nucleic-acid-specific predictors and substantially higher antibody–antigen prediction accuracy compared with AlphaFold-Multimer v.2.37,8. Together, these results show that high-accuracy modelling across biomolecular space is possible within a single unified deep-learning framework.

Sudarev V.V., Gette M.S., Bazhenov S.V., Tilinova O.M., Zinovev E.V., Manukhov I.V., Kuklin A.I., Ryzhykau Y., Vlasov A.V.

Ferritin is a universal protein complex responsible for iron perception in almost all living organisms and has applications from fundamental biophysics to drug delivery and structure-based immunogen design. Different platforms based on ferritin share similar technological challenges limiting their development – control of self-assembling processes of ferritin itself as well as ferritin-based chimeric recombinant protein complexes. In our research, we studied self-assembly processes of ferritin-based protein complexes under different expression conditions. We fused a ferritin subunit with a SMT3 protein tag, a homolog of human Small Ubiquitin-like Modifier (SUMO-tag), which was taken to destabilize ferritin 3-fold channel contacts and increase ferritin-SUMO subunits solubility. We first obtained the octameric protein complex of ferritin-SUMO (8xFer-SUMO) and studied its structural organization by small-angle X-ray scattering (SAXS). Obtained SAXS data correspond well with the high-resolution models predicted by AlphaFold and CORAL software of an octameric assembly around the 4-fold channel of ferritin without formation of 3-fold channels. Interestingly, three copies of 8xFer-SUMO do not assemble into 24-meric globules. Thus, we first obtained and structurally characterized ferritin-based self-assembling oligomers in a deadlock state. Deadlock oligomeric states of ferritin extend the known scheme of its self-assembly process, being new potential tools for a number of applications. Finally, our results might open new directions for various biotechnological platforms utilizing ferritin-based tools.

Meng E.C., Goddard T.D., Pettersen E.F., Couch G.S., Pearson Z.J., Morris J.H., Ferrin T.E.

AbstractAdvances in computational tools for atomic model building are leading to accurate models of large molecular assemblies seen in electron microscopy, often at challenging resolutions of 3‐4 Å. We describe new methods in the UCSF ChimeraX molecular modeling package that take advantage of machine‐learning structure predictions, provide likelihood‐based fitting in maps, and compute per‐residue scores to identify modeling errors. Additional model‐building tools assist analysis of mutations, post‐translational modifications, and interactions with ligands. We present the latest ChimeraX model‐building capabilities, including several community‐developed extensions. ChimeraX is available free of charge for noncommercial use at https://www.rbvi.ucsf.edu/chimerax.This article is protected by copyright. All rights reserved.

Osipov S.D., Ryzhykau Y.L., Zinovev E.V., Minaeva A.V., Ivashchenko S.D., Verteletskiy D.P., Sudarev V.V., Kuklina D.D., Nikolaev M.Y., Semenov Y.S., Zagryadskaya Y.A., Okhrimenko I.S., Gette M.S., Dronova E.A., Shishkin A.Y., et. al.

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 FOF1-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 FOF1-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 FOF1 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 FOF1-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 FOF1 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.

Sudarev V.V., Dolotova S.M., Bukhalovich S.M., Bazhenov S.V., Ryzhykau Y.L., Uversky V.N., Bondarev N.A., Osipov S.D., Mikhailov A.E., Kuklina D.D., Murugova T.N., Manukhov I.V., Rogachev A.V., Gordeliy V.I., Gushchin I.Y., et. al.

Ferritin is a vital protein complex responsible for storing iron in almost all living organisms. It plays a crucial role in various metabolic pathways, inflammation processes, stress response, and pathogenesis of cancer and neurodegenerative diseases. In this review we discuss the role of ferritin in diseases, cellular iron regulation, its structural features, and its role in biotechnology. We also show that molecular mechanisms of ferritin self-assembly are key for a number of biotechnological and pharmaceutical applications. The assembly pathways strongly depend on the interface context of ferritin monomers and the stability of its different intermediate oligomers. To date, several schemes of self-assembly kinetics have been proposed. Here, we compare different self-assembly mechanisms and discuss the possibility of self-assembly control by switching between deadlock intermediate states.

Dayhoff G.W., Uversky V.N.

Protein intrinsic disorder is found in all kingdoms of life and is known to underpin numerous physiological and pathological processes. Computational methods play an important role in characterizing and identifying intrinsically disordered proteins and protein regions. Herein, we present a new high-efficiency web-based disorder predictor named Rapid Intrinsic Disorder Analysis Online (RIDAO) that is designed to facilitate the application of protein intrinsic disorder analysis in genome-scale structural bioinformatics and comparative genomics/proteomics. RIDAO integrates six established disorder predictors into a single, unified platform that reproduces the results of individual predictors with near-perfect fidelity. To demonstrate the potential applications, we construct a test set containing more than one million sequences from one hundred organisms comprising over 420 million residues. Using this test set, we compare the efficiency and accessibility (i.e. ease of use) of RIDAO to five well-known and popular disorder predictors, namely: AUCpreD, IUPred3, metapredict V2, flDPnn, and SPOT-Disorder2. We show that RIDAO yields per-residue predictions at a rate two to six orders of magnitude greater than the other predictors and completely processes the test set in under an hour. RIDAO can be accessed free of charge at https://ridao.app. This article is protected by copyright. All rights reserved.

Wang W., Liu X., Wang Y., Wang Y., Fu D., Xi H., Zhao Y., Wang H.

Ferritins can generally be divided into four subfamilies based on their structural characteristics, namely, the classic ferritins (Ftns), bacterioferritins (Bfrs), DNA-binding proteins from starved cells (Dps’), and encapsulated ferritins (EncFtns). However, the ferritin from Mycoplasma penetrans (Mpef) possesses a particular ferroxidase center with an extreme low activity and exhibits unusual characteristics, indicating that it could be a member of a quite different subfamily of ferritins. Hereby, the crystal structure of the ferritin from Ureaplasma urealyticum (Uurf) is presented, Mpef and Uurf have very similar properties, though they display very low sequence similarity. Thus, ferritins from Mycoplasma with these unique properties do not belong to any known subfamily, but they should rather be placed in a novel ferritin subfamily, which we term Mycoplasma Ferritin (Mfr). Ferritin from Mycoplasma represents some unique characteristics, including a unique ferroxidase center and much lower iron oxidation activity, as well as the channel for iron passthrough. Therefore, it should be classified into a new ferritin subfamily, Mycoplasma ferritin (Mfr).

Wang X., Liu Y., Xue C., Hu Y., Zhao Y., Cai K., Li M., Luo Z.

cGAS-STING pathway is a key DNA-sensing machinery and emerges as a promising target to overcome the immunoresistance of solid tumors. Here we describe a bovine serum albumin (BSA)/ferritin-based nanoagonist incorporating manganese (II) ions and β-lapachone, which cooperatively activates cGAS-STING signaling in dendritic cells (DCs) to elicit robust adaptive antitumor immunity. Mn2+-anchored mannose-modified BSAs and β-lapachone-loaded ferritins are crosslinked to afford bioresponsive protein nanoassemblies, which dissociate into monodispersive protein units in acidic perivascular tumor microenvironment (TME), thus enabling enhanced tumor penetration and spatiotemporally controlled Mn2+ and β-lapachone delivery to DCs and tumor cells, respectively. β-lapachone causes immunogenic tumor cell apoptosis and releases abundant dsDNA into TME, while Mn2+ enhances the sensitivity of cGAS to dsDNA and augments STING signaling to trigger downstream immunostimulatory signals. The cGAS-STING nanoagonist enhances the tumor-specific T cell-mediated immune response against poorly immunogenic solid tumors in vivo, offering a robust approach for immunotherapy in the clinics. Manganese has a crucial role in cGAS-STING-mediated DNA sensing and has emerged as a STING agonist. Here the authors report the design and characterization of a nanosystem incorporating manganese ions and the chemotherapeutic drug β-lapachone, inducing T-cell mediated anti-tumor immune responses in preclinical cancer models.

Tsoraev G.V., Protasova E.A., Klimanova E.A., Ryzhykau Y.L., Kuklin A.I., Semenov Y.S., Ge B., Li W., Qin S., Friedrich T., Sluchanko N.N., Maksimov E.G.

The structural organization of natural pigment-protein complexes provides a specific environment for the chromophore groups. Yet, proteins are inherently dynamic and conformationally mobile. In this work, we demonstrate the heterogeneity of chromophores of C-phycocyanin (C-PC) from Arthrospira platensis. Part of the population of trimeric C-PC is subject to spontaneous disturbances of protein–protein interactions resulting in increased conformational mobility of the chromophores. Upon fluorescence excitation in the visible range, the spectral signatures of these poorly populated states are masked by bulk chromophore states, but the former could be clearly discriminated when the fluorescence is excited by near-infrared quanta. Such selective excitation of conformationally mobile C-PC chromophores is due to the structure of their S1 level, which is characterized by a significantly broadened spectral line. We demonstrate that the anti-Stokes C-PC fluorescence is the result of single-photon absorption. By combining spectral and structural methods, we characterize four distinct states of C-PC chromophores emitting at 620, 650, 665, and 720 nm and assigned the fast component in the anti-Stokes fluorescence decay kinetics in the range of 690–750 nm to the chromophores with increased conformational mobility. Our data suggest that the spectral and temporal characteristics of the anti-Stokes fluorescence can be used to study protein dynamics and develop methods to visualize local environment parameters such as temperature.

Krausova K., Charousova M., Kratochvil Z., Takacsova P., Tesarova B., Sivak L., Peskova M.K., Sukupova M., Zivotska H., Makovicky P., Yamashita I., Okamoto N., Hynek D., Haddad Y., Pekarik V., et. al.

• Reversible reassembly properties of four different types of ferritins were studied. • For all examined ferritins, disassembly is faster in acidic pH than in basic pH. • Archaeal Pfu ferritin is the most stable in both, acidic and basic conditions. • All ferritins form protein corona hampering their cellular uptake. • For all ferritins, kidney/liver are the preferential bioaccumulation sites. Understanding the reversible reassembly properties of ferritins (FRTs) is crucial for enabling their applicability in nanomedicine. These properties include drug loading capabilities and also subsequent payload release in desired site-of-action. Thus, the presented study is focused on understanding the disassembly of recombinantly produced FRTs of varying origin and subunit composition, i.e . equine FRT composed of 22 L - and 2 H -subunits (EcaLH) or 24 L -subunits (EcaL), human FRT composed of 24 H -subunits (HsaH) and archaeal Pyrococcus furiosus FRT (Pfu). Disassembly was distinctly influenced by pH and ionic strength. Except for Pfu, the disassembly kinetics in acidic pH is rapid upon reaching an innate threshold, reaching the final state within minutes. Disassembly kinetics in basic pH is slower. Pfu is partially disassembled within the entire pH range. While equine FRT disassembles into free subunits, HsaH disassembles into subunit clusters. EcaL and Pfu form large aggregates during disassembly in mildly acidic pH, although basic pH causes partial disassembly without aggregation, suggesting usability for basic pH-triggered drug loading. We show that in human serum/plasma, FRTs readily form protein corona, hampering their uptake. Interestingly, we found out that archaeal Pfu likely exhibits similar receptor affinity as mammalian FRTs. Further, in vivo toxicity and biodistribution study of a single dose of FRTs demonstrated very low toxicity of FRTs and their preferential liver/kidney bioaccumulation.

Kurakin S., Ivankov O., Skoi V., Kuklin A., Uhríková D., Kučerka N.

Combining small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and densitometric measurements, we have studied the interactions of the divalent cations Ca2+ and Mg2+ with the lipid vesicles prepared of a mixed-chain palmitoyl-oleoyl-phosphatidylcholine (POPC) at 25°C. The structural parameters of the POPC bilayer, such as the bilayer thickness, lateral area, and volume per lipid, displayed no changes upon the ion addition at concentrations up to 30 mM and minor changes at > 30 mM Ca2+ and Mg2+, while some decrease in the vesicle radius was observed over the entire concentration range studied. This examination allows us to validate the concept of lipid–ion interactions governed by the area per lipid suggested previously and to propose the mixed mode of those interactions that emerge in the POPC vesicles. We speculate that the average area per POPC lipid that corresponds to the cutoff length of lipid–ion interactions generates an equal but opposite impact on ion bridges and separate lipid–ion pairs. As a result of the dynamic equilibrium, the overall structural properties of bilayers are not affected. As the molecular mechanism proposed is affected by the structural properties of a particular lipid, it might help us to understand the fundamentals of processes occurring in complex multicomponent membrane systems.

Murugova T.N., Ivankov O.I., Ryzhykau Y.L., Soloviov D.V., Kovalev K.V., Skachkova D.V., Round A., Baeken C., Ishchenko A.V., Volkov O.A., Rogachev A.V., Vlasov A.V., Kuklin A.I., Gordeliy V.I.

AbstractDespite remarkable progress, mainly due to the development of LCP and ‘bicelle’ crystallization, lack of structural information remains a bottleneck in membrane protein (MP) research. A major reason is the absence of complete understanding of the mechanism of crystallization. Here we present small-angle scattering studies of the evolution of the “bicelle” crystallization matrix in the course of MP crystal growth. Initially, the matrix corresponds to liquid-like bicelle state. However, after adding the precipitant, the crystallization matrix transforms to jelly-like state. The data suggest that this final phase is composed of interconnected ribbon-like bilayers, where crystals grow. A small amount of multilamellar phase appears, and its volume increases concomitantly with the volume of growing crystals. We suggest that the lamellar phase surrounds the crystals and is critical for crystal growth, which is also common for LCP crystallization. The study discloses mechanisms of “bicelle” MP crystallization and will support rational design of crystallization.

Mirdita M., Schütze K., Moriwaki Y., Heo L., Ovchinnikov S., Steinegger M.

AbstractColabFold offers accelerated prediction of protein structures and complexes by combining the fast homology search of MMseqs2 with AlphaFold2 or RoseTTAFold. ColabFold’s 40−60-fold faster search and optimized model utilization enables prediction of close to 1,000 structures per day on a server with one graphics processing unit. Coupled with Google Colaboratory, ColabFold becomes a free and accessible platform for protein folding. ColabFold is open-source software available at https://github.com/sokrypton/ColabFold and its novel environmental databases are available at https://colabfold.mmseqs.com.

Yin S., Davey K., Dai S., Liu Y., Bi J.

Ferritin stores and releases iron ions in mammals. It is globally important as a drug nanocarrier. This is because of its unique hollow-spherical structure, desirable stability and biological properties. Novel drug-loading approaches plus various functionalization approaches have been developed to improve ferritin in response to differing demands in disease treatments. Here, we critically review ferritin drug delivery and evaluate its diverse drug-loading and functionalization approaches, we: (1) Introduce basic structural and property information related to ferritin as a drug nanocarrier; (2) Contrast in detail the different means to load drugs and the selection of drug loading means; (3) Discuss multiple ferritin functionalization approaches, together with related advantages and potential risks; and, (4) Compare ferritin with alternative, commonly-used drug nanocarriers. We conclude that despite that no drugs based on ferritin are commercially available, the market potential for it is significant, and evaluate future research directions. Findings from this work will be of immediate benefit and interest to a wide range of researchers and manufacturers for drug delivery using ferritin.