Biochemistry [JOURNAL]
Sun
200 papers
Mitochondrial Ubiquitination as a Signaling Hub: Balancing Mitophagy, Inflammation, and Cell Death.
Kumar A, Zacharioudakis E
Biochemistry · 2026 Apr · PMID 41941306 · Publisher ↗
Mitochondria are increasingly recognized as signaling organelles that coordinate cell-fate decisions during stress. Because outer mitochondrial membrane (OMM) proteins are exposed to the cytosol, they are prominent subst...
Mitochondria are increasingly recognized as signaling organelles that coordinate cell-fate decisions during stress. Because outer mitochondrial membrane (OMM) proteins are exposed to the cytosol, they are prominent substrates for ubiquitination, a dynamic post-translational modification that encodes information through diverse chain architectures and linkage types. In this review, we examine how ubiquitination of OMM proteins functions as a molecular switch that integrates mitochondrial stress signals and engages three major, often antagonistic, stress-response mechanisms: mitophagy, cell death, and innate immune signaling. We highlight an emerging concept that a stress-responsive "ubiquitin code" is written on OMM substrates, in which pathway selection is coordinated by the identity of ubiquitinated OMM proteins together with the linkage type and branching of attached polyubiquitin chains. We provide an updated overview of the E3 ubiquitin ligases and deubiquitinases (DUBs) that write and erase this code and summarize ubiquitin linkage types reported on key OMM substrates across these pathways. For mitophagy, we cover both PARKIN-dependent and PARKIN-independent mechanisms mediated by other E3 ligases and counteracted by DUBs. For innate immunity, we discuss how ubiquitination of OMM proteins regulates the MDA5/RIG-I-MAVS axis and NF-κB signaling. For cell death, we describe how ubiquitination of anti- and pro-apoptotic BCL-2 family proteins can either lower or increase the threshold for the induction of apoptosis. We also highlight the newfound role of PARKIN to drive apoptosis through a BAX/BAK-independent mechanism. Finally, we discuss therapeutic opportunities to reprogram OMM ubiquitination by targeting E3 ligases or DUBs directly, or by using PROTAC- and DUBTAC-based strategies.
Transient Helicity in the Intrinsically Disordered Protein ACTR Measured by Hydrogen Exchange.
Cuciurean IS, Parsbæk CB, Rand KD … +2 more , Mulder FAA, Teilum K
Biochemistry · 2026 Apr · PMID 41937577 · Publisher ↗
Intrinsically disordered proteins (IDPs) play essential roles in cellular signaling and regulation, often relying on transient structural elements to mediate interactions. NMR chemical shifts are widely used to detect se...
Intrinsically disordered proteins (IDPs) play essential roles in cellular signaling and regulation, often relying on transient structural elements to mediate interactions. NMR chemical shifts are widely used to detect secondary structures in IDPs, but complementary methods are needed to validate and refine these measurements. Hydrogen exchange is a powerful probe of local structure and dynamics in folded proteins, yet its accuracy for detecting small differences in transient helicity in IDPs remains understudied. Here, we systematically evaluate hydrogen exchange measured by NMR and MS (HDX-MS) in four variants of the activator for thyroid hormone and retinoid receptors (ACTR) activation domain that differ in helical propensity. Using NMR-based exchange rates, we introduce pseudo-protection factors referenced to the wild-type protein, enabling a robust comparison among variants without relying on "intrinsic" peptide-based chemical exchange rates. These pseudo-protection factors correlate strongly with helicity derived from chemical shifts, demonstrating that hydrogen exchange can resolve subtle structural differences in highly dynamic regions and vice versa. Our findings establish hydrogen exchange as a sensitive and reproducible method for characterizing transient structure in IDPs, complementing NMR chemical shift analysis.
Two-Stage Purification of Recombinant Antibodies Using Non-Ionic Detergent Prevents Endotoxin-Induced Macrophage Activation: The Case of Bispecific Mini-Antibody MYSTI-2.
Khodak YA, Litvinova AM, Sysonov FA … +4 more , Drutskaya MS, Safenkova IV, Nedospasov SA, Vorobiev II
Biochemistry (Mosc) · 2026 Mar · PMID 41936564 · Publisher ↗
is one of the most common producers of recombinant proteins, including therapeutic antibody fragments. However, the outer membrane of contains high levels of lipopolysaccharide (LPS, also known as endotoxin), which can...
is one of the most common producers of recombinant proteins, including therapeutic antibody fragments. However, the outer membrane of contains high levels of lipopolysaccharide (LPS, also known as endotoxin), which can activate innate immune receptors, trigger immune responses, and induce systemic inflammation that may progress to septic shock. Ensuring extremely low endotoxin levels in preparations intended for applications is critically important. In this study, we investigated the endotoxin content in preparations of the bispecific mini-antibody MYSTI-2 produced in two strains: the Rosetta strain, which synthesizes conventional LPS, and the ClearColi strain, which synthesizes potentially non-toxic form of LPS. Our results demonstrate that near-complete removal of LPS can be achieved only through the use of a non-ionic detergent during purification, regardless of the bacterial strain used for protein production.
Effect of Mutations in the N-Terminal Peptide of the Coat Protein on the Structure of Potato Virus X According to Small-Angle X-Ray Scattering and Molecular Dynamics.
Ksenofontov AL, Petoukhov MV, Arutyunyan AM … +7 more , Oleynikov IP, Peters GS, Baratova LA, Arkhipenko MV, Nikitin NA, Karpova OV, Shtykova EV
Biochemistry (Mosc) · 2026 Mar · PMID 41936563 · Publisher ↗
Mutations in the N-terminal peptide (Ser-Thr to Ala-Gly substitution) of the coat protein (CP) of potato virus X (PVX-ST) render its genomic RNA translationally competent, unlike in the wild-type PVX virions. Consequentl...
Mutations in the N-terminal peptide (Ser-Thr to Ala-Gly substitution) of the coat protein (CP) of potato virus X (PVX-ST) render its genomic RNA translationally competent, unlike in the wild-type PVX virions. Consequently, RNA within the PVX-ST virions can be translated without additional triggers (such as phosphorylation or interaction with the triple gene block 1 protein), unlike the encapsidated RNA of the wild-type virus. Comprehensive structural analysis using molecular dynamics (MD), small-angle X-ray scattering (SAXS), and tritium planigraphy revealed differences in the virion organization. The mutations were shown to increase hydrophobicity and induce partial folding of the N-terminal peptides. This triggers structural rearrangement in the PVX-ST virion: packing density of the coat proteins within the helical capsid is altered. This conclusion is supported by the SAXS data, increased accessibility for tritium labeling of the key CP domains (including the RNA-binding region), and reduced stability against the action of the sodium dodecyl sulfate detergent. The obtained results provide explanation for the mechanism by which the encapsidated RNA of the PVX-ST mutant becomes accessible to ribosomes. This mechanism is associated with structural rearrangement of the N-terminal coat protein peptide and change in the packing density of the helical capsid.
Bispecific Mini-Antibody with an Anti-CD14 Module Effectively Controls Bioavailability of the Human Tumor Necrosis Factor Produced by Human Monocytes.
Konev IY, Korneev KV, Kozlovsky SV … +8 more , Dianov DV, Litvinova AM, Drize NI, Rybtsov SA, Gubernatorova EO, Sysonov FA, Drutskaya MS, Nedospasov SA
Biochemistry (Mosc) · 2026 Mar · PMID 41936562 · Publisher ↗
Systemic blockade of proinflammatory cytokines such as IL-1, TNF, and IL-6 using therapeutic antibodies has proven effective in treating a wide range of autoimmune and other chronic inflammatory diseases. However, such b...
Systemic blockade of proinflammatory cytokines such as IL-1, TNF, and IL-6 using therapeutic antibodies has proven effective in treating a wide range of autoimmune and other chronic inflammatory diseases. However, such blockade also suppresses non-redundant protective and homeostatic functions of cytokines, leading to a number of undesirable side effects. In this study, a novel bispecific mini-antibody featuring modules targeting human TNF and CD14 demonstrated efficacy in controlling TNF secretion from human peripheral blood monocytes. Administration of this antibody protected humanized TNF mice from lethal hepatotoxicity induced by a combination of LPS and D-galactosamine.
The Structure of the Nucleosomal DNA Repair Intermediate Affects the HPF1-Independent Automodification Activity of PARP2.
Kurgina TA, Shtanov DM, Kutuzov MM … +2 more , Moor NA, Lavrik OI
Biochemistry (Mosc) · 2026 Mar · PMID 41936561 · Publisher ↗
DNA-dependent nuclear enzymes poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) are involved in the regulation of multiple DNA repair pathways, including base excision repair (BER). After activation by binding to da...
DNA-dependent nuclear enzymes poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) are involved in the regulation of multiple DNA repair pathways, including base excision repair (BER). After activation by binding to damaged DNA, these enzymes synthesize negatively charged poly(ADP-ribose) (PAR) and covalently attach to amino acid residues of target proteins, including PARPs themselves. PARP2 activity is influenced by the nature of DNA lesion; for example, it is efficiently stimulated by DNA breaks flanked by phosphate group. However, it remains unclear which stages of the auto-PARylation reaction are most sensitive to the structure of damaged DNA. In this study, we investigated how PARP2 activity depends on the presence and position of a single-nucleotide gap in DNA (either free or in the context of nucleosome) at different stages of the automodification reaction conducted in the absence of the histone PARylation factor HPF1. The obtained results suggest that the presence of the gap affects the affinity of PARP2 for DNA/nucleosomes, thereby determining the number of catalytically active enzyme molecules and the efficiency of PARylation initiation. In contrast, PAR elongation was affected by the lesion location in the DNA/nucleosome structure, namely, its distance from the blunt DNA ends, and the environment of histone tails. Therefore, the damaged DNA structure can influence both the amount and the length of PAR synthesized by PARP2.
Disruption of Lam Family Sterol Transporters Results in Sporulation Defects in the Yeast .
Surikova ED, Golyshev SA, Smirnova EA … +4 more , Bartysh EA, Knorre DA, Severin FF, Sokolov SS
Biochemistry (Mosc) · 2026 Mar · PMID 41936560 · Publisher ↗
The primary role of sterols in the cell is to support plasma membrane function, and for this reason their concentration in this compartment is the highest among all cell membranes. In the yeast , sterol transport between...
The primary role of sterols in the cell is to support plasma membrane function, and for this reason their concentration in this compartment is the highest among all cell membranes. In the yeast , sterol transport between membranes is mediated by proteins of Osh and Lam families. The Lam1-Lam4 proteins are reported to transport sterols passively from plasmalemma to endoplasmic reticulum. The Lam5-Lam6 proteins transport sterols at the ER interface with vacuoles and mitochondria. Deletion of the family genes does not impair cell growth under standard conditions, which makes their biological role unclear. We hypothesized that the Lam family proteins may play a role in yeast sporulation, as the spore plasma membrane is formed from the ER-derived vesicles, which contain less sterol than the plasma membrane, necessitating sterol transport into the newly forming spore plasma membrane. To test this hypothesis, we generated diploid strains with the and deletions. We demonstrated that double deletion of the genes reduced both percentage of the sporulating cells and number of the spores per ascus. Conversely, deletion of the genes reduced proportion of the full asci but did not inhibit sporulation initiation. We demonstrated that deletion of the genes induces cell wall thickening and structural defects. Clusters of osmiophilic granules were detected at the cell wall surface of these spores. Spores with the deletions of the family genes demonstrated reduced resistance to heat shock and alkali. Taken together, our data indirectly support our hypothesis and point out that sterol transport by the LAM family proteins is necessary for the sterol redistribution during sporulation.
Analysis of Purity and Quality of Small Extracellular Vesicles Isolated from Blood Plasma by Proteomics and Raman Spectroscopy.
Chernyshev VS, Starodubtseva NL, Rimskaya EN … +14 more , Bugrova AE, Kononikhin AS, Silachev DN, Tokareva AO, Evtushenko EA, Yakovlev AA, Yurin AM, Kepsha MA, Mezhevitinova EA, Nikolaev EN, Frankevich VE, Nazarova NM, Prilepskaya VN, Sukhikh GT
Biochemistry (Mosc) · 2026 Mar · PMID 41936559 · Publisher ↗
Ultracentrifugation (UC) has long been considered the "gold standard" for extracellular vesicle (EV) isolation. However, due to its drawbacks such as high cost of an ultracentrifuge and rotors, time-consuming and labor-i...
Ultracentrifugation (UC) has long been considered the "gold standard" for extracellular vesicle (EV) isolation. However, due to its drawbacks such as high cost of an ultracentrifuge and rotors, time-consuming and labor-intensive protocol, low yield considering initial biofluid volume and low throughput, development of new EV isolation approaches is still ongoing. Here we compare three methods for isolating the most studied EV subtype, small extracellular vesicles (sEVs), from human plasma: ultracentrifugation (UC), express asymmetric depth filtration (ExADFi), and anti-CD9 immunoaffinity capture (AS-CD9) with focus on their Raman and proteomic profiles. For all three methods, purity and quality of the sEV isolation were assessed based on the level of contamination of the sEV fraction with major plasma proteins such as albumin and apolipoproteins (APOA1, APOH, APOA4, APOC2, APOC1, and APOC4). UC showed the highest ratio of protein to nanoparticle concentration. AS-CD9 and ExADFi provided comparable to UC purity and levels of non-vesicular contaminants with AS-CD9 requiring minimal time and labor. ExADFi showed characteristics including purity of the sEV samples, yield, and isolation time that is between the UC and AS-CD9 methods. Raman spectroscopy provided more details about characteristics of the isolated sEVs and confirmed differences observed in the proteomic profiles. The findings demonstrate that the AS-CD9 and ExADFi methods could be appropriate substitutes of the classical UC-based isolation method and be chosen depending on the final requirements and use of the purified sEVs such as further functional and biomarker studies.
Comparative Bioinformatics Analysis of Physicochemical Properties of the Mouse Protein Epitopes.
Lobanov MY, Dovidchenko NV, Zakharov ME … +2 more , Panfilov AV, Galzitskaya OV
Biochemistry (Mosc) · 2026 Mar · PMID 41936558 · Publisher ↗
Major histocompatibility complex class I (MHC I) plays a crucial role in immune functions. This complex typically binds short fragments of protein chains, 8-9 amino acid residues in length, referred to as epitopes. In th...
Major histocompatibility complex class I (MHC I) plays a crucial role in immune functions. This complex typically binds short fragments of protein chains, 8-9 amino acid residues in length, referred to as epitopes. In this study, we investigated differences between the peptides that bind to this complex (dataset N) and those that do not (dataset N). To compare the datasets N and N, Z-score analysis using the Z-score function was applied to identify statistically significant differences in physicochemical properties under study: aliphatic index (α), charge (Z), hydrophobicity (H), isoelectric point (pI), molecular weight (M), and instability index (II). All properties except for the instability index depend solely on amino acid composition of the peptides and not on the sequence-specific features. For the evaluated physicochemical properties, the Z-score values indicated no significant differences between the datasets N and N. Maximum Z-score values were 0.30 for the aliphatic index and 0.29 for hydrophobicity. The most robust and reliable separation between the datasets N and N was achieved using the r-value method, yielding classification accuracy of approximately 70% and Z-score of 0.63. This result is close to the separation accuracy of 75% obtained using the MHCflurry program. Analysis of amino acid distributions in the datasets N and N showed that the residues such as tyrosine, phenylalanine, isoleucine, leucine, and valine occur more frequently than cysteine, tryptophan, arginine, and lysine in the octa- and nonapeptide epitopes that noncovalently bind to MHC class I. Using bioinformatics analysis and artificial intelligence approaches, we demonstrated the extent to which binding and non-binding peptides can be discriminated based solely on amino acid composition.
Epigallocatechin Gallate as an Anti-Fibrotic Agent.
Tarahovsky YS, Gaidin SG, Kim YA
Biochemistry (Mosc) · 2026 Mar · PMID 41936557 · Publisher ↗
Epigallocatechin gallate (EGCG), a major polyphenolic compound in green tea, exhibits preventive and therapeutic effects in many fibrotic diseases. Tissue fibrosis is characterized by excessive deposition of collagen fib...
Epigallocatechin gallate (EGCG), a major polyphenolic compound in green tea, exhibits preventive and therapeutic effects in many fibrotic diseases. Tissue fibrosis is characterized by excessive deposition of collagen fibrils in the extracellular matrix, primarily due to dysregulation of cellular signaling pathways. However, we have previously demonstrated that EGCG directly inhibits the formation of collagen fibrils from collagen monomers under experimental conditions that excluded involvement of cellular signaling systems. This review explores the antifibrotic action of EGCG, which may occur through (i) its influence on cellular signaling and (ii) direct binding to collagen monomers, leading to the inhibition of pathological fibrillogenesis, as well as discuss the prospects for targeting the collagen assembly process.
Circular RNAs Fifty Years After Their Discovery.
Filippenkov IB, Tsareva EV, Mozgovoy IV … +3 more , Sudarkina OY, Dergunova LV, Limborska SA
Biochemistry (Mosc) · 2026 Mar · PMID 41936556 · Publisher ↗
Circular RNAs (circRNAs) are a unique class of covalently closed molecules formed through non-canonical splicing and characterized by a markedly greater stability compared to linear RNAs. Although the first circRNA was d...
Circular RNAs (circRNAs) are a unique class of covalently closed molecules formed through non-canonical splicing and characterized by a markedly greater stability compared to linear RNAs. Although the first circRNA was discovered half a century ago in 1976 in a viroid, they had remained largely overlooked for several decades. Over the past ten years, the however, interest in circRNAs has grown substantially, even as their biological functions and overall significance continue to be debated. It is now well established that circRNAs constitute a large and diverse group of molecules with varied origins and properties. They have been identified across a wide range of organisms, from prokaryotes to plants and mammals, where they participate in the regulation of numerous cellular processes. The unique properties of circRNAs are beginning to be exploited for practical applications, including their use as disease biomarkers and platforms for the development of novel therapeutic strategies. This review summarizes the knowledge accumulated on circRNAs since their discovery and highlights recent advances in understanding their biology and potential applications.
Phosphatidylinositol Interactions with the SARS-CoV-2 Envelope Protein Investigated by Lipid C Labeling and Solid-State NMR.
Medeiros-Silva J, Zhang Y, Hong M
Biochemistry · 2026 Apr · PMID 41931714 · Publisher ↗
Membrane protein structure and function are intimately influenced by the surrounding lipids. Solid-state NMR spectroscopy is an important approach for investigating site-specific protein-lipid interactions under physiolo...
Membrane protein structure and function are intimately influenced by the surrounding lipids. Solid-state NMR spectroscopy is an important approach for investigating site-specific protein-lipid interactions under physiological conditions. To observe protein-lipid contacts with high spectral sensitivity and lipid selectivity, here we describe an efficient protocol for producing and purifying C-labeled phospholipids from yeast. We focused on phosphatidylinositol (PI), an important lipid involved in cellular signaling and membrane trafficking. Using C-labeled PI, we investigated its interaction with the SARS-CoV-2 envelope protein E. C chemical shifts, relaxation times and cross-polarization buildup times indicate that the E transmembrane domain (ETM) rigidified the inositol headgroup and the acyl chains without causing significant chemical shift perturbations to the lipid, indicating that ETM-PI interaction is weak. Despite this weak interaction, protein-lipid cross peaks are observed in two-dimensional C-C correlation spectra, indicating that a subpopulation of PI lipids has specific interactions with the protein. These protein-bound annular PI lipids interact with Thr, Asn and Ser residues at the N- and C-terminal ends of the transmembrane helix, likely through hydrogen bonding and other electrostatic interactions. These results provide direct evidence that the SARS E protein interact with anionic PI lipids, and this interaction may modulate cation conduction by this pathogenic viroporin.
Enhancing Inhibitory Potency and Serum Stability of Peptide Inhibitors of the EphB2 Receptor.
Garcia BE, Epstein SR, Legarreta SA … +3 more , Tennett JC, Cain JM, Sawyer N
Biochemistry · 2026 Apr · PMID 41930744 · Publisher ↗
Ephrin (Eph) receptors play key regulatory roles in physiological processes, such as tissue development, cell migration, and angiogenesis. Upregulation of specific Eph receptors has been identified in many different canc...
Ephrin (Eph) receptors play key regulatory roles in physiological processes, such as tissue development, cell migration, and angiogenesis. Upregulation of specific Eph receptors has been identified in many different cancers, highlighting Eph receptors as promising targets for new cancer therapeutics. Though targeting individual Eph receptors is challenging because of the high sequence homology among the 14 Eph receptors in humans, peptides have been isolated through phage display that target specific receptors (EphA2, EphA4, EphB2, EphB4). While many of these peptides have been further optimized based on the original phage display hits, the EphB2 receptor-targeting SNEW peptide has received less attention. Here we describe parallel strategies to modify SNEW, leading to improved affinity for the EphB2 receptor and greater stability in human serum while retaining SNEW's high specificity for the EphB2 receptor. Specifically, replacement of the N-terminal serine residue with cyclic α-amino acids, particularly those with saturated, six-membered rings, increased inhibitory potency against the EphB2 receptor-ephrin B2 interaction. Replacement of a central proline residue with 4,4-difluoroproline led to a significant increase in serum stability in the context of SNEW and its more potent N-terminally modified variants. SNEW variants with greater potency and serum stability offer lead candidates for targeting the EphB2 receptor in associated cancers and other diseases. Additionally, the modification approaches employed for SNEW may be extensible for N-terminal serine/threonine/cysteine substitution and/or proline substitution to improve protein targeting by other peptides.
Prion Protein-Derived Cell-Penetrating Peptide Inhibits Type II Diabetes-Associated Islet Amyloid Polypeptide Aggregation and Cytotoxicity.
Oh Y, Palanikumar L, Howarth M … +6 more , Maity D, Ali L, Mustafa M, Kumar S, Hamilton AD, Magzoub M
Biochemistry · 2026 Apr · PMID 41926749 · Full text
Islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone copackaged and cosecreted with insulin by pancreatic β-cells. A pathological hallmark of type II diabetes is the self-assembly of IAPP into β-sheet rich am...
Islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone copackaged and cosecreted with insulin by pancreatic β-cells. A pathological hallmark of type II diabetes is the self-assembly of IAPP into β-sheet rich amyloid fibers, which is associated with β-cell impairment. Previously, we showed that a cell-penetrating peptide (CPP) construct, consisting of a hydrophobic signal sequence coupled to a polycationic nuclear localization signal (NLS)-like sequence, exhibited potent antiprion activity and antagonism of Alzheimer's disease-associated amyloid-β (Aβ) peptide aggregation and neurotoxicity. Here, we have extended this approach toward type II diabetes by assessing the efficacy of the CPP construct, designated as neural cell adhesion molecule-1 (NCAM1)-prion protein (PrP), in inhibiting IAPP oligomerization, fiber formation, and associated cytotoxicity. Using complementary and experiments, we show that NCAM1-PrP effectively modulates IAPP's toxic structures into nontoxic conformations. This study underlines the potential of our designed CPP-based therapeutic approach as a versatile tool in the battle against amyloid-associated pathologies.
Amino Acid Variants at the P94 Position in Class a Sortase Modulate Substrate Binding and Enzyme Activity.
Cox-Tigre N, Stewart ME, Tucker J … +4 more , Walkenhauer EG, Wilce CS, Antos JM, Amacher JF
Biochemistry · 2026 Apr · PMID 41926714 · Full text
The surface of Gram-positive bacteria is a highly regulated environment with specific attachment of proteins required for viability. Sortase enzymes recognize and ligate substrates to the peptidoglycan layer in these mic...
The surface of Gram-positive bacteria is a highly regulated environment with specific attachment of proteins required for viability. Sortase enzymes recognize and ligate substrates to the peptidoglycan layer in these microorganisms, which can be highly pathogenic (e.g., ). As such, sortases represent a potentially novel target for antibiotic development. In addition, the catalytic activity of sortase enzymes is utilized in sortase-mediated ligation (SML) engineering approaches for a variety of uses. In SML experiments, engineered variants of sortase A (saSrtA) are the most widely used enzymes. Structural analyses of experimental saSrtA structures revealed that the P94 position interacts directly with Y187 when saSrtA is in its inactive conformation. Interestingly, P94 is mutated in the previously engineered pentamutant (or saSrtA5M), to P94R. We wanted to interrogate single mutations at P94 further to characterize its effect on activity and/or substrate specificity. We created 18 P94X mutations (except cysteine) and tested relative activity for 4 substrate sequences: LPATG, LPETG, LPKTG, and LPSTG. We identified several P94 variants that both outperformed and contained differing specificity as compared to P94R. We tested P94A and P94D saSrtA5M variants and found that, depending on the substrate, these variants could outperform saSrtA5M in activity >3-fold. Finally, we compared saSrtA5M and P94D saSrtA5M in a model sortase-mediated ligation reaction using a LPKTG substrate and saw ∼2-fold greater product formation. We argue that future studies which combine rational design and high throughput approaches, e.g., directed evolution, may result in sortase variants with increased SML potential.
Mapping the Structural Determinants of Quorum Sensing Activity in Via Mutational Analysis of Its Competence Stimulating Peptide.
Renshaw CP, Basnet A, Yeh CR … +2 more , Wang A, Tal-Gan Y
Biochemistry · 2026 Apr · PMID 41923425 · Publisher ↗
is an oral commensal bacterium implicated in the severe cardiovascular disease, infective endocarditis. In Mitis group Streptococci, including , colonization, competition, and virulence are regulated by the ComABCDE quor...
is an oral commensal bacterium implicated in the severe cardiovascular disease, infective endocarditis. In Mitis group Streptococci, including , colonization, competition, and virulence are regulated by the ComABCDE quorum sensing (QS) system. This pathway depends on secretion of the competence stimulating peptide (CSP), which activates the membrane-bound histidine kinase receptor, ComD, to induce competence gene expression through the alternative sigma factor, ComX. Targeting this system offers a promising strategy for nonlethal inhibition of bacterial communication and virulence. This study investigates the structure-activity relationship (SAR) between the CSP and its ComD receptor. Alanine- and d-amino acid-substituted CSP libraries were synthesized and screened using a -luciferase reporter strain to assess receptor activation. Key residues required for ComD binding and activation were identified. The -terminal aspartic acid was essential for activation, and its substitution (CSP-D1A) yielded a potent competitive inhibitor. Circular dichroism spectroscopy revealed that CSP and most analogs adopt weak or transient α-helical structures in membrane-mimicking environments. AlphaFold 3 Multimer modeling of CSP/ComD and CSP-D1A/ComD interactions corroborated the SAR competitive inhibition findings, yet superimposition of the two predicted complexes did not indicate major differences in the binding orientation or receptor conformation, illustrating the limitations of static models when predicting structural dynamics. Overall, our findings define key molecular determinants of CSP signaling and provide a foundation for designing peptide-based QS inhibitors as novel antivirulence therapeutics.
Recombinant Expression and Automated Flow Synthesis of Fold-Switching Proteins Derived from Protein G.
Kjerfve C, Kwao JK, Wolfe J … +4 more , Glover J, Wood B, Golden C, Truex NL
Biochemistry · 2026 Apr · PMID 41921064 · Publisher ↗
Fold-switching proteins are an emerging class of biomolecules that respond to subtle cues to regulate biological processes. Expanding our understanding of fold-switching proteins requires reliable methods for preparing n...
Fold-switching proteins are an emerging class of biomolecules that respond to subtle cues to regulate biological processes. Expanding our understanding of fold-switching proteins requires reliable methods for preparing new variants with high purity and well-defined thermodynamic properties, but currently these methods remain limited. Here, we describe complementary methods for preparing fold-switching proteins by recombinant expression and, to our knowledge, the first reported total chemical synthesis. We prepared eight proteins derived from protein G: 56-residue G and G, with 95 and 98% sequence identity. Four variants were produced by bacterial expression in as SUMO fusion proteins, followed by traceless on-column cleavage and size-exclusion chromatography to generate the native sequences. The same four variants were also produced by automated flow protein synthesis, followed by RP-HPLC and anion-exchange chromatography purification. Both workflows give highly pure proteins that adopt folded conformations upon chromatographic desalting into phosphate buffer. Circular dichroism shows that the proteins attain the anticipated 3-α or 4β+α topologies. Each pair of recombinant and synthetic proteins adopts a similar conformation, as corroborated by trypsin digestion and H NMR studies. Thermodynamic studies show that the melting temperatures are within 1 °C of each other and the energetic differences are less than ±0.1 kcal mol. Together, these methods provide benchmarks for dual chemical and biological preparations of fold-switching proteins, facilitating access to broader chemical space to study their structural and thermodynamic properties and to design new proteins with switchable properties for applications in science and medicine.
Probing the Role of Accessory FeS Clusters in Putative Sensory Group D [FeFe] Hydrogenase: Influence of the C-Terminal [4Fe-4S] Cluster on the Reactivity of HydS.
Schumann C, Böhm M, Bhowmik P … +4 more , Huang P, Cabotaje PR, Land H, Berggren G
Biochemistry · 2026 Apr · PMID 41919677 · Full text
Hydrogenases are metalloenzymes that play key roles in H metabolism. Beyond their catalytic function, [FeFe] hydrogenases from phylogenetic groups C and D have been proposed to act as H sensors. These putative sensory en...
Hydrogenases are metalloenzymes that play key roles in H metabolism. Beyond their catalytic function, [FeFe] hydrogenases from phylogenetic groups C and D have been proposed to act as H sensors. These putative sensory enzymes contain, in addition to the canonical H-activating H-cluster, N-terminal [4Fe-4S] clusters as well as an atypical C-terminal [4Fe-4S] cluster, the latter being further associated with a Per-Arnt-Sim (PAS) domain in group C enzymes. The functional significance of these C-terminal clusters and their influence on enzymatic activity, however, remain poorly understood. Here we studied the accessory [4Fe-4S] clusters in the model group D enzyme from (HydS), by disrupting cluster formation in the N- and C-terminal domains, respectively. Spectroscopic and biochemical investigations indicated that one of the N-terminal [4Fe-4S] clusters is critical for the structural integrity of HydS. In contrast, disrupting the C-terminal [4Fe-4S] cluster through a cysteine to alanine mutation (C379A) resulted in a stable enzyme variant with a modified cluster. The C379A variant retained catalytic activity similar to the WT enzyme, although with a 2-fold enhancement of the H oxidation rate observed under high-driving-force conditions. Based on electron paramagnetic resonance spectroscopy, we found the oxidation state of the C-terminal cluster to be unresponsive to the presence of H gas. We inferred that the H-sensing or signaling function is unlikely to involve redox state changes in the C-terminal [4Fe-4S] cluster. Still, highly conserved charged residues around the [4Fe-4S] clusters of group D [FeFe] hydrogenases indicate a functional role of the C-terminal region.
Computations Reveal How Firefly Luciferase Governs Side Reactivity of Superoxide.
Blinova AR, Domratcheva T, Grigorenko BL
Biochemistry · 2026 Jun · PMID 41919442 · Publisher ↗
The dual nature of superoxide (O•) as both a damaging oxidant and a common intermediate in oxygen-dependent enzymatic reactions raises a fundamental question about how enzymes control its reactivity. Firefly luciferase (...
The dual nature of superoxide (O•) as both a damaging oxidant and a common intermediate in oxygen-dependent enzymatic reactions raises a fundamental question about how enzymes control its reactivity. Firefly luciferase (FLuc), which productively utilizes O• to drive its light-emitting reaction, provides an excellent model system to elucidate these control mechanisms. In this work, using a combination of quantum chemical, QM/MM, and QM/MM MD simulations, we demonstrate that this selectivity is governed by a preorganized active-site architecture. We show that a hydrogen-bonding network, primarily with the F246 backbone and active-site water molecules, stabilizes O•. At the same time, the G245-F246 backbone and F246 side chain sterically block unproductive additions of O• to the deprotonated luciferyl adenylate radical and orient O• for selective formation of the dioxetanone─the key intermediate of the light-emitting pathway. We also identify how the H244 side chain, protonated in the preceding reaction steps, can trigger a major unproductive side reaction producing the hydroperoxyl radical and subsequently hydrogen peroxide, and propose that this pathway may be potentially regulated by the pyrophosphate cofactor acting as a scavenger for excessive protons in the active site. Finally, we establish that the Mg ion is essential for the final dioxetanone ring closure by charge neutralization, which reduces the electrostatic repulsion in the AMP cleavage step and prevents the formation of an adenylated dioxetanone analogue unproductive in light emission.
Thermodynamic Basis of Temperature Adaptation in Three Outward Proton Pump Rhodopsins Distributed Across Diverse Thermal Environments.
Ohtake R, Kondo K, Nakano S … +3 more , Demura M, Kikukawa T, Tsukamoto T
Biochemistry · 2026 Apr · PMID 41915759 · Publisher ↗
Microbial rhodopsins with light-driven outward proton pump activity are widely distributed across Earth's diverse environments and contribute to solar energy conversion in global ecosystems. Yet, the thermodynamic princi...
Microbial rhodopsins with light-driven outward proton pump activity are widely distributed across Earth's diverse environments and contribute to solar energy conversion in global ecosystems. Yet, the thermodynamic principles enabling their function across broad temperature ranges remain poorly understood. Here, we examined the photocycles of three outward proton pump rhodopsins originating from low-, moderate-, and high-temperature environments─HnPR, PR, and TR─and analyzed their kinetics over a wide temperature range using flash photolysis. Thermodynamic activation parameters were determined for each transition state based on transition-state theory. Comparative analysis revealed that the balance of enthalpic and entropic contributions in the early P1 → P2 transition strongly correlates with the environmental temperature in which each rhodopsin is distributed, reflecting distinct adaptation strategies among the three proteins. TR exhibited temperature-dependent alterations in its photocycle, including a shift in the linearity of the Eyring plot for the P2 → P3 transition, consistent with a structural rearrangement previously observed by time-resolved FTIR spectroscopy. In contrast, HnPR displayed hallmark features of cold-adapted proteins in the P4 → P0 transition, including a reduced activation enthalpy and a pronounced decrease in activation entropy, enabling efficient turnover at low temperatures. Together, these findings provide a thermodynamic framework for understanding how outward proton pump rhodopsins function across diverse thermal habitats and illustrate the distinct molecular strategies by which they balance functional dynamics and structural stability to support light-driven proton transport in nature.
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