Protein Environment Research Articles

Mutual Dependence between Membrane Phase Separation and Bacterial Division Protein Dynamics in Synthetic Cell Models

Cell membranes in bacteria are laterally polarized to produce specific environments for membrane proteins, e.g., proteins involved in cell division which accumulate at mid‐cell or the cell poles. An interesting result of such membrane‐lipid interplay is the reorganization of lipid domains together with membrane‐bound proteins at the onset of cell division, suggesting functional significance of membrane compartments in the cell cycle. Here, by adopting the key bacterial division proteins MinC, MinD, MinE, FtsA and FtsZ as an archetypal spatial patterning system, we present a simple vesicle‐based in vitro model to explore the mutual dependence of protein pattern formation and membrane heterogeneity. Like many other peripheral membrane proteins, Min proteins exhibit preferential binding and macro‐scale pattern formation at Ld domains, which leads to altered oscillation mode selection in phase‐separated membrane compartments (GUVs). Moreover, incorporating bacterial division proteins within phase‐separated GUVs leads to blebbing‐like membrane deformations followed by the reorganization of Lo domains aligning at the neck region of the bleb, which agrees well with the domain rearrangement in bacterial membranes immediately preceding the radial constriction process. Overall, the presented in vitro model system showcases a basic framework to better comprehend the cellular division mechanism in consideration of complex cellular lipid environments.

Absorption and Excited-State Coherences of Cryogenically Cold Retinal Protonated Schiff Base in Vacuo.

Retinal protonated Schiff base (RPSB), found in its all-trans conformer in Bacteriorhodopsin, undergoes barrier-controlled isomerization upon photoabsorption through polyene- chain torsion. The effects of the protein environment on the active vibrations during photoabsorption and their redistribution are still not understood. This paper reports on femtosecond time-resolved action-absorption measurements of cryogenically cooled gas-phase all-trans RPSB, which exhibit two coherent vibrational oscillations, 167(14) cm-1 and 117(1) cm-1, of the first excited state with dephasing times of ∼ 1 ps. The absence of the high-frequency vibration in solution and the low-frequency vibration in the protein indicates that these vibrations are sensitive to environments. An action-absorption spectrum of cryogenically cold all-trans RPSB, reveals a ∼ 310 cm-1 active vibration when using a hole-burning technique and 1500 cm-1 C=Cstretching modes.

Modelling the Repair of Carbon-Centered Protein Radicals by Phenolic Antioxidants.

Oxidative stress is a biological process that has been linked to many diseases, hence understanding how to prevent and repair it is essential to medicine. The thermodynamics and kinetics of the repair reactions of radically damaged leucine (a lateral chain in a simplified protein environment) by twenty phenolic antioxidants are studied at the M06-2X(SMD)/6-31++G(d,p) level of theory in water and pentyl ethanoate. The two repair mechanisms modelled are formal-hydrogen atom transfer (f-HAT) and single electron transfer (SET). Although all f-HAT reactions are thermodynamically favourable, only one of the phenols produced rate constants in the diffusion limit, exhibiting biological relevance. SET is not suspected to be an important repair pathway for the phenols studied. We show that the Bell-Evans-Polanyi principle, which relates thermodynamics and kinetics properties for a reaction, breaks down when comparing between the solvents, protein repair sites, and the phenolic antioxidants. While thermodynamic data can be used as valuable screening tools, the kinetic calculation of rate constants in solution is crucial for enhancing the biological relevance of theoretical studies.

Separation and Purification of CHO Secretome and Extracellular Vesicles for Proteome Analysis.

For decades, host cell proteins (HCPs) have been investigated as putative contaminants in downstream processing of biopharmaceutical products of Chinese hamster ovary (CHO) cells. However, little is still known about the composition of the entire protein and vesicle environment in CHO cultivations. Ever evolving mass spectrometry techniques allow more and more insights into cell-cell communication processes and the composition of extracellular matrix, proteases, and further actively segregated compounds such as extracellular vesicles (EVs). EVs themselves are a heterologous group consisting of exosomes, ectosomes, and apoptotic vesicles. To specifically analyze these subsets of the secretome and determine beneficial and detrimental factors for a production process, targeted separation and purification techniques are necessary.In this chapter, we present our optimized workflows for a clear differentiation between directly secreted proteins and the vesicular protein content of different fractions (especially exosomal small EVs) from CHO cell supernatant for proteomic analysis by NanoLC ESI-MS.

The beta 2 adrenergic receptor cross-linked interactome identifies 14-3-3 proteins as regulating the availability of signaling-competent receptors.

The emerging picture of G protein-coupled receptor function suggests that the global signaling response is an integrated sum of a multitude of individual receptor responses, each regulated by their local protein environment. The beta 2 adrenergic receptor (B2AR) has long served as an example receptor in the development of this model. But the mechanism and the identity of the protein-protein interactions that govern the availability of receptors competent for signaling remains incompletely characterized. To address this question, we characterized the interactome of agonist-stimulated B2AR in HEK293 cells using FLAG co-immunoprecipitation coupled to SILAC labeling and mass spectrometry. Our B2AR cross-linked interactome identified 190 high-confidence proteins, including almost all known interacting proteins and six out of seven isoforms of the 14-3-3 family of scaffolding proteins. Inhibiting 14-3-3 proteins with the peptide difopein enhanced isoproterenol-stimulated adrenergic signaling via cAMP approximately three-fold, and increased both miniGs and arrestin recruitment to B2AR more than two fold each, without noticeably changing EC50 with respect to cAMP signaling or effector recruitment upon stimulation. Our results show that 14-3-3 proteins negatively regulate downstream signaling by inhibiting access of B2AR to effector proteins. We propose that 14-3-3 proteins maintain a dynamic pool of B2AR that has reduced signaling efficacy in response to acute agonist stimulation, limiting the amount of signaling-competent receptors at the plasma membrane. Significance Statement This study presents a new interactome of the agonist-stimulated beta 2 adrenergic receptor (B2AR), a paradigmatic GPCR that is both a model system for members of this class and an important signaling protein in respiratory, cardiovascular, and metabolic regulation. We identify 14-3-3 proteins as responsible for restricting B2AR access to signaling effectors and maintaining a receptor population that is insensitive to acute stimulation by agonists.

Reactivity of a Morita-Baylis-Hillman Adduct Derivative Bearing a Triphenylamine Moiety with Lysine Models.

The reactivity of Morita-Baylis-Hillman Adduct (MBHA) derivative 7 was studied with different primary amine derivatives such as n-butylamine, Nα-acetyl-L-lysine methyl ester, and a poly-(L-lysine) derivative as lysine models to obtain information about the possible reactions in complex protein environments. MBHA derivative 7 reacted with n-butylamine or Nα-acetyl-L-lysine methyl ester producing monoadducts 9a or 9c, which showed bright emission features in the green region at 526-535 nm with photoluminescence quantum yield values in solutions of 73 % and 51 %, respectively. Based on these results, MBHA derivative 7 can be considered an interesting new fluorogenic probe potentially useful in the labelling of basic amino acid residues. Furthermore, similar to other MBHA derivatives, compound 7 showed the tendency to produce diadducts especially in polar solvents system where specific interactions between the extended aromatic moieties may play a major role.

Arginine Accelerates Sulfur Fluoride Exchange and Phosphorus Fluoride Exchange Reactions between Proteins.

Sulfur fluoride exchange (SuFEx) and phosphorus fluoride exchange (PFEx) click chemistries are advancing research across multiple disciplines. By genetically incorporating latent bioreactive unnatural amino acids (Uaas), these chemistries have been integrated into proteins, enabling precise covalent linkages with biological macromolecules and paving the way for new applications. However, their suboptimal reaction rates in proteins limit effectiveness, and traditional catalytic methods for small molecules are often incompatible with biological systems or in vivo applications. We demonstrated that introducing an arginine adjacent to the latent bioreactive Uaa significantly boosts SuFEx and PFEx reaction rates between proteins. This method is effective across various Uaas, target residues, and protein environments. Notably, it also enables efficient SuFEx reactions in acidic conditions, common in certain cellular compartments and tumor microenvironments, which typically hinder SuFEx reactions. Furthermore, we developed the first covalent cell engager that substantially enhances natural killer cell activation through improved covalent interaction facilitated by arginine. These findings provide mechanistic insights and offer a biocompatible strategy to harness these robust chemistries for advancing biological research and developing new biotherapeutics.

Arginine Accelerates Sulfur Fluoride Exchange and Phosphorus Fluoride Exchange Reactions between Proteins

AbstractSulfur fluoride exchange (SuFEx) and phosphorus fluoride exchange (PFEx) click chemistries are advancing research across multiple disciplines. By genetically incorporating latent bioreactive unnatural amino acids (Uaas), these chemistries have been integrated into proteins, enabling precise covalent linkages with biological macromolecules and paving the way for new applications. However, their suboptimal reaction rates in proteins limit effectiveness, and traditional catalytic methods for small molecules are often incompatible with biological systems or in vivo applications. We demonstrated that introducing an arginine adjacent to the latent bioreactive Uaa significantly boosts SuFEx and PFEx reaction rates between proteins. This method is effective across various Uaas, target residues, and protein environments. Notably, it also enables efficient SuFEx reactions in acidic conditions, common in certain cellular compartments and tumor microenvironments, which typically hinder SuFEx reactions. Furthermore, we developed the first covalent cell engager that substantially enhances natural killer cell activation through improved covalent interaction facilitated by arginine. These findings provide mechanistic insights and offer a biocompatible strategy to harness these robust chemistries for advancing biological research and developing new biotherapeutics.

Switchable protection and exposure of a sensitive squaraine dye within a redox active rotaxane

In nature, molecular environments in proteins can sterically protect and stabilize reactive species such as organic radicals through non-covalent interactions. Here, we report a near-infrared fluorescent rotaxane in which the stabilization of a chemically labile squaraine fluorophore by the coordination of a tetralactam macrocycle can be controlled chemically and electrochemically. The rotaxane can be switched between two co-conformations in which the wheel either stabilizes or exposes the fluorophore. Coordination by the wheel affects the squaraine’s stability across four redox states and renders the radical anion significantly more stable—by a factor of 6.7—than without protection by a mechanically bonded wheel. Furthermore, the fluorescence properties can be tuned by the redox reactions in a stepwise manner. Mechanically interlocked molecules provide an excellent scaffold to stabilize and selectively expose reactive species in a co-conformational switching process controlled by external stimuli.

Discovery of Multiple Light-Harvesting States of the Photosynthetic Protein PE545.

Cryptophytes are photosynthetic microalga that flourish in a remarkable diversity of natural environments by using pigment-containing proteins with absorption maxima tuned to each ecological niche. While this diversity in the absorption has been well established, the subsequent photophysics is highly sensitive to the local protein environment and so may exhibit similar variation. Thermal fluctuations of the protein conformation are expected to introduce photophysical heterogeneity of the pigments that may have evolved important functional properties in a manner similar to that of the absorption. However, such heterogeneity is averaged out in ensemble measurements and, therefore, has not yet been probed. Here, we report single-molecule measurements of phycoerythrin 545 (PE545), the prototypical cryptophyte antenna protein, in its native dimeric form. A conformational ensemble was resolved consisting of distinct photophysical states with different light-harvesting properties. Proteins that did not quench, partially quenched, or fully quenched absorbed light were observed. Light intensity increased the quenched-state population of the dimer, potentially as a mechanism to deal with the extreme light intensities found in aqueous environments. Cross-linking, which mimics local interactions, introduces this light-dependent functionality while also suppressing other conformational dynamics. The cellular organization can, therefore, actively modulate the protein conformation and dynamics, selecting for distinct levels of light harvesting. Thus, the complex conformational equilibrium provides an additional mechanism for cryptophytes and likely other photosynthetic organisms to optimize solar energy capture and conversion.

Vinyl Ether Maleic Acid Polymers: Tunable Polymers for Self-Assembled Lipid Nanodiscs and Environments for Membrane Proteins.

Native lipid bilayer mimetics, including those that use amphiphilic polymers, are important for the effective study of membrane-bound peptides and proteins. Copolymers of vinyl ether monomers and maleic anhydride were developed with controlled molecular weights and hydrophobicity through reversible addition-fragmentation chain-transfer polymerization. After polymerization, the maleic anhydride units can be hydrolyzed, giving dicarboxylates. The vinyl ether and maleic anhydride copolymerized in a close to alternating manner, giving essentially alternating hydrophilic maleic acid units and hydrophobic vinyl ether units along the backbone after hydrolysis. The vinyl ether monomers and maleic acid polymers self-assembled with lipids, giving vinyl ether maleic acid lipid particles (VEMALPs) with tunable sizes controlled by either the vinyl ether hydrophobicity or the polymer molecular weight. These VEMALPs were able to support membrane-bound proteins and peptides, creating a new class of lipid bilayer mimetics.

Tunneling Mechanisms of Quinones in Photosynthetic Reaction Center–Light Harvesting 1 Supercomplexes

In photosynthesis, light energy is absorbed and transferred to the reaction center, ultimately leading to the reduction of quinone molecules through the electron transfer chain. The oxidation and reduction of quinones generate an electrochemical potential difference used for adenosine triphosphate synthesis. The trafficking of quinone/quinol molecules between electron transport components has been a long‐standing question. Here, an atomic‐level investigation into the molecular mechanism of quinol dissociation in the photosynthetic reaction center–light‐harvesting complex 1 (RC–LH1) supercomplexes from Rhodopseudomonas palustris, using classical molecular dynamics (MD) simulations combined with self‐random acceleration MD‐MD simulations and umbrella sampling methods, is conducted. Results reveal a significant increase in the mobility of quinone molecules upon reduction within RC–LH1, which is accompanied by conformational modifications in the local protein environment. Quinol molecules have a tendency to escape from RC–LH1 in a tail‐first mode, exhibiting channel selectivity, with distinct preferred dissociation pathways in the closed and open LH1 rings. Furthermore, comparative analysis of free energy profiles indicates that alternations in the protein environment accelerate the dissociation of quinol molecules through the open LH1 ring. In particular, aromatic amino acids form π‐stacking interactions with the quinol headgroup, resembling the key components in a conveyor belt system. This study provides insights into the molecular mechanisms that govern quinone/quinol exchange in bacterial photosynthesis and lays the framework for tuning electron flow and energy conversion to improve metabolic performance.

Exploring the binding interaction of Bis-benzimidazoles with BSA and relocation of bound drug from BSA to DNA

In recent years Bis-benzimidazole derivatives have gained much research interest due to their regulatory role in biological reactions to control diseases. Therefore, probing the interaction of the bioactive derivatives with biomolecules is crucial for both physiological and pharmacological aspects. Consequently, we studied the interaction between Bis-benzimidazoles and bovine serum albumin (BSA) as model transport protein by multi-spectroscopic techniques. It was also evident that substitutions of the phenolic OH group of Hoechst-33258 by -OMe and -NEt2 group have negligible impact on the binding phenomenon with BSA. Additionally, we have shown that the bound derivatives can be successfully relocated from serum albumin to calf-thymus DNA (ct-DNA). The observed higher binding affinity of the Hoechst-33258 to ct-DNA than BSA was responsible for the relocation of dyes from BSA to DNA. The competitive displacement assay with known site markers revealed that the probable binding location of the Hoechst-33258 within the serum protein was site IB. Molecular docking study was also performed to support the experimental findings and to obtain the possible interaction of drug molecule in the protein environment. Our findings might be helpful in overcoming the challenges associated with the delivery of bis-benzimidazole derivatives.

Superexchange Electron Transfer and Protein Matrix in the Charge-Separation Process of Photosynthetic Reaction Centers.

In type-II reaction centers, such as photosystem II (PSII) and reaction centers from purple bacteria (PbRC), light-induced charge separation involves electron transfer from pheophytin (PheoD1) to quinone (QA), occurring near a conserved tryptophan residue (D2-Trp253 in PSII and Trp-M252 in PbRC). This study investigates the route of the PheoD1-to-QA electron transfer, focusing on the superexchange coupling (|HPheoD1···QA|) in the PSII protein environment. |HPheoD1···QA| is significantly larger for the PheoD1-to-QA electron transfer via the unoccupied molecular orbitals of D2-Trp253 ([Trp]•--like intermediate state, 0.73 meV) compared to direct electron transfer (0.13 meV), suggesting that superexchange is the dominant mechanism in the PSII protein environment. While the overall impact of the protein environment is limited, local interactions, particularly H-bonds, enhance superexchange electron transfer by directly affecting the delocalization of molecular orbitals. The D2-W253F mutation significantly decreases the electron transfer rate. The conservation of D2-Trp253/D1-Phe255 (Trp-M252/Phe-L216 in PbRC) in the two branches appears to differentiate superexchange coupling, contributing to the branches being either active or inactive in electron transfer.

Polar Interactions between Substrate and Flavin Control Iodotyrosine Deiodinase Function.

Flavin cofactors offer a wide range of chemical mechanisms to support a great diversity in catalytic function. As a corollary, such diversity necessitates careful control within each flavoprotein to limit its function to an appropriate subset of possible reactions and substrates. This task falls to the protein environment surrounding the flavin in most enzymes. For iodotyrosine deiodinase that catalyzes a reductive dehalogenation of halotyrosines, substrates can dictate the chemistry available to the flavin. Their ability to stabilize the necessary one-electron reduced semiquinone form of flavin strictly depends on a direct coordination between the flavin and α-ammonium and carboxylate groups of its substrates. While perturbations to the carboxylate group do not significantly affect binding to the resting oxidized form of the deiodinase, dehalogenation (kcat/Km) is suppressed by over 2000-fold. Lack of the α-ammonium group abolishes detectable binding and dehalogenation. Substitution of the ammonium group with a hydroxyl group does not restore measurable binding but does support dehalogenation with an efficiency greater than those of the carboxylate derivatives. Consistent with these observations, the flavin semiquinone does not accumulate during redox titration in the presence of inert substrate analogues lacking either the α-ammonium or carboxylate groups. As a complement, a nitroreductase activity based on hydride transfer is revealed for the appropriate substrates with perturbations to their zwitterion.

Facile incorporation of non-canonical heme ligands in myoglobin through chemical protein synthesis

The incorporation of non-canonical amino acids (ncAAs) into the metal coordination environments of proteins has endowed metalloproteins with enhanced properties and novel activities, particularly in hemoproteins. In this work, we disclose a scalable synthetic strategy that enables the production of myoglobin (Mb) variants with non-canonical heme ligands, i.e., HoCys and f4Tyr. The ncAA-containing Mb* variants (with H64V/V68A mutations) were obtained through two consecutive native chemical ligations and a subsequent desulfurization step, with overall isolated yield up to 28.6 % in over 10-milligram scales. After refolding and heme b cofactor reconstitution, the synthetic Mb* variants showed typical electronic absorption bands. When subjected to the catalysis of the cyclopropanation of styrene, both synthetic variants, however, were not as competent as the His-ligated Mb*. We envisioned that the synthetic method reported herein would be useful for incorporating a variety of ncAAs with diverse structures and properties into Mb for varied purposes.

A Polarizable QM/MM Model That Combines the State-Averaged CASSCF and AMOEBA Force Field for Photoreactions in Proteins.

This study presents the polarizable quantum mechanics/molecular mechanics (QM/MM) embedding of the state-averaged complete active space self-consistent field (SA-CASSCF) in the atomic multipole optimized energetics for biomolecular applications (AMOEBA) force field for the purpose of studying photoreactions in protein environments. We describe two extensions of our previous work that combine SA-CASSCF with AMOEBA water models, allowing it to be generalized to AMOEBA models for proteins and other macromolecules. First, we discuss how our QM/MM model accounts for the discrepancy between the direct and polarization electric fields that arises in the AMOEBA description of intramolecular polarization. A second improvement is the incorporation of link atom schemes to treat instances in which the QM/MM boundary goes through covalent bonds. A single-link atom scheme and double-link atom scheme are considered in this work, and we will discuss how electrostatic interaction, van der Waals interaction, and various kinds of valence terms are treated across the boundary. To test the accuracy of the link atom scheme, we will compare QM/MM with full QM calculations and study how the errors in ground state properties, excited state properties, and excitation energies change when tuning the parameters in the link atom scheme. We will also test the new SA-CASSCF/AMOEBA method on an elementary reaction step in NanoLuc, an artificial bioluminescence luciferase. We will show how the reaction mechanism is different when calculated in the gas phase, in polarizable continuum medium (PCM), versus in protein AMOEBA models.

Guanidinium and spermidinium decavanadates: as small biomimetic models to understand non-covalent interactions between decavanadate and arginine and lysine side chains in proteins

During the last three decades, numerous investigations have been conducted on polyoxidovanadates to treat several illnesses and inhibit enzymes. Numerous decavanadate compounds have been proposed as potential therapies for Diabetes mellitus, Cancer, and Alzheimer’s disease. Only six relevant functional proteins interacting with decavanadate, V10, have been deposited in the PDB. These are acid phosphatase, tyrosine kinase, two ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), the human transient receptor potential cation channel (TRPM4), and the human cell cycle protein CksHs1. The interaction sites in these proteins mainly consist of Arginine and Lysine, side chains binding to the decavanadate anion. To get further knowledge regarding non-covalent interactions of decavanadate in protein environments, guanidinium and spermidinium decavanadates were synthesized, crystallized, and subjected to analysis utilizing various techniques, including FTIR, Raman, 51V-NMR, TGA, and X-ray diffraction. The DFT calculations were employed to calculate the interaction energy between the decavanadate anion and the organic counterions. Furthermore, the Quantum Theory of Atoms in Molecules (QTAIM) and Non-covalent Interaction-Reduced Density Gradient (NCI-RDG) analyses were conducted to understand the non-covalent interactions present in these adducts. Decavanadate can engage in electrostatic forces, van der Waals, and hydrogen bond interactions with guanidinium and spermidinium, as shown by their respective interaction energies. Both compounds were highly stabilized by strong hydrogen bond interactions N−H···O and weak non-covalent interactions C−H···O. In addition, the interactions between guanidinium and spermidinium cations and decavanadate anion form several stable rings. This study provides new information on non-covalent intermolecular interactions between decavanadate and small biomimetic models of arginine and lysine lateral chains in protein environments.

Environment-dependent chlorophyll-chlorophyll charge transfer states in Lhca4 pigment-protein complex.

Photosystem I (PSI) light-harvesting antenna complexes LHCI contain spectral forms that absorb and emit photons of lower energy than that of its primary electron donor, P700. The most red-shifted fluorescence is associated with the Lhca4 complex. It has been suggested that this red emission is related to the inter-chlorophyll charge transfer (CT) states. In this work we present a systematic quantum-chemical study of the CT states in Lhca4, accounting for the influence of the protein environment by estimating the electrostatic interactions. We show that significant energy shifts result from these interactions and propose that the emission of the Lhca4 complex is related not only to the previously proposed a603+-a608- state, but also to the a602+-a603- state. We also investigate how different protonation patterns of protein amino acids affect the energetics of the CT states.

A Comprehensive Study of Factors Affecting the Prediction of the pKa Shift of Asp26 in Thioredoxin Protein.

The stable protonation state of ionizable amino acids in a protein can be predicted by computing the pKa shift of that residue within the protein environment. Thermodynamic Integration (TI) is an ideal molecular dynamics-based approach for predicting the pKa shift of ionizable protein residues. Here, we probe TI-based simulation protocols for their ability to accurately predict the pKa shift of Asp26 in thioredoxin. While implicit solvent models can predict the pKa shift accurately, explicit solvent models result in substantial errors. To understand the underlying reason for this surprising discrepancy, we investigate the role of various factors such as solvent models, conformational sampling, background charges, and polarization.