Intrinsic apoptosis is a form of cell death which is activated, executed, and inhibited by the Bcl-2 protein family. The structural basis of the inhibition mechanisms remains elusive. Here, we characterize the ensemble structural model of the inhibitory Bcl-xL/tBid complex at the mitochondrial membrane by probing interresidue distances and dynamic solvent accessibilities complemented by integrative modeling and molecular dynamics simulations. We show that Bcl-xL and tBid form a heterodimer anchored to the membrane by the C-terminal helix of Bcl-xL. The BH3 domain of tBid is wedged between the exposed hydrophobic groove of Bcl-xL and the membrane headgroups, while tBid's C-terminal helices remain dynamically engaged with the bilayer. This dynamic architecture sheds light on the mechanism of indirect inhibition of apoptosis.

Liquid-liquid phase separation of biomolecules can be induced in vitro by selecting specific concentrations and environmental conditions. Phase diagrams relating the temperature, pressure, etc. to the concentrations of proteins and co-solutes aid in identifying the specific phase separation conditions for target proteins. However, it remains challenging to determining complete phase diagrams for protein liquid-liquid phase separation. Here, we combine optical microscopy and in situ Raman spectroscopy to quantify the temperature-dependent concentrations of the light and dense phases of human γD-crystallin and construct a complete phase diagram. The method can be extended to systems in the presence of co-solutes, which enables the quantitative description of the partitioning of a co-solute in the light and dense phase of proteins.

The Bcl-2 protein Bcl-xL is an inhibitor of intrinsic apoptosis which either directly inhibits the pore-forming Bcl-2 proteins, like Bax or Bak, or indirectly inhibits pore formation by sequestering the pro-apoptotic BH3-only activators. The structural basis of the inhibition of pore formation in the outer mitochondrial membrane is still largely unknown due to the lack of atomic resolution structures of the relevant inhibitory complexes at the membrane. Herein, a protocol to obtain high-yield recombinant monomeric full-length Bcl-xL proteins is presented. The monomeric Bcl-xL retains the ability to shuttle between membrane and aqueous environments and can successfully inhibit Bcl-2-induced membrane permeabilization via both modes of action, as proven by in vitro and in organelle assays with a minimal Bcl-2 interactome constituted by Bcl-xL, cBid, and Bax.

Intrinsic apoptosis is mainly regulated through a network of conserved interactions between Bcl-2 proteins involving hydrophobic binding grooves and BH3 domains. Despite these conserved interfaces, family members exhibit distinct binding affinities and play opposing roles in apoptosis. While static structural differences partially account for this divergence, it remains unclear how opposing apoptotic function reflects in BH3 helix engagement of individual members. Here, we investigate how a BidBH3 peptide engages with the hydrophobic groove of full-length membrane-anchored Bcl-xL and Bax to identify shared and unique features of binding that may relate to distinct apoptotic functions. Using state-of-the-art enhanced-sampling simulations, we mapped the complete binding and folding landscapes of these critical cell-death regulators in membranes. Our simulations align with experimental measurements in terms of predicted absolute binding affinities, and also capture the dynamic, atomistic details of the conformational changes induced by BH3 helices. Together, these details highlight the structural principles of BH3 in-groove engagement that determine apoptotic function, paving the way towards the modulation of the interactions among the Bcl-2 family members.

Reactive oxygen species (ROS) are promising green candidates for tackling challenges ranging from antimicrobial resistance to water decontamination. Metal oxide nanomaterials structured as thin films, deposited at room temperature (RT) using plasma technology, can deliver ROS to the environment by catalyzing oxygen and water following a chemodynamics approach. This study proposes thin film plasma polymerization as a strategy to precisely control ROS delivery, unravel ROS formation mechanism at the catalytic interface, and ensure ROS‐driven chemistry. A proper combination of semiconductors, specifically silver oxide and titanium oxide, is used as a model system for ROS production. This specific coupling of semiconductors produces ROS in the dark due to charge separation without ion leaching. Plasma surface functionalization with nanoporous SiOx‐like films in the 1–100 nm range allows selective control of the delivery of radicals with different characteristic lifetimes such as superoxide anion and singlet oxygen based on the thickness of the functional layer. As proof of promising applications, results regarding radicals' detection are correlated with the antimicrobial activity of the ROS‐releasing system. Thin film plasma surface functionalization allows control of ROS delivery, ensuring that the material efficacy is due to ROS and not by other direct redox chemistry or leaching processes.

Tc toxins are pore-forming virulence factors of many pathogenic bacteria. Following pH-induced conformational changes, they perforate the target membrane like a syringe to translocate toxic enzymes into a cell. Although this complex transformation has been structurally well studied, the reaction pathway and the resulting temporal evolution have remained elusive. We used an integrated biophysical approach to monitor prepore-to-pore transition and found a reaction time of ~30 hours for a complete transition. We show two asynchronous general steps of the process, shell opening and channel ejection, with the overall reaction pathway being a slow multistep process involving three intermediates. Liposomes, an increasingly high pH, or receptors facilitate shell opening, which is directly correlated with an increased rate of the prepore-to-pore transition. Channel ejection is a near-instantaneous process which occurs with a transition time of <60 milliseconds. Understanding the mechanism of action of Tc toxins and unveiling modulators of the kinetics are key steps toward their application as biomedical devices or biopesticides.

The formation of protein condensates (droplets) via liquid-liquid phase separation (LLPS) is a commonly observed phenomenon in vitro. Changing the environmental properties with cosolutes, molecular crowders, protein partners, temperature, pressure, etc. has been shown to favor or disfavor the formation of protein droplets by fine-tuning the water-water, water-protein, and protein-protein interactions. Therefore, these environmental properties and their spatiotemporal fine-tuning are likely to be important also in a cellular context at the existing protein expression levels. One of the key physicochemical properties of biomolecules impacted by molecular crowding is diffusion, which determines the viscoelastic behavior of the condensates. Here, we investigate the change in the rotational diffusion of γD-crystallin, undergoing LLPS in vitro in aqueous solutions in the absence and presence of cosolutes. We studied its rotational dynamics using molecular dynamics simulations (MD), electron paramagnetic resonance (EPR) spectroscopy, and fluorescence spectroscopy. MD simulations performed under dilute and crowded conditions show that the rotational diffusion of crystallin in water is retarded by 1 to 2 orders of magnitude in the condensed phase. To obtain the rotational dynamics in the dilute phase, we used fluorescence anisotropy and to extract the retardation factor in the condensed phase, we used spin-labeled γD-crystallin proteins as EPR viscosity nanoprobes. Aided by a viscosity nanoruler calibrated with solutions at increasing sucrose concentrations, we validated the rotational diffusion retardation predicted by MD simulations. This study underlines the predictive power of MD simulations and showcases the use of a sensitive EPR nanoprobe to extract the viscosity of biomolecular condensates.

The mycobacterial ABC transporter IrtAB features an ABC exporter fold, yet it imports iron-charged siderophores called mycobactins. Here, we present extensive cryo-EM analyses and DEER measurements, revealing that IrtAB alternates between an inward-facing and an outward-occluded conformation, but does not sample an outward-facing conformation. When IrtAB is locked in its outward-occluded conformation in nanodiscs, mycobactin is bound in the middle of the lipid bilayer at a membrane-facing crevice opening at the heterodimeric interface. Mutations introduced at the crevice abrogate mycobactin import and in corresponding structures, the crevice is collapsed. A conserved triple histidine motif coordinating a zinc ion is present below the mycobactin binding site. Substitution of these histidine residues with alanine results in a decoupled transporter, which hydrolyzes ATP, but lost its capacity to import mycobactins. Our data suggest that IrtAB imports mycobactin via a credit-card mechanism in a transport cycle that is coupled to the presence of zinc. Mycobacteria import life-essential iron using capturing molecules called mycobactins. In this work, the authors uncover how an ATP-fuelled transporter imports mycobactin across the inner membrane in an unusual “credit card” mechanism, during which mycobactin slides down a crevice at the transporter surface.

The Bcl-2 protein Bcl-xL is a well-known inhibitor of apoptosis which can either directly inhibit the pore-forming Bcl-2 proteins, as Bax or Bak, or indirectly inhibit pore formation by sequestering the proapoptotic BH3-only proteins, as cBid or Bim. The structural basis of the inhibition of pore formation in the outer mitochondrial membrane by Bcl-xL is still largely unknown. Up to now, challenges in obtaining full-length Bcl-xL have hampered the full understanding of the inhibitory interactome at the membrane. Here we present an optimized protocol for the purification of full-length Bcl-xL, which allows separation of monomeric Bcl-xL from large oligomers which could not be otherwise eliminated in the purification procedure. The obtained monomeric Bcl-xL successfully inhibited pore formation via both modes of action, based on assays performed on large unilamellar vesicles in the presence of a minimal Bcl-2 interactome constituted by Bcl-xL, cBid and Bax. In contrast, the oligomeric fraction retained only the ability to indirectly inhibit pore formation by sequestering cBid. The presented protocol will pave the way towards biophysical studies aimed to unveil the structural details of apoptosis inhibition. ### Competing Interest Statement The authors have declared no competing interest. SNSF Sinergia grant, CRSII5_216587

Protonatable nitroxides are electron paramagnetic resonance (EPR) molecular probes employed for pH measurements in bulk aqueous media. The change in the protonation state of the molecule induces a measurable change in the g- and hyperfine (A) parameters used as pH indicators. The quantitative understanding of the origin of the change of the EPR parameters in terms of electronic structure and different solvation patterns is still lacking. Here, we delve into the origins of the changes in the g- and hyperfine (A) parameters of 14N upon protonation of the heterocyclic nitrogen of the pH-sensitive nitroxide probe HMI (2,2,3,4,5,5-hexamethylimidazolidin-1-oxyl, C9H19N2O) by means of combined experimental and theoretical techniques that have been developed and extensively validated in previous works. To establish a molecular-level understanding of the dependency of EPR parameters on the pH of the medium, we considered two limiting cases of deprotonated (pH ≫ pKa) and protonated (pH ≪ pKa) states of HMI. We found that, upon protonation of the heterocyclic nitrogen, the change in the electronic structure dominates the pH dependency of isotropic g and A values. Supporting this prominent role of electronic structure modulation, the average shift of EPR observables between the corresponding hydrogen-bonding states of the protonated and unprotonated forms remains constant. Furthermore, the results establish that the hydrogen bonding network structures around the nucleus of interest only marginally change upon protonation, although the populations of corresponding states with given H-bond numbers strongly do. This feature entails an additional, smaller contribution to the relative pH dependency of giso and Aiso values over electronic structure modulation upon protonation in a given H-bond state. The findings of this study pave the way to investigating HMI-based labels in peptides and other pH-sensitive EPR probes in protic polar solvents.

Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labelling provides information on structure and dynamics of biomolecules. Increasing the availability of spin labels with different properties is an elegant way to foster a more accurate analysis of the EPR data in relation to the biological problem investigated. In this study, we present a comparative investigation of labelling efficiency, surface accessibility, site specificity and width of the distance distributions obtained on two proteins with the nitroxide-based bromoacrylaldehyde spin label (BASL) versus the two commercial spin labels MTSL (methanethiosulfonate spin label) and MAP (maleimido proxyl). Based on the predicted distances from a rotamer library approach and on the experimental distance distributions, BASL is shown to provide generally narrower distance distributions compared to the other nitroxide labels. The exquisite surface specificity of BASL with respect to MAP could be successfully exploited to selectively label surface cysteines in proteins containing a high number of native cysteines. In addition, the distinct site-reactivity of BASL and MAP towards two surface-exposed cysteines was leveraged for orthogonal labelling strategies with nitroxide and gadolinium labels.

Modulation of absorbance and emission is key for the design of chiral chromophores. Accessing a series of compounds absorbing and emitting (circularly polarized) light over a wide spectral window and often toward near-infrared is of practical value in (chir)optical applications. Herein, by late-stage functionalization on derivatives bridging triaryl methyl and helicene domains, we have achieved the regioselective triple introduction of para electron-donating or electron-withdrawing substituents. Extended tuning of electronic (e.g., E-1/2(red) -1.50 V -> -0.68 V) and optical (e.g., emission covering from 550 to 850 nm) properties is achieved for the cations and neutral radicals; the latter compounds being easily prepared by mono electron reductions under electrochemical or chemical conditions. While luminescence quantum yields can be increased up to 70% in the cationic series, strong Cotton effects are obtained for certain radicals at low energies (lambda(abs) similar to 700-900 nm) with g(abs) values above 10(-3). The open-shell electronic nature of the radicals was further characterized by electron paramagnetic resonance revealing an important spin density delocalization that contributes to their persistence

Liquid–liquid phase separation (LLPS) plays a key role in the compartmentalization of cells via the formation of biomolecular condensates. Here, we combined atomistic molecular dynamics (MD) simulations and terahertz (THz) spectroscopy to determine the solvent entropy contribution to the formation of condensates of the human eye lens protein γD-crystallin. The MD simulations reveal an entropy tug-of-war between water molecules that are released from the protein droplets and the ones that are retained within the condensates, two categories of water molecules that were also assigned spectroscopically. A recently developed THz calorimetry method enables a quantitative comparison of the experimental and computational entropy changes of the released water molecules. The strong correlation mutually validates the two approaches and opens the way to a detailed atomic-level understanding of the different driving forces underlying LLPS.

Tc toxins are virulence factors of many insects and human pathogenic bacteria. They attach as soluble prepores to receptors on host cells and following acidification in the late endosome, perforate the cell membrane like a syringe to translocate toxic enzymes into the host cell through their pore-forming channel. Although this complex transformation has been structurally well studied, the functional aspects of this large-scale rearrangement, such as the reaction pathway with possible intermediate states and the resulting temporal evolution have remained elusive. Here, we used an integrated biophysical approach to monitor the prepore-to-pore transition and found that it takes ∼28 h when induced by high pH in the absence of other factors. In the presence of liposomes, an increasingly high pH or receptors, such as heparin or Vsg, the probability to transform prepores to pores increases by a factor of up to 4. This effect can also be mimicked by biotinylation or site-directed mutagenesis of the shell, demonstrating that shell destabilization is a crucial step in prepore-to-pore transition. We show that shell opening is a heterogeneous process with transition times ranging from 60 ms to 1.6 s and resolve three sequential intermediate states: an initial transient intermediate during shell destabilization, a first stable intermediate where the receptor-binding domains on the shell rearrange and a second stable intermediate with an open shell. In contrast, the ejection of the pore-forming channel from the open shell is highly cooperative with a transition time of < 60 ms. This detailed knowledge of the Tc toxin mechanism of action, even in the absence of receptors, is important for the future application of Tc toxins as biomedical devices or biopesticides.### Competing Interest StatementThe authors have declared no competing interest.

Nitroxides are common EPR sensors of microenvironmentalpropertiessuch as polarity, numbers of H-bonds, pH, and so forth. Their solvationin an aqueous environment is facilitated by their high propensityto form H-bonds with the surrounding water molecules. Their g- and A-tensor elements are key parametersto extracting the properties of their microenvironment. In particular,the g ( xx ) value of nitroxidesis rich in information. It is known to be characterized by discretevalues representing nitroxide populations previously assigned to havedifferent H-bonds with the surrounding waters. Additionally, thereis a large g-strain, that is, a broadening of g-values associated with it, which is generally correlatedwith environmental and structural micro-heterogeneities. The g-strain is responsible for the frequency dependence ofthe apparent line width of the EPR spectra, which becomes evidentat high field/frequency. Here, we address the molecular origin ofthe g ( xx ) heterogeneityand of the g-strain of a nitroxide moiety (HMI: 2,2,3,4,5,5-hexamethylimidazolidin-1-oxyl,C9H19N2O) in water. To treat thesolvation effect on the g-strain, we combined a multi-frequencyexperimental approach with ab initio molecular dynamics simulationsfor structural sampling and quantum chemical EPR property calculationsat the highest realistically affordable level, including an explicitlymicro-solvated HMI ensemble and the embedded cluster reference interactionsite model. We could clearly identify the distinct populations ofthe H-bonded nitroxides responsible for the g ( xx ) heterogeneity experimentally observed,and we dissected the role of the solvation shell, H-bond formation,and structural deformation of the nitroxide in the creation of the g-strain associated with each nitroxide subensemble. Twocontributions to the g-strain were identified inthis study. The first contribution depends on the number of hydrogenbonds formed between the nitroxide and the solvent because this hasa large and well-understood effect on the g ( xx )-shift. This contribution can only be resolved athigh resonance frequencies, where it leads to distinct peaks in the g ( xx ) region. The second contributionarises from configurational fluctuations of the nitroxide that necessarilylead to g-shift heterogeneity. These contributionscannot be resolved experimentally as distinct resonances but add tothe line broadening. They can be quantitatively analyzed by studyingthe apparent line width as a function of microwave frequency. Interestingly,both theory and experiment confirm that this contribution is independent of the number of H-bonds. Perhaps even moresurprisingly, the theoretical analysis suggests that the configurationalfluctuation broadening is not induced by the solventbut is inherently present even in the gas phase. Moreover, the calculationspredict that this broadening decreases upon solvationof the nitroxide.

Electron paramagnetic resonance spectroscopy (EPR) is mostly used in structural biology in conjunction with pulsed dipolar spectroscopy (PDS) methods to monitor interspin distances in biomacromolecules at cryogenic temperatures both in vitro and in cells. In this context, spectroscopically orthogonal spin labels were shown to increase the information content that can be gained per sample. Here, we exploit the characteristic properties of gadolinium and nitroxide spin labels at physiological temperatures to study side chain dynamics via continuous wave (cw) EPR at X band, surface water dynamics via Overhauser dynamic nuclear polarization at X band and short-range distances via cw EPR at high fields. The presented approaches further increase the accessible information content on biomolecules tagged with orthogonal labels providing insights into molecular interactions and dynamic equilibria that are only revealed under physiological conditions.

Macromolecular protein assemblies are of fundamental importance for many processes inside the cell, as they perform complex functions and constitute central hubs where reactions occur. Generally, these assemblies undergo large conformational changes and cycle through different states that ultimately are connected to specific functions further regulated by additional small ligands or proteins. Unveiling the 3D structural details of these assemblies at atomic resolution, identifying the flexible parts of the complexes, and monitoring with high temporal resolution the dynamic interplay between different protein regions under physiological conditions is key to fully understanding their properties and to fostering biomedical applications. In the last decade, we have seen remarkable advances in cryo-electron microscopy (EM) techniques, which deeply transformed our vision of structural biology, especially in the field of macromolecular assemblies. With cryo-EM, detailed 3D models of large macromolecular complexes in different conformational states became readily available at atomic resolution. Concomitantly, nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy (EPR) have benefited from methodological innovations which also improved the quality of the information that can be achieved. Such enhanced sensitivity widened their applicability to macromolecular complexes in environments close to physiological conditions and opened a path towards in-cell applications. In this review we will focus on the advantages and challenges of EPR techniques with an integrative approach towards a complete understanding of macromolecular structures and functions.

Juvenile visceral steatosis (JVS) mice are associated with systemic carnitine deficiency (Kuwajima, et al., 1991). In order to investigate the cause of this deficiency, we compared fibroblast carnitine transport activities in normal mice and JVS mice. The kinetic analysis showed that in normal fibroblasts, the Km and Vmax values for saturable uptake was 15.6μM and 2.56 pmol/min/mg protein, respectively. In JVS fibroblasts, however, saturable uptake was not observed. There was no great difference in the linear component of uptake between normal and JVS fibroblasts. At the physiological concentration (50 μ M) of carnitine, the fibroblast carnitine transport activity in JVS mice was decreased to 18 % of that in the normal mice. Thus there is hardly any carnitine transport activity in the fibroblasts of JVS mice, indicating that the JVS mouse can be regarded as an animal model of primary carnitine deficiency.

The multi-subunit membrane protein complex Photosystem II (PSII) catalyzes the light-driven oxidation of water and with this the initial step of photosynthetic electron transport in plants, algae, and cyanobacteria. Its biogenesis is coordinated by a network of auxiliary proteins that facilitate the stepwise assembly of individual subunits and cofactors, forming various intermediate complexes until fully functional mature PSII is present at the end of the process. In the current study, we purified PSII complexes from a mutant line of the thermophilic cyanobacterium Thermosynechococcus vestitus BP-1 in which the extrinsic subunit PsbO, characteristic for active PSII, was fused with an N-terminal Twin-Strep-tag. Three distinct PSII complexes were separated by ion-exchange chromatography after the initial affinity purification. Two complexes differ in their oligomeric state (monomeric and dimeric) but share the typical subunit composition of mature PSII. They are characterized by the very high oxygen-evolving activity of approx. 6,000 µmol O 2 · (mg Chl·h) -1 . Analysis of the third (heterodimeric) PSII complex revealed lower oxygen-evolving activity of approx. 3,000 µmol O 2 · (mg Chl·h) -1 and manganese content of 2.7 (± 0.2) per reaction center compared to 3.7 (± 0.2) of fully active PSII. Mass spectrometry and time-resolved fluorescence spectroscopy further indicated that PsbO is partially replaced by Psb27 in this PSII fraction, thus implying a role in the repair of the complex.