Solution Structures of Two Different FRP-OCP Complexes as Revealed via SEC-SANS.

Photosynthetic organisms have established photoprotective mechanisms in order to dissipate excess light energy into heat, which is commonly known as non-photochemical quenching. Cyanobacteria utilize the orange carotenoid protein (OCP) as a high-light sensor and quencher to regulate the energy flow in the photosynthetic apparatus. Triggered by strong light, OCP undergoes conformational changes to form the active red state (OCPR). In many cyanobacteria, the back conversion of OCP to the dark-adapted state is assisted by the fluorescence recovery protein (FRP). However, the exact molecular events involving OCP and its interaction with FRP remain largely unraveled so far due to their metastability. Here, we use small-angle neutron scattering combined with size exclusion chromatography (SEC-SANS) to unravel the solution structures of FRP-OCP complexes using a compact mutant of OCP lacking the N-terminal extension (∆NTEOCPO) and wild-type FRP. The results are consistent with the simultaneous presence of stable 2:2 and 2:1 FRP-∆NTEOCPO complexes in solution, where the former complex type is observed for the first time. For both complex types, we provide ab initio low-resolution shape reconstructions and compare them to homology models based on available crystal structures. It is likely that both complexes represent intermediate states of the back conversion of OCP to its dark-adapted state in the presence of FRP, which are of transient nature in the photocycle of wild-type OCP. This study demonstrates the large potential of SEC-SANS in revealing the solution structures of protein complexes in polydisperse solutions that would otherwise be averaged, leading to unspecific results.

Freeze-Induced Phase Transition and Local Pressure in a Phospholipid/Water System: Novel Insights Were Obtained from a Time/Temperature Resolved Synchrotron X-ray Diffraction Study.

Water-to-ice transformation results in a 10% increase in volume, which can have a significant impact on biopharmaceuticals during freeze-thaw cycles due to the mechanical stresses imparted by the growing ice crystals. Whether these stresses would contribute to the destabilization of biopharmaceuticals depends on both the magnitude of the stress and sensitivity of a particular system to pressure and sheer stresses. To address the gap of the "magnitude" question, a phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), is evaluated as a probe to detect and quantify the freeze-induced pressure. DPPC can form several phases under elevated pressure, and therefore, the detection of a high-pressure DPPC phase during freezing would be indicative of a freeze-induced pressure increase. In this study, the phase behavior of DPPC/water suspensions, which also contain the ice nucleation agent silver iodide, is monitored by synchrotron small/wide-angle X-ray scattering during the freeze-thaw transition. Cooling the suspensions leads to heterogeneous ice nucleation at approximately -7 °C, followed by a phase transition of DPPC between -11 and -40 °C. In this temperature range, the initial gel phase of DPPC, Lß', gradually converts to a second phase, tentatively identified as a high-pressure Gel III phase. The Lß'-to-Gel III phase transition continues during an isothermal hold at -40 °C; a second (homogeneous) ice nucleation event of water confined in the interlamellar space is detected by differential scanning calorimetry (DSC) at the same temperature. The extent of the phase transition depends on the DPPC concentration, with a lower DPPC concentration (and therefore a higher ice fraction), resulting in a higher degree of Lß'-to-Gel III conversion. By comparing the data from this study with the literature data on the pressure/temperature Lß'/Gel III phase boundary and the lamellar lattice constant of the Lß' phase, the freeze-induced pressure is estimated to be approximately 0.2-2.6 kbar. The study introduces DPPC as a probe to detect a pressure increase during freezing, therefore addressing the gap between a theoretical possibility of protein destabilization by freeze-induced pressure and the current lack of methods to detect freeze-induced pressure. In addition, the observation of a freeze-induced phase transition in a phospholipid can improve the mechanistic understanding of factors that could disrupt the structure of lipid-based biopharmaceuticals, such as liposomes and mRNA vaccines, during freezing and thawing.

Biophysical Determinants for the Viscosity of Concentrated Monoclonal Antibody Solutions.

Monoclonal antibodies (mAbs) are particularly relevant for therapeutics due to their high specificity and versatility, and mAb-based drugs are hence used to treat numerous diseases. The increased patient compliance of self-administration motivates the formulation of products for subcutaneous (SC) administration. The associated challenge is to formulate highly concentrated antibody solutions to achieve a significant therapeutic effect, while limiting their viscosity and preserving their physicochemical stability. Protein-protein interactions (PPIs) are in fact the root cause of several potential problems concerning the stability, manufacturability, and delivery of a drug product. The understanding of macroscopic viscosity requires an in-depth knowledge on protein diffusion, PPIs, and self-association/aggregation. Here, we study the self-diffusion of different mAbs of the IgG1 subtype in aqueous solution as a function of the concentration and temperature by quasi-elastic neutron scattering (QENS). QENS allows us to probe the short-time self-diffusion of the molecules and therefore to determine the hydrodynamic mAb cluster size and to gain information on the internal mAb dynamics. Small-angle neutron scattering (SANS) is jointly employed to probe structural details and to understand the nature and intensity of PPIs. Complementary information is provided by molecular dynamics (MD) simulations and viscometry, thus obtaining a comprehensive picture of mAb diffusion.

Structural and Dynamical Properties of Elastin-Like Peptides near Their Lower Critical Solution Temperature.

Elastin-like peptides (ELPs) are artificially derived intrinsically disordered proteins (IDPs) mimicking the hydrophobic repeat unit in the protein elastin. ELPs are characterized by a lower critical solution temperature (LCST) in aqueous media. Here, we investigate the sequence GVG(VPGVG)3 over a wide range of temperatures (below, around, and above the LCST) and peptide concentrations employing all-atom molecular dynamics simulations, where we focus on the role of intra- and interpeptide interactions. We begin by investigating the structural properties of a single peptide that demonstrates a hydrophobic collapse with temperature, albeit moderate, because the sequence length is short. We observe a change in the interaction between two peptides from repulsive to attractive with temperature by evaluating the potential of mean force, indicating an LCST-like behavior. Next, we explore dynamical and structural properties of peptides in multichain systems. We report the formation of dynamical aggregates with coil-like conformation, in which valine central residues play an important role. Moreover, the lifetime of contacts between chains strongly depends on the temperature and can be described by a power-law decay that is consistent with the LCST-like behavior. Finally, the peptide translational and internal motion are slowed by an increase in the peptide concentration and temperature.

Stages of OCP-FRP Interactions in the Regulation of Photoprotection in Cyanobacteria, Part 2: Small-Angle Neutron Scattering with Partial Deuteration.

We used small-angle neutron scattering partially coupled with size-exclusion chromatography to unravel the solution structures of two variants of the Orange Carotenoid Protein (OCP) lacking the N-terminal extension (OCP-ΔNTE) and its complex formation with the Fluorescence Recovery Protein (FRP). The dark-adapted, orange form OCP-ΔNTEO is fully photoswitchable and preferentially binds the pigment echinenone. Its complex with FRP consists of a monomeric OCP component, which closely resembles the compact structure expected for the OCP ground state, OCPO. In contrast, the pink form OCP-ΔNTEP, preferentially binding the pigment canthaxanthin, is mostly nonswitchable. The pink OCP form appears to occur as a dimer and is characterized by a separation of the N- and C-terminal domains, with the canthaxanthin embedded only into the N-terminal domain. Therefore, OCP-ΔNTEP can be viewed as a prototypical model system for the active, spectrally red-shifted state of OCP, OCPR. The dimeric structure of OCP-ΔNTEP is retained in its complex with FRP. Small-angle neutron scattering using partially deuterated OCP-FRP complexes reveals that FRP undergoes significant structural changes upon complex formation with OCP. The observed structures are assigned to individual intermediates of the OCP photocycle in the presence of FRP.

Process Dependent Complexity in Multicomponent Gels.

Mixing low molecular weight gelators (LMWGs) can be used to combine favorable properties of the individual components within a multifunctional gel. Such multicomponent systems are complex enough in themselves but the method of combining components is not commonly considered something to influence self-assembly. Herein, two multicomponent systems comprising of a naphthalene-based dipeptide hydrogelator and one of two modified naphthalene diimides (NDIs), one of which forms gels, and the other does not, are investigated. These systems are probed, examining the structures formed and their gel properties (when preparing a solution from either a mixed powder of both components or by mixing pre-formed solutions of each component) using rheology, small angle neutron scattering (SANS), and absorbance spectroscopy. It is found that by altering the method of preparation, it is can either induce self-sorting or co-assembly within the fibers formed that underpin the gel network.

Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.

Double dynamic hydrogels formed by wormlike surfactant micelles and cross-linked polymer.

HYPOTHESIS: Interpenetrating networks consisting of a polymer network with dynamic cross-links and a supramolecular network allow obtaining hydrogels with significantly enhanced mechanical properties. EXPERIMENTS: Binary hydrogels composed of a dynamically cross-linked poly(vinyl alcohol) (PVA) network and a transient network of entangled highly charged mixed wormlike micelles (WLMs) of surfactants (potassium oleate and n-octyltrimethylammonium bromide) were prepared and studied by rheometry, SANS, USANS, cryo-TEM, and NMR spectroscopy. FINDINGS: Binary hydrogels show significantly enhanced rheological properties (a 3400-fold higher viscosity and 27-fold higher plateau modulus) as compared to their components taken separately. This is due to the microphase separation leading to local concentrating of PVA and WLMs providing larger number of polymer-polymer contacts for cross-linking and longer WLMs with more entanglements. Such materials are very promising for the application in many areas, ranging from enhanced oil recovery to biomedical uses.

Comparison of Cyclic and Linear Poly(lactide)s Using Small-Angle Neutron Scattering.

Small-angle neutron scattering (SANS) experiments were conducted on cyclic and linear polymers of racemic and l-lactides (PLA) with the goal of comparing chain configurations, scaling, and effective polymer-solvent interactions of the two topologies in acetone-d 6 and THF-d 8. There are limited reports of SANS results on cyclic polymers due to the lack of substantial development in the field until recently. Now that pure, well-defined cyclic polymers are accessible, unanswered questions about their rheology and physical conformations can be better investigated. Previously reported SANS experiments have used cyclic and linear polystyrene samples; therefore, our work allowed for direct comparison using a contrasting (structurally and sterically) polymer. We compared SANS results of cyclic and linear PLA samples with various microstructures and molecular weights at two different temperatures, allowing for comparison with a wide range of variables. The results followed the trends of previous experiments, but much greater differences in the effective polymer-solvent interaction parameters between cyclic and linear forms of PLA were observed, implying that the small form factor and hydrogen bonding in PLA allowed for much more compact conformations in the cyclic form only. Also, the polymer microstructure was found to influence polymer-solvent interaction parameters substantially. These results illustrate how the difference in polymer-solvent interactions between cyclic and linear polymers can vary greatly depending on the polymer in question and the potential of neutron scattering as a tool for identification and characterization of the cyclic topology.

Dual Transient Networks of Polymer and Micellar Chains: Structure and Viscoelastic Synergy.

Dual transient networks were prepared by mixing highly charged long wormlike micelles of surfactants with polysaccharide chains of hydroxypropyl guar above the entanglement concentration for each of the components. The wormlike micelles were composed of two oppositely charged surfactants potassium oleate and n-octyltrimethylammonium bromide with a large excess of anionic surfactant. The system is macroscopically homogeneous over a wide range of polymer and surfactant concentrations, which is attributed to a stabilizing effect of surfactants counterions that try to occupy as much volume as possible in order to gain in translational entropy. At the same time, by small-angle neutron scattering (SANS) combined with ultrasmall-angle neutron scattering (USANS), a microphase separation with the formation of polymer-rich and surfactant-rich domains was detected. Rheological studies in the linear viscoelastic regime revealed a synergistic 180-fold enhancement of viscosity and 65-fold increase of the longest relaxation time in comparison with the individual components. This effect was attributed to the local increase in concentration of both components trying to avoid contact with each other, which makes the micelles longer and increases the number of intermicellar and interpolymer entanglements. The enhanced rheological properties of this novel system based on industrially important polymer hold great potential for applications in personal care products, oil recovery and many other fields.

Molecular Flexibility of Antibodies Preserved Even in the Dense Phase after Macroscopic Phase Separation.

Antibody therapies are typically based on high-concentration formulations that need to be administered subcutaneously. These conditions induce several challenges, inter alia a viscosity suitable for injection, sufficient solution stability, and preservation of molecular function. To obtain systematic insights into the molecular factors, we study the dynamics on the molecular level under strongly varying solution conditions. In particular, we use solutions of antibodies with poly(ethylene glycol), in which simple cooling from room temperature to freezing temperatures induces a transition from a well-dispersed solution into a phase-separated and macroscopically arrested system. Using quasi-elastic neutron scattering during in situ cooling ramps and in prethermalized measurements, we observe a strong decrease in antibody diffusion, while internal flexibility persists to a significant degree, thus ensuring the movement necessary for the preservation of molecular function. These results are relevant for a more dynamic understanding of antibodies in high-concentration formulations, which affects the formation of transient clusters governing the solution viscosity.

Temperature and salt controlled tuning of protein clusters.

The formation of molecular assemblies in protein solutions is of strong interest both from a fundamental viewpoint and for biomedical applications. While ordered and desired protein assemblies are indispensable for some biological functions, undesired protein condensation can induce serious diseases. As a common cofactor, the presence of salt ions is essential for some biological processes involving proteins, and in aqueous suspensions of proteins can also give rise to complex phase diagrams including homogeneous solutions, large aggregates, and dissolution regimes. Here, we systematically study the cluster formation approaching the phase separation in aqueous solutions of the globular protein BSA as a function of temperature (T), the protein concentration (cp) and the concentrations of the trivalent salts YCl3 and LaCl3 (cs). As an important complement to structural, i.e. time-averaged, techniques we employ a dynamical technique that can detect clusters even when they are transient on the order of a few nanoseconds. By employing incoherent neutron spectroscopy, we unambiguously determine the short-time self-diffusion of the protein clusters depending on cp, cs and T. We determine the cluster size in terms of effective hydrodynamic radii as manifested by the cluster center-of-mass diffusion coefficients D. For both salts, we find a simple functional form D(cp, cs, T) in the parameter range explored. The calculated inter-particle attraction strength, determined from the microscopic and short-time diffusive properties of the samples, increases with salt concentration and temperature in the regime investigated and can be linked to the macroscopic behavior of the samples.

Enhancement of the mechanical properties of lysine-containing peptide-based supramolecular hydrogels by chemical cross-linking.

Exposure of lysine-containing peptide-based gelators to the cross-linking agent glutaraldehyde allows tuning of gel mechanical properties. The effect of cross-linking depends on the position of the lysine residue in the peptide chain, the concentration of gelator and the conditions under which cross-linking takes place. Through control of these factors, cross-linking leads to increased gel strength.

Bulk Phase Behavior vs Interface Adsorption: Specific Multivalent Cation and Anion Effects on BSA Interactions.

Proteins are ubiquitous and play a critical role in many areas from living organisms to protein microchips. In humans, serum albumin has a prominent role in the foreign body response since it is the first protein which will interact with, e.g., an implant or stent. In this study, we focused on the influence of salts (i.e., different cations (Y3+, La3+) and anions (Cl-, I-) on bovine serum albumin (BSA) in terms of its bulk behavior as well as the role of charges for protein adsorption at the solid-liquid interface in order to understand and control the underlying molecular mechanisms and interactions. This is part of our group's effort to gain a deeper understanding of protein-protein and protein-surface interactions in the presence of multivalent ions. In the bulk, we established two new phase diagrams and found not only multivalent cation-triggered phase transitions, but also a dependence of the protein behavior on the type of anion. The attractive interactions between proteins were observed to increase from Cl- < NO3- < I-, resulting in iodide preventing re-entrant condensation and promoting liquid-liquid phase separation in bulk. Using ellipsometry and a quartz-crystal microbalance with dissipation (QCM-D), we obtained insight into the growth of the protein adsorption layer. Importantly, we found that phase transitions at the substrate can be triggered by certain interface properties, whether they exist in the bulk solution or not. Through the use of a hydrophilic, negatively charged surface (native silica), the direct binding of anions to the interface was prevented. Interestingly, this led to re-entrant adsorption even in the absence of re-entrant condensation in bulk. However, the overall amount of adsorbed protein was enhanced through stronger attractive protein-protein interactions in the presence of iodide salts. These findings illustrate how carefully chosen surface properties and salts can directly steer the binding of anions and cations, which guide protein behavior, thus paving the way for specific/triggered protein-protein, protein-salt, and protein-surface interactions.

Interplay between Glass Formation and Liquid-Liquid Phase Separation Revealed by the Scattering Invariant.

The interplay of the glass transition with liquid-liquid phase separation (LLPS) is a subject of intense debate. We use the scattering invariant Q to probe how approaching the glass transition affects the shape of LLPS boundaries in the temperature/volume fraction plane. Two protein systems featuring kinetic arrest with a lower and an upper critical solution temperature phase behavior, respectively, are studied varying the quench depth. Using Q we noninvasively identify system-dependent differences for the effect of glass formation on the LLPS boundary. The glassy dense phase appears to enter the coexistence region for the albumin-YCl3 system, whereas it follows the equilibrium binodal for the γ-globulin-PEG system.

Evolution of the structure and dynamics of bovine serum albumin induced by thermal denaturation.

Protein denaturation in concentrated solutions consists of the unfolding of the native protein structure, and subsequent cross-linking into clusters or gel networks. While the kinetic evolution of structure has been studied for some cases, the underlying microscopic dynamics of proteins has so far been neglected. However, protein dynamics is essential to understand the specific nature of assembly processes, such as diffusion-limited growth, or vitrification of dense liquids. Here, we present a study on thermal denaturation of concentrated solutions of bovine serum albumin (BSA) in D2O with and without NaCl. Using small-angle scattering, we provide information on structure before, during and after denaturation. Using quasi-elastic neutron scattering, we monitor in real-time the microscopic dynamics and dynamical confinement throughout the entire denaturation process covering protein unfolding and cross-linking. After denaturation, the protein dynamics is slowed down in salty solutions compared to those in pure water, while the stability and dynamics of the native solution appears unaffected by salt. The approach presented here opens opportunities to link microscopic dynamics to emerging structural properties, with implications for assembly processes in soft and biological matter.

Packing and dynamics of a protein solution approaching the jammed state.

The packing of proteins and their collective behavior in crowded media is crucial for the understanding of biological processes. Here we study the structural and dynamical evolution of solutions of the globular protein bovine serum albumin with increasing concentration via drying using small angle X-ray scattering and dynamic light scattering. We probe an evolving correlation peak on the scattering profile, corresponding to the inter-protein distance, ξ, which decreases following a power law of the protein volume fraction, φ. The rate of decrease in ξ becomes faster above a protein concentration of ∼200 mg ml-1 (φ = 0.15). The power law exponent changes from 0.33, which is typical of colloidal or protein solutions, to 0.41. During the entire drying process, we observe the development and the growth of two-step relaxation dynamics with increasing φ as revealed by dynamic light scattering. We find three different regimes of the dependence of ξ as a function of φ. In the dilute regime (φ < 0.22), protein molecules are far apart from each other compared to their size. In this case, the dynamics mainly corresponds to Brownian motion. At an intermediate concentration (0.22 < φ < 0.47), inter-protein distances become comparable to the size of protein molecules, leading to a preferential orientation of the ellipsoidal protein molecules along with a possible deformation. In this regime, the dynamics shows two distinct relaxation times. At a very high concentration (φ > 0.47), the system reaches a jammed state. Subsequently, the secondary relaxation time in this state becomes extremely slow. In this state, the protein molecules have approximately one hydration layer. This study contributes to the understanding of protein molecular packing in crowded environments and the phenomenon of density-driven jamming for soft matter systems.

Multivalent ions and biomolecules: Attempting a comprehensive perspective.

Ions are ubiquitous in nature. They play a key role for many biological processes on the molecular scale, from molecular interactions, to mechanical properties, to folding, to self-organisation and assembly, to reaction equilibria, to signalling, to energy and material transport, to recognition etc. Going beyond monovalent ions to multivalent ions, the effects of the ions are frequently not only stronger (due to the obviously higher charge), but qualitatively different. A typical example is the process of binding of multivalent ions, such as Ca2+ , to a macromolecule and the consequences of this ion binding such as compaction, collapse, potential charge inversion and precipitation of the macromolecule. Here we review these effects and phenomena induced by multivalent ions for biological (macro)molecules, from the "atomistic/molecular" local picture of (potentially specific) interactions to the more global picture of phase behaviour including, e. g., crystallisation, phase separation, oligomerisation etc. Rather than attempting an encyclopedic list of systems, we rather aim for an embracing discussion using typical case studies. We try to cover predominantly three main classes: proteins, nucleic acids, and amphiphilic molecules including interface effects. We do not cover in detail, but make some comparisons to, ion channels, colloidal systems, and synthetic polymers. While there are obvious differences in the behaviour of, and the relevance of multivalent ions for, the three main classes of systems, we also point out analogies. Our attempt of a comprehensive discussion is guided by the idea that there are not only important differences and specific phenomena with regard to the effects of multivalent ions on the main systems, but also important similarities. We hope to bridge physico-chemical mechanisms, concepts of soft matter, and biological observations and connect the different communities further.

Unification of lower and upper critical solution temperature phase behavior of globular protein solutions in the presence of multivalent cations.

In globular protein systems, upper critical solution temperature (UCST) behavior is common, but lower critical solution temperature (LCST) phase transitions are rare. In addition, the temperature sensitivity of such systems is usually difficult to tune. Here we demonstrate that the charge state of globular proteins in aqueous solutions can alter their temperature-dependent phase behavior. We show a universal way to tune the effective protein interactions and induce both UCST and LCST-type transitions in the system using trivalent salts. We provide a phase diagram identifying LCST and UCST regimes as a function of protein and salt concentrations. We further propose a model based on an entropy-driven cation binding mechanism to explain the experimental observations.

A neutron scattering perspective on the structure, softness and dynamics of the ligand shell of PbS nanocrystals in solution.

Small-angle neutron and X-ray scattering, neutron backscattering and neutron time-of-flight spectroscopy are applied to reveal the structure of the ligand shell, the temperature-dependent diffusion properties and the phonon spectrum of PbS nanocrystals functionalized with oleic acid in deuterated hexane. The nanocrystals decorated with oleic acid as well as the desorbed ligand molecules exhibit simple Brownian diffusion with a Stokes-Einstein temperature-dependence and inhibited freezing. Ligand molecules desorbed from the surface show strong spatial confinement. The phonon spectrum of oleic acid adsorbed to the nanocrystal surface exhibits hybrid modes with a predominant Pb-character. Low-energy surface modes of the NCs are prominent and indicate a large mechanical softness in solution. This work provides comprehensive insights into the ligand-particle interaction of colloidal nanocrystals in solution and highlights its effect on the diffusion and vibrational properties as well as their mechanical softness.