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Fluorescent RNA aptamers have the potential to enable routine quantitation and localization of RNA molecules and serve as models for understanding biologically active aptamers. In recent years, several fluorescent aptamers have been selected and modified to improve their properties, revealing that small changes to the RNA or the ligands can modify significantly their fluorescent properties. Although structural biology approaches have revealed the bound, ground state of several fluorescent aptamers, characterization of low-abundance, excited states in these systems is crucial to understanding their folding pathways. Here we use pressure as an alternative variable to probe the suboptimal states of the Mango III aptamer with both fluorescence and NMR spectroscopy approaches. At moderate KCl concentrations, increasing pressure disrupted the G-quadruplex structure of the Mango III RNA and led to an intermediate with lower fluorescence. These observations indicate the existence of suboptimal RNA structural states that still bind the TO1-biotin fluorophore and moderately enhance fluorescence. At higher KCl concentration as well, the intermediate fluorescence state was populated at high pressure, but the G-quadruplex remained stable at high pressure, supporting the notion of parallel folding and/or binding pathways. These results demonstrate the usefulness of pressure for characterizing RNA folding intermediates.

In order to achieve a comprehensive understanding of protein aggregation processes, an exploration of solvation dynamics, a key yet intricate component of biological phenomena, is mandatory. In the present study, we used Fourier Transform Infrared Spectroscopy (FT-IR) and Terahertz(THz)-spectroscopy complemented by Atomic Force Microscopy and kinetic experiments utilizing Thioflavin T (ThT) fluorescence to elucidate the changes in solvation dynamics during liquid-liquid phase separation and subsequent amyloid fibril formation, the latter representing a transition from liquid to solid phase separation. These processes are pivotal in the pathology of neurodegenerative disorders such as Alzheimer's and Parkinson's disease. We focus on the ACC1-13K24-ATP protein complex, which undergoes fibril formation followed by droplet generation. Our investigation reveals the importance of hydration as a driving force in these processes, offering new insights into the molecular mechanisms at play.

The interactions of exenatide, a Trp-containing peptide used as a drug to treat diabetes, with liposomes were studied by isothermal titration calorimetry (ITC), tryptophan (Trp) fluorescence, and microscale thermophoresis measurements. The results are not only important for better understanding the release of this specific drug from vesicular phospholipid gel formulations but describe a general scenario as described before for various systems. This study introduces a model to fit these data on the basis of primary and secondary peptide-lipid interactions. Finally, resolving apparent inconsistencies between different methods aids the design and critical interpretation of binding experiments in general. Our results show that the net cationic exenatide adsorbs electrostatically to liposomes containing anionic diacyl phosphatidylglycerol lipids (PG); however, the ITC data could not properly be fitted by any established model. The combination of electrostatic adsorption of exenatide to the membrane surface and its self-association (Kd = 46 μM) suggested the possibility of secondary binding of peptide to the first, primarily (i.e., lipid-) bound peptide layer. A global fit of the ITC data validated this model and suggested one peptide to bind primarily per five PG molecules with a Kd≈ 0.2 μM for PC/PG 1:1 and 0.6 μM for PC/PG 7:3 liposomes. Secondary binding shows a weaker affinity and a less exothermic or even endothermic enthalpy change. Depending on the concentration of liposomes, secondary binding may also lead to liposomal aggregation as detected by dynamic light-scattering measurements. ITC quantifies primary and secondary binding separately, whereas microscale thermophoresis and Trp fluorescence represent a summary or average of both effects, possibly with the fluorescence data showing somewhat greater weighting of primary binding. Systems with secondary peptide-peptide association within the membrane are mathematically analogous to the adsorption discussed here.

For over 50 years, it has been known that the mitosis of eukaryotic cells is inhibited already at high hydrostatic pressure conditions of 30 MPa. This effect has been attributed to the disorganization of microtubules, the main component of the spindle apparatus. However, the structural details of the depolymerization and the origin of the pressure sensitivity have remained elusive. It has also been a puzzle how complex organisms could still successfully inhabit extreme high-pressure environments such as those encountered in the depth of oceans. We studied the pressure stability of microtubules at different structural levels and for distinct dynamic states using high-pressure Fourier-transform infrared spectroscopy and Synchrotron small-angle x-ray scattering. We show that microtubules are hardly stable under abyssal conditions, where pressures up to 100 MPa are reached. This high-pressure sensitivity can be mainly attributed to the internal voids and packing defects in the microtubules. In particular, we show that lateral and longitudinal contacts feature different pressure stabilities, and they define also the pressure stability of tubulin bundles. The intactness of both contact types is necessary for the functionality of microtubules in vivo. Despite being known to dynamically stabilize microtubules and prevent their depolymerization, we found that the anti-cancer drug taxol and the accessory protein MAP2c decrease the pressure stability of microtubule protofilaments. Moreover, we demonstrate that the cellular environment itself is a crowded place and accessory proteins can increase the pressure stability of microtubules and accelerate their otherwise highly pressure-sensitive de novo formation.

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other rigid lipids and cholesterol into liquid-ordered domains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral organization is of utmost significance. Hence, we made use of the light-induced trans-cisisomerization of azobenzene-modified acyl chains to develop a set of photoswitchable sphingolipids with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran-blocked sphingosine) that are able to shuttle between liquid-ordered and liquid-disordered regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photoisomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that the sphingosine-based (Azo-β-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photoswitchable lipids promote a reduction in liquid-ordered microdomain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having tetrahydropyran groups that block H-bonding at the sphingosine backbone (lipids named Azo-THP-SM, Azo-THP-Cer) induce an increase in the liquid-ordered domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable liquid-ordered domains.

Phosphoinositols are an important class of phospholipids that are involved in a myriad of cellular processes, from cell signaling to motility and adhesion. Vinculin (Vn) is a major adaptor protein that regulates focal adhesions in conjunction with PIP2in lipid membranes and other cytoskeletal components. The binding and unbinding transitions of Vn at the membrane interface are an important link to understanding the coordination of cell signaling and motility. Using different biophysical tools, including atomic force microscopy combined with confocal fluorescence microscopy and Fourier transform infrared spectroscopy, we studied the nanoscopic interactions of activated and autoinhibited states of Vn with lipid membranes. We hypothesize that a weak interaction occurs between Vn and lipid membranes, which leads to binding of autoinhibited Vn to supported lipid bilayers, and to unbinding in freestanding lipid vesicles. Likely driving forces may include tethering of the C-terminus to the lipid membrane, as well as hydrophobic helix-membrane interactions. Conversely, activated Vn binds strongly to membranes through specific interactions with clusters of PIP2embedded in lipid membranes. Activated Vn harbored on PIP2clusters may form small oligomeric interaction platforms for further interaction partners, which is necessary for the proper function of focal adhesion points.

K-Ras4B is a membrane-bound small GTPase with a prominent role in cancer development. It contains a polybasic farnesylated C-terminus that is required for the correct localization and clustering of K-Ras4B in distinct membrane domains. PDEδ and the Ca2+-binding protein calmodulin (CaM) are known to function as potential binding partners for farnesylated Ras proteins. However, they differ in the number of interaction sites with K-Ras4B, leading to different modes of interaction, and thus affect the subcellular distribution of K-Ras4B in different ways. Although it is clear that Ca2+-bound CaM can play a role in the dynamic spatial cycle of K-Ras4B in the cell, the exact molecular mechanism is only partially understood. In this biophysical study, we investigated the effect of Ca2+/CaM on the interaction of GDP- and GTP-loaded K-Ras4B with heterogeneous model biomembranes by using a combination of different spectroscopic and imaging techniques. The results show that Ca2+/CaM is able to extract K-Ras4B from negatively charged membranes in a nucleotide-independent manner. Moreover, the data demonstrate that the complex of Ca2+/CaM and K-Ras4B is stable in the presence of anionic membranes and shows no membrane binding. Finally, the influence of Ca2+/CaM on the interaction of K-Ras4B with membranes is compared with that of PDEδ, which was investigated in a previous study. Although both CaM and PDEδexhibit a hydrophobic binding pocket for farnesyl, they have different effects on membrane binding of K-Ras4B and hence should be capable of regulating K-Ras4B plasma membrane localization in the cell.

Membrane type 1-matrix metalloproteinase (MT1-MMP or MMP-14) is a zinc-transmembrane metalloprotease involved in the degradation of extracellular matrix and tumor invasion. While changes in solvation of MT1-MMP have been recently studied, little is known about the structural and energetic changes associated with MT1-MMP while interacting with substrates. Steady-state kinetic and thermodynamic data (including activation energies and activation volumes) were measured over a wide range of temperatures and pressures by means of a stopped-flow fluorescence technique. Complementary temperature- and pressure-dependent Fourier-transform infrared measurements provided corresponding structural information of the protein. MT1-MMP is stable and active over a wide range of temperatures (10–55°C). A small conformational change was detected at 37°C, which is responsible for the change in activity observed at the same temperature. Pressure decreases the enzymatic activity until complete inactivation occurs at 2 kbar. The inactivation is associated with changes in the rate-limiting step of the reaction caused by additional hydration of the active site upon compression and/or minor conformational changes in the active site region. Based on these data, an energy and volume diagram could be established for the various steps of the enzymatic reaction.

The small GTP-binding proteins Arl2 and Arl3, which are close homologs, share a number of interacting partners and act as displacement factors for prenylated and myristoylated cargo. Nevertheless, both proteins have distinct biological functions. Whereas Arl3 is considered a ciliary protein, Arl2 has been reported to be involved in tubulin folding, mitochondrial function, and Ras signaling. How these different roles are attained by the two homolog proteins is not fully understood. Recently, we showed that the N-terminal amphipathic helix of Arl3, but not that of Arl2, regulates the release of myristoylated ciliary proteins from the GDI-like solubilizing factor UNC119a/b. In the biophysical study presented here, both proteins are shown to exhibit a preferential localization and clustering in liquid-disordered domains of phase-separated membranes. However, the membrane interaction behavior differs significantly between both proteins with regard to their nucleotide loading. Whereas Arl3 and other Arf proteins with an N-terminal amphipathic helix require GTP loading for the interaction with membranes, Arl2 binds to membranes in a nucleotide-independent manner. In contrast to Arl2, the N-terminal helix of Arl3 increases the binding affinity to UNC119a. Furthermore, UNC119a impedes membrane binding of Arl3, but not of Arl2. Taken together, these results suggest an interplay among the nucleotide status of Arl3, the location of the N-terminal helix, membrane fluidity and binding, and the release of lipid modified cargos from carriers such as UNC119a. Since a specific Arl3-GEF is postulated to reside inside cilia, the N-terminal helix of Arl3•GTP would be available for allosteric regulation of UNC119a cargo release only inside cilia.

Actin is the main component of the microfilament system in eukaryotic cells and can be found in distinct morphological states. Global (G)-actin is able to assemble into highly organized, supramolecular cellular structures known as filamentous (F)-actin and bundled (B)-actin. To evaluate the structure and stability of G-, F-, and B-actin over a wide range of temperatures and pressures, we used Fourier transform infrared spectroscopy in combination with differential scanning and pressure perturbation calorimetry, small-angle x-ray scattering, laser confocal scanning microscopy, and transmission electron microscopy. Our analysis was designed to provide new (to our knowledge) insights into the stabilizing forces of actin self-assembly and to reveal the stability of the actin polymorphs, including in conditions encountered in extreme environments. In addition, we sought to explain the limited pressure stability of actin self-assembly observed in vivo.G-actin is not only the least temperature-stable but also the least pressure-stable actin species. Under abyssal conditions, where temperatures as low as 1–4°C and pressures up to 1 kbar are reached, G-actin is hardly stable. However, the supramolecular assemblies of actin are stable enough to withstand the extreme conditions usually encountered on Earth. Beyond ∼3–4 kbar, filamentous structures disassemble, and beyond ∼4 kbar, complete dissociation of F-actin structures is observed. Between ∼1 and 2 kbar, some disordering of actin assemblies commences, in agreement with in vivo observations. The limited pressure stability of the monomeric building block seems to be responsible for the suppression of actin assembly in the kbar pressure range.

Understanding the intermolecular interaction potential, V(r), of proteins under the influence of temperature, pressure, and salt concentration is essential for understanding protein aggregation, crystallization, and protein phase behavior in general. Here, we report small-angle x-ray scattering studies on dense lysozyme solutions of high ionic strength as a function of temperature and pressure. We show that the interaction potential changes in a nonlinear fashion over a wide range of temperatures, salt, and protein concentrations. Neither temperature nor protein and salt concentration lead to marked changes in the pressure dependence of V(r), indicating that changes of the water structure dominate the pressure dependence of the intermolecular forces. Furthermore, by analysis of the temperature, pressure, and ionic strength dependence of the normalized second virial coefficient, b2, we show that the interaction can be fine-tuned by pressure, which can be used to optimize b2values for controlled protein crystallization.

We report x-ray reflectivity and grazing incidence x-ray diffraction measurements of lipopolysaccharide (LPS) monolayers at the water-air interface. Our investigations reveal that the structure and lateral ordering of the LPS molecules is very different from phospholipid systems and can be modulated by the ionic strength of the aqueous subphase in an ion-dependent manner. Our findings also indicate differential effects of monovalent and divalent ions on the two-dimensional ordering of lipid domains. Na+ions interact unspecifically with LPS molecules based on their ability to efficiently screen the negative charges of the LPS molecules, whereas Ca2+ions interact specifically by cross-linking adjacent molecules in the monolayer. At low lateral pressures, Na+ions present in the subphase lead to a LPS monolayer structure ordered over large areas with high compressibility, nearly hexagonal packing of the hydrocarbon chains, and high density in the LPS headgroup region. At higher film pressures, the LPS monolayer becomes more rigid and results in a less perfect, oblique packing of the LPS hydrocarbon chains as well as a smaller lateral size of highly ordered domains on the monolayer. Furthermore, associated with the increased surface pressure, a conformational change of the sugar headgroups occurs, leading to a thickening of the entire LPS monolayer structure. The effect of Ca2+ions in the subphase is to increase the rigidity of the LPS monolayer, leading to an oblique packing of the hydrocarbon chains already at low film pressures, an upright orientation of the sugar moieties, and much smaller sizes of ordered domains in the plane of the monolayer. In the presence of both Na+- and Ca2+ions in the subphase, the screening effect of Na+is predominant at low film pressures, whereas, at higher film pressures, the structure and lateral organization of LPS molecules is governed by the influence of Ca2+ions. The unspecific charge-screening effect of the Na+ions on the conformation of the sugar moiety becomes less dominant at biologically relevant lateral pressures.

Bipolar tetraether lipids (BTLs) are abundant in crenarchaeota, which thrive in both thermophilic and nonthermophilic environments, with wide-ranging growth temperatures (4–108°C). BTL liposomes can serve as membrane models to explore the role of BTLs in the thermal stability of the plasma membrane of crenarchaeota. In this study, we focus on the liposomes made of the polar lipid fraction E (PLFE). PLFE is one of the main BTLs isolated from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Using molecular acoustics (ultrasound velocimetry and densimetry), pressure perturbation calorimetry, and differential scanning calorimetry, we have determined partial specific adiabatic and isothermal compressibility, their respective compressibility coefficients, partial specific volume, and relative volume fluctuations of PLFE large unilamellar vesicles (LUVs) over a wide range of temperatures (20–85°C). The results are compared with those obtained from liposomes made of dipalmitoyl-L-α-phosphatidylcholine (DPPC), a conventional monopolar diester lipid. We found that, in the entire temperature range examined, compressibilities of PLFE LUVs are low, comparable to those found in gel state of DPPC. Relative volume fluctuations of PLFE LUVs at any given temperature examined are 1.6–2.2 times more damped than those found in DPPC LUVs. Both compressibilities and relative volume fluctuations in PLFE LUVs are much less temperature-sensitive than those in DPPC liposomes. The isothermal compressibility coefficient (βTlipid) of PLFE LUVs changes from 3.59 × 10−10Pa−1at 25°C to 4.08 × 10−10Pa−1at 78°C. Volume fluctuations of PLFE LUVs change only 0.25% from 30°C to 80°C. The highly damped volume fluctuations and their low temperature sensitivity, echo that PLFE liposomes are rigid and tightly packed. To our knowledge, the data provide a deeper understanding of lipid packing in PLFE liposomes than has been previously reported, as well as a molecular explanation for the low solute permeation and limited membrane lateral motion. The obtained results may help to establish new strategies for rational design of stable BTL-based liposomes for drug/vaccine delivery.

A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil.

The volumetric properties of proteins yield information about the changes in packing and hydration between various states along the folding reaction coordinate and are also intimately linked to the energetics and dynamics of these conformations. These volumetric characteristics can be accessed via pressure perturbation methods. In this work, we report high-pressure unfolding studies of the ankyrin domain of the Notch receptor (Nank1–7) using fluorescence, small-angle x-ray scattering, and Fourier transform infrared spectroscopy. Both equilibrium and pressure-jump kinetic fluorescence experiments were consistent with a simple two-state folding/unfolding transition under pressure, with a rather small volume change for unfolding compared to proteins of similar molecular weight. High-pressure fluorescence, Fourier transform infrared spectroscopy, and small-angle x-ray scattering measurements revealed that increasing urea over a very small range leads to a more expanded pressure unfolded state with a significant decrease in helical content. These observations underscore the conformational diversity of the unfolded-state basin. The temperature dependence of pressure-jump fluorescence relaxation measurements demonstrated that at low temperatures, the folding transition state ensemble (TSE) lies close in volume to the folded state, consistent with significant dehydration at the barrier. In contrast, the thermal expansivity of the TSE was found to be equivalent to that of the unfolded state, indicating that the interactions that constrain the folded-state thermal expansivity have not been established at the folding barrier. This behavior reveals a high degree of plasticity of the TSE of Nank1–7.

Ras GTPases play a crucial role in signal transduction cascades involved in cell differentiation and proliferation, and membrane binding is essential for their proper function. To determine the influence of the nature of the lipid anchor motif and the difference between the active (GTP) and inactive (GDP) forms of N-Ras on partitioning and localization in the lipid membrane, five different N-Ras constructs with different lipid anchors and nucleotide loading (Far/Far (GDP), HD/Far (GDP), HD/HD (GDP), Far (GDP), and HD/Far (GppNHp)) were synthesized. Using the surface plasmon resonance technique, we were able to follow the insertion and dissociation process of the lipidated proteins into and out of model membranes consisting of pure liquid-ordered (lo) or liquid-disordered (ld) phase and a heterogeneous two-phase mixture, i.e., a raft mixture with lo+ ldphase coexistence. In addition, we examined the influence of negatively charged headgroups and stored curvature elastic stress on the binding properties of the lipidated N-Ras proteins. In most cases, significant differences were found for the various anchor motifs. In general, N-Ras proteins insert preferentially into a fluidlike, rather than a rigid, ordered lipid bilayer environment. Electrostatic interactions with lipid headgroups or stored curvature elastic stress of the membrane seem to have no drastic effect on the binding and dissociation processes of the lipidated proteins. The monofarnesylated N-Ras exhibits generally the highest association rate and fastest dissociation process in fluidlike membranes. Double lipidation, especially including farnesylation, of the protein leads to drastically reduced initial binding rates but strong final association. The change in the nucleotide loading of the natural N-Ras HD/Far induces a slightly different binding and dissociation kinetics, as well as stability of association, and seems to influence the tendency to segregate laterally in the membrane plane. The GDP-bound inactive form of N-Ras with an HD/Far anchor shows stronger membrane association, which might be due to a more pronounced tendency to self-assemble in the membrane matrix than is seen with the active GTP-bound form.

The islet amyloid polypeptide (IAPP) and insulin are coproduced by the β-cells of the pancreatic islets of Langerhans. Both peptides can interact with negatively charged lipid membranes. The positively charged islet amyloid polypeptide partially inserts into these membranes and subsequently forms amyloid fibrils. The amyloid fibril formation of insulin is also accelerated by the presence of negatively charged lipids, although insulin has a negative net charge at neutral pH-values. We used water-polymer model interfaces to differentiate between the hydrophobic and electrostatic interactions that can drive these peptides to adsorb at an interface. By applying neutron reflectometry, the scattering-length density profiles of IAPP and insulin, as adsorbed at three different water-polymer interfaces, were determined. The islet amyloid polypeptide most strongly adsorbed at a hydrophobic poly-(styrene) surface, whereas at a hydrophilic, negatively charged poly-(styrene sulfonate) interface, the degree of adsorption was reduced by 50%. Almost no IAPP adsorption was evident at this negatively charged interface when we added 100 mM NaCl. On the other hand, negatively charged insulin was most strongly attracted to a hydrophilic, negatively charged interface. Our results suggest that IAPP is strongly attracted to a hydrophobic surface, whereas the few positive charges of IAPP cannot warrant a permanent immobilization of IAPP at a hydrophilic, negatively charged surface at an ionic strength of 100 mM. Furthermore, the interfacial accumulation of insulin at a hydrophilic, negatively charged surface may represent a favorable precondition for nucleus formation and fibril formation.

Universal features of the peptide aggregation process suggest a common mechanism, with a first-order phase transition in aqueous solutions of the peptides being the driving force. Small system sizes strongly affect the stability of the minor phase in the two-phase region. We show manifestations of this effect in aqueous solutions of fragments of the islet amyloid polypeptide, using computer simulation methods and invoking various approaches in characterizing clustering and aggregate formation. These systems with peptide concentrations deeply inside the immiscibility region show two distinct stable states, which interchange with time: one state contains a peptide aggregate; and the other state has an aggregate that is noticeably dissolved. The first state is relevant for macroscopic systems, whereas the second one is artificial. At a fixed concentration, the occurrence probability of the aggregate state vanishes upon decreasing the system size, thus indicating the necessity to apply a finite size-scaling for meaningful studies of peptide aggregation by simulations. The effect observed may be one of the factors responsible for the difference between intracellular and extracellular aggregation and fibrillization of polypeptides. The finite size of biological cells or their compartments may be playing a decisive role in hampering intracellular aggregation of highly insoluble amyloidogenic proteins, whereas aggregation is unavoidable in the extracellular space at the same peptide concentration.

We present a general-purpose model for biomolecular simulations at the molecular level that incorporates stochasticity, spatial dependence, and volume exclusion, using diffusing and reacting particles with physical dimensions. To validate the model, we first established the formal relationship between the microscopic model parameters (timestep, move length, and reaction probabilities) and the macroscopic coefficients for diffusion and reaction rate. We then compared simulation results with Smoluchowski theory for diffusion-limited irreversible reactions and the best available approximation for diffusion-influenced reversible reactions. To simulate the volumetric effects of a crowded intracellular environment, we created a virtual cytoplasm composed of a heterogeneous population of particles diffusing at rates appropriate to their size. The particle-size distribution was estimated from the relative abundance, mass, and stoichiometries of protein complexes using an experimentally derived proteome catalog from Escherichia coliK12. Simulated diffusion constants exhibited anomalous behavior as a function of time and crowding. Although significant, the volumetric impact of crowding on diffusion cannot fully account for retarded protein mobility in vivo, suggesting that other biophysical factors are at play. The simulated effect of crowding on barnase-barstar dimerization, an experimentally characterized example of a bimolecular association reaction, reveals a biphasic time course, indicating that crowding exerts different effects over different timescales. These observations illustrate that quantitative realism in biosimulation will depend to some extent on mesoscale phenomena that are not currently well understood.

Although sterol-phospholipid interactions have been of interest for many years now, a complete thermodynamic profile of these systems is still missing. To contribute to a better understanding of the thermodynamic functions of these systems, we determined isothermal compressibility coefficient data for dipalmitoylphosphocholine (DPPC) and DPPC-containing cholesterol and ergosterol vesicles by means of molecular acoustics (ultrasound velocimetry and densimetry) and differential scanning and pressure perturbation calorimetric techniques. A particular focus was on the influence of the differential structural properties of the two sterols on the thermodynamic properties of lipid bilayers, and on the nature of the critical point region of phospholipid-sterol systems by determining thermodynamic fluctuation parameters. Contrary to significant changes in conformational and dynamical properties of the DPPC-sterol membranes, no marked differences were found in the various thermodynamic properties studied, including the adiabatic (βSlipid) and isothermal (βTlipid) compressibility, as well as the volume fluctuations. Differences in βTlipidandβSlipid become dramatic in the gel-fluid transition region only, due to a significant degree of slow relaxational processes in the microsecond time range in the transition region. Our data show no evidence for the existence of a typical critical point phenomenon in the concentration and temperature range where a critical point in the DPPC-sterol phase diagram is expected to appear. Hence, on a macroscopic level, it seems more appropriate to describe the sterol-phospholipid binary mixtures in the liquid-ordered/liquid-disordered coexistence region as a phase region consisting essentially of small nanodomains only. Such small-domain dimensions, with a series of particular properties such as increased line energy, spontaneous curvature, and limited lifetime, seem also to be typical of raftlike domains in cell membranes.

In this work, we performed a detailed thermodynamic study, including ultrasound velocimetry, densimetry, calorimetry, and FTIR spectroscopy, of an aggregation-prone protein (insulin) under different salt-screening conditions to gain a deeper insight into the scenario of physicochemical events during its temperature-induced unfolding and aggregation reactions. Differences in aggregation and fibrillization pathways are reflected in changes of the partial molar volume, the coefficients of thermal expansion and compressibility, and the infrared spectral properties of the protein. Combining all experimental data allows setting up a scheme for the temperature-dependent insulin aggregation reaction in the presence and absence of NaCl. As revealed by complementary atomic force microscopy studies, under charge-screening conditions, a process involving structural reorganization, ripening, and formation of more compact nuclei from amorphous oligomers is involved in the formation of mature fibrillar morphologies. In this work, our focus was to put forward a comprehensive discussion of the use of ultrasound velocimetry in disentangling different aggregation pathways. In fact, ultrasound velocimetry proved to be very sensitive to changes in aggregation pathway, highlighting the importance of density and compressibility changes in the different aggregation and fibrillization reactions of the protein.

Islet amyloid polypeptide (IAPP) is a pancreatic hormone and one of a number of proteins that are involved in the formation of amyloid deposits in the islets of Langerhans of type II diabetes mellitus patients. Though IAPP-membrane interactions are known to play a major role in the fibrillation process, the mechanism and the peptide's conformational changes involved are still largely unknown. To obtain new insights into the conformational dynamics of IAPP upon its aggregation at membrane interfaces and to relate these structures to its fibril formation, we studied the association of IAPP at various interfaces including neutral as well as charged phospholipids using infrared reflection absorption spectroscopy. The results obtained reveal that the interaction of human IAPP with the lipid interface is driven by the N-terminal part of the peptide and is largely driven by electrostatic interactions, as the protein is able to associate strongly with negatively charged lipids only. A two-step process is observed upon peptide binding, involving a conformational transition from a largely α-helical to a β-sheet conformation, finally forming ordered fibrillar structures. As revealed by simulations of the infrared reflection absorption spectra and complementary atomic force microscopy studies, the fibrillar structures formed consist of parallel intermolecular β-sheets lying parallel to the lipid interface but still contain a significant number of turn structures. We may assume that these dynamical conformational changes observed for negatively charged lipid interfaces play an important role as the first steps of IAPP-induced membrane damage in type II diabetes.

Analysis of antigen dissociation provides insight into peptide presentation modes of folded human leukocyte antigen (HLA) molecules, which consist of a heavy chain, β2-microglobulin (β2m), and an antigenic peptide. Here we have monitored peptide-HLA interactions and peptide dissociation kinetics of two HLA-B27 subtypes by fluorescence depolarization techniques. A single natural amino-acid substitution distinguishes the HLA-B*2705 subtype that is associated with the autoimmune disease ankylosing spondylitis from the non-disease-associated HLA-B*2709 subtype. Peptides with C-terminal Arg or Lys represent 27% of the natural B*2705 ligands. Our results show that dissociation of a model peptide with a C-terminal Lys (GRFAAAIAK) follows a two-step mechanism. Final peptide release occurs in the second step for both HLA-B27 subtypes. However, thermodynamics and kinetics of peptide-HLA interactions reveal different molecular mechanisms underlying the first step, as indicated by different activation energies of 95±8kJ/mol (HLA-B*2705) and 150±10kJ/mol (HLA-B*2709). In HLA-B*2709, partial peptide dissociation probably precedes fast final peptide release, while in HLA-B*2705 an allosteric mechanism based on long-range interactions between β2m and the peptide binding groove controls the first step. The resulting peptide presentation mode lasts for days at physiological temperature, and determines the peptide-HLA-B*2705 conformation, which is recognized by cellular ligands such as T-cell receptors.

Ras proteins have to be associated with the inner leaflet of the plasma membrane to perform their signaling functions. This membrane targeting and binding is controlled by post-translational covalent attachment of farnesyl and palmitoyl chains to cysteines in the membrane anchor region of the N- and H-Ras isoforms. Two N-Ras lipoproteins were investigated, namely a farnesylated and hexadecylated protein, presenting the natural hydrophobic modifications and a doubly hexadecylated construct, respectively. The proteins are surface active and form a Gibbs monolayer at the air-D2O interface. The contours of the amide-I bands were analyzed using infrared reflection absorption spectroscopy (IRRAS). Langmuir monolayers of a mixture of POPC, brain sphingomyelin, and cholesterol were used as half of a model biomembrane to study the insertion of these N-Ras proteins. They insert with their hydrophobic anchors into lipid monolayers but at higher surface pressures (30mN/m); the farnesylated and hexadecylated protein desorbs completely from the monolayer, whereas the doubly hexadecylated protein remains incorporated. During the insertion process, changes in the orientation of the protein secondary structure were detected by comparison with simulated IRRA spectra, based on the information on the relative orientation of the secondary structure elements from the protein crystal structure data.

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for several familial amyloid diseases. Here, we use FTIR to describe the secondary structural changes that take place when wt TTR undergoes heat- or high-pressure-induced denaturation, as well as fibril formation. Upon thermal denaturation, TTR loses part of its intramolecular β-sheet structure followed by an increase in nonnative, probably antiparallel β-sheet contacts (bands at 1616 and 1686cm−1) and in the light scattering, suggesting its aggregation. Pressure-induced denaturation studies show that even at very elevated pressures (12kbar), TTR loses only part of its β-sheet structure, suggesting that pressure leads to a partially unfolded species. On comparing the FTIR spectrum of the TTR amyloid fibril produced at atmospheric pressure upon acidification (pH 4.4) with the one presented by the native tetramer, we find that the content of β-sheets does not change much upon fibrillization; however, the alignment of β-sheets is altered, resulting in the formation of distinct β-sheet contacts (band at 1625cm−1). The random-coil content also decreases in going from tetramers to fibrils. This means that, although part of the tertiary- and secondary-structure content of the TTR monomers has to be lost before fibril formation, as previously suggested, there must be a subsequent reorganization of part of the random-coil structure into a well-organized structure compatible with the amyloid fibril, as well as a readjustment of the alignment of the β-sheets. Interestingly, the infrared spectrum of the protein recovered from a cycle of compression-decompression at pD 5, 37°C, is quite similar to that of fibrils produced at atmospheric pressure (pH 4.4), which suggests that high hydrostatic pressure converts the tetramers of TTR into an amyloidogenic conformation.

The influence of Gramicidin D (GD) incorporation on the structure and phase behavior of aqueous dispersions of DMPC lipid bilayers has been studied using small-angle x-ray scattering (SAXS) and 2H-NMR spectroscopy. The experiments covered a temperature range from −10°C to 60°C and a pressure range of 0.001–4kbar. Pressure was used to be able to tune the lipid bilayer conformational order and phase state and because high pressure is an important feature of certain natural biotopes. The data show that, depending on the GD concentration, the structure of the temperature- and pressure-dependent lipid phases is significantly altered by the insertion of the polypeptide, and a p,T-phase diagram could be obtained for intermediate GD concentrations. Upon gramicidin insertion, a rather narrow fluid-gel coexistence regions is formed. Two gel phases are induced which are different from those of the pure lipid bilayer system and which separate at low temperatures/high pressures. For both the temperature- and pressure-induced fluid-to-gel transition, a similar pseudocritical transitional behavior is observed, which is even more pronounced upon incorporation of the peptide.

The main hypothesis for prion diseases is that the cellular protein (PrPC) can be altered into a misfolded, β-sheet-rich isoform (PrPSc), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we investigate the effects of amino-terminal deletion mutations, rPrPΔ51–90 and rPrPΔ32–121, on the stability and the packing properties of recombinant murine PrP. The region lacking in rPrPΔ51–90 is involved physiologically in copper binding and the other construct lacks more amino-terminal residues (from 32 to 121). The pressure stability is dramatically reduced with decreasing N-domain length and the process is not reversible for rPrPΔ51–90 and rPrPΔ32–121, whereas it is completely reversible for the wild-type form. Decompression to atmospheric pressure triggers immediate aggregation for the mutants in contrast to a slow aggregation process for the wild-type, as observed by Fourier-transform infrared spectroscopy. The temperature-induced transition leads to aggregation of all rPrPs, but the unfolding temperature is lower for the rPrP amino-terminal deletion mutants. The higher susceptibility to pressure of the amino-terminal deletion mutants can be explained by a change in hydration and cavity distribution. Taken together, our results show that the amino-terminal region has a pivotal role on the development of prion misfolding and aggregation.

Differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) were used to characterize thermal phase transitions, membrane packing, and volumetric properties in multilamellar vesicles (MLVs) composed of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at different temperatures. For PLFE MLVs derived from cells grown at 78°C, the first DSC heating scan exhibits an endothermic transition at 46.7°C, a small hump near 60°C, and a broad exothermic transition at 78.5°C, whereas the PPC scan reveals two transitions at ∼45°C and 60°C. The endothermic peak at 46.7°C is attributed to a lamellar-to-lamellar phase transition and has an unusually low ΔH (3.5kJ/mol) and ΔV/V (0.1%) value, as compared to those for the main phase transitions of saturated diacyl monopolar diester lipids. This result may arise from the restricted trans-gauche conformational changes in the dibiphytanyl chain due to the presence of cyclopentane rings and branched methyl groups and due to the spanning of the lipid molecules over the whole membrane. The exothermic peak at 78.5°C probably corresponds to a lamellar-to-cubic phase transition and exhibits a large and negative ΔH value (−23.2kJ/mol), which is uncommon for normal lamellar-to-cubic phospholipid phase transformations. This exothermic transition disappears in the subsequent heating scans and thus may involve a metastable phase, which is irreversible at the scan rate used. Further, there is no distinct peak in the plot of the thermal expansion coefficient α versus temperature near 78.5°C, indicating that this lamellar-to-cubic phase transition is not accompanied by any significant volume change. For PLFE MLVs derived from cells grown at 65°C, similar DSC and PPC profiles and thermal history responses were obtained. However, the lower growth temperature yields a higher ΔV/V (∼0.25%) and ΔH (14kJ/mol) value for the lamellar-to-lamellar phase transition measured at the same pH (2.1). A lower growth temperature also generates a less negative temperature dependence of α. The changes in ΔV/V, ΔH, and the temperature dependence of α can be attributed to the decrease in the number of cyclopentane rings in PLFE at the lower growth temperature. The relatively low ΔV/V and small ΔH involved in the phase transitions help to explain why PLFE liposomes are remarkably thermally stable and also echo the proposal that PLFE liposomes are generally rigid and tightly packed. These results help us to understand why, despite the occurrence of thermal-induced phase transitions, PLFE liposomes exhibit a remarkably low temperature sensitivity of proton permeation and dye leakage.

As the application of high-resolution atomic force microscopy (AFM) has led us recently to the discovery of a unique pressure-induced circular amyloid, we used the same approach to examine morphological events accompanying insulin aggregation under ambient conditions. This study presents the multistage, hierarchical character of the spontaneous fibrillation of insulin at low pH and at 60 and 70°C, and—due to the marked enhancement of image resolution achieved—brings new clues as to the fibrils’ ultrastructure and mechanisms of its assembly. Specifically, focusing on the prefibrillar amorphous aggregates occurring 30s after elevating temperature to the nucleation-enhancing 60°C, revealed the tendency of the globule-shaped oligomers to queue and assembly into elongated forms. This suggests that the shape of the nuclei itself predetermines—in part—the fibrillar architecture of the amyloid. Among first fibrillar features, short but relatively thick (8-nm) seedlike forms appeared on a very short timescale within the first minute of incubation. It has been shown that such fibrils are likely to act as lateral scaffolds for the growth of amyloid. By using phase-image AFM as a nanometer-resolved probe of visco-elastic surface properties, we were able to show that bundles of early protofilaments associated into parallel fibrils are capable of a cooperative transformation into twisted, highly ordered superhelices of the mature amyloid. Independently from producing evidence for the step-resolved character of the process, intermediate and morphologically heterogeneous forms were trapped and characterized, which yields direct evidence for the multipathway character of the amyloidogenesis of insulin. Apart from the faster kinetics, the increased temperature of 70°C leads to a higher degree of morphological variability: along straight rods, twisted ribbonlike structures, rod bundles, and ropelike structures become prominent in the corresponding AFM data.

We investigated the temperature- and pressure-dependent structure and phase behavior of a solvated oligopeptide, GVG(VPGVG), which serves as a minimalistic elastin-like model system, over a large region of the thermodynamic phase field, ranging from 2 to 120°C and from ambient pressure up to ∼10 kbar, applying various spectroscopic (CD, FT-IR) and thermodynamic (DSC, PPC) measurements. We find that this octapeptide behaves as a two-state system which undergoes the well-known inverse-temperature folding transition occurring at T ≈ 36°C, and, in addition, a slow trend reversal at higher temperatures, finally leading to a reentrant unfolding close to the boiling point of water. Furthermore, the pressure-dependence of the folding/unfolding transition was studied to yield a more complete picture of the p, T-stability diagram of the system. A molecular-level picture of these processes, in particular on the role of water for the folding and unfolding events of the peptide, presented with the help of molecular-dynamics simulations, is presented in a companion article in this issue.

The pressure dependence of the photocycle kinetics of bacteriorhodopsin from Halobacterium salinariumwas investigated at pressures up to 4kbar at 25°C and 40°C. The kinetics can be adequately modeled by nine apparent rate constants, which are assigned to irreversible transitions of a single relaxation chain of nine kinetically distinguishable states P1to P9. All states except P1and P9consist of two or more spectral components. The kinetic states P2to P6comprise only the two fast equilibrating spectral states Land M. From the pressure dependence, the volume differences ΔVLM0between these two spectral states could be determined that range from ΔVLM0=−11.4±0.7ml/mol (P2) to ΔVLM0=14.6±2.8mL/mol (P6). A model is developed that explains the dependence of ΔVLM0on the kinetic state by the electrostriction effect of charges, which are formed and neutralized during the L/Mtransition.

The kinetics of chain disruption and collapse of staphylococcal nuclease after positive or negative pressure jumps was monitored by real-time small-angle x-ray scattering under pressure. We used this method to probe the overall conformation of the protein by measuring its radius of gyration and pair-distance-distribution function p(r) which are sensitive to the spatial extent and shape of the particle. At all pressures and temperatures tested, the relaxation profiles were well described by a single exponential function. No fast collapse was observed, indicating that the rate limiting step for chain collapse is the same as that for secondary and tertiary structure formation. Whereas refolding at low pressures occurred in a few seconds, at high pressures the relaxation was quite slow, ∼1h, due to a large positive activation volume for the rate-limiting step for chain collapse. A large increase in the system volume upon folding implies significant dehydration of the transition state and a high degree of similarity in terms of the packing density between the native and transition states in this system. This study of the time-dependence of the tertiary structure in pressure-induced folding/unfolding reactions demonstrates that novel information about the nature of protein folding transitions and transition states can be obtained from a combination of small-angle x-ray scattering using high intensity synchrotron radiation with the high pressure perturbation technique.

Differential scanning calorimetry, x-ray diffraction, and infrared and 31P-nuclear magnetic resonance (31P-NMR) spectroscopy were used to examine the thermotropic phase behavior and organization of cationic model membranes composed of the P-O-ethyl esters of a homologous series of n-saturated 1,2-diacyl phosphatidylcholines (Et-PCs). Differential scanning calorimetry studies indicate that on heating, these lipids exhibit single highly energetic and cooperative endothermic transitions whose temperatures and enthalpies are higher than those of the corresponding phosphatidylcholines (PCs). Upon cooling, these Et-PCs exhibit two exothermic transitions at temperatures slightly below the single endotherm observed upon heating. These cooling exotherms have both been assigned to transitions between the liquid-crystalline and gel phases of these lipids by x-ray diffraction. The x-ray diffraction data also show that unlike the parent PCs, the chain-melting phase transition of these Et-PCs involves a direct transformation of a chain-interdigitated gel phase to the lamellar liquid-crystalline phase for the homologous series of n≥14. Our 31P-NMR spectroscopic studies indicate that the rates of phosphate headgroup reorientation in both gel and liquid-crystalline phases of these lipids are comparable to those of the corresponding PC bilayers. However, the shape of the 31P-NMR spectra observed in the interdigitated gel phase indicates that phosphate headgroup reorientation is subject to constraints that are not encountered in the non-interdigitated gel phases of parent PCs. The infrared spectroscopic data indicate that the Et-PCs adopt a very compact form of hydrocarbon chain packing in the interdigitated gel phase and that the polar/apolar interfacial regions of these bilayers are less hydrated than those of corresponding PC bilayers in both the gel and liquid-crystalline phases. Our results indicate that esterification of PC phosphate headgroups results in many alterations of bilayer physical properties aside from the endowment of a positively charged surface. This fact should be considered in assessing the interactions of these compounds with naturally occurring lipids and with other biological materials.