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Complex glycans serve important functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates, such as un-decaprenol phosphate in bacteria. In this study we focus on a small enzyme, PglC fromCampylobacter, structurally characterized for the first time in 2018, as a detergent solubilized construct. PglC is a monotopic phosphoglycosyl transferase (PGT), that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results reveal a specific interaction of undecaprenol phosphate with PGT cationic residues and suggest a role for critical conformational transitions and electrostatic steering in substrate recognition, overcoming significant energetic barriers to binding. The study highlights that computation, guided by fundamental chemical principles, can advance the study of biochemical processes at membrane bilayers and provide chemical insight at a molecular level that cannot be derived by experiment alone.Insert Table of Contents artwork here

The preponderance of Intrinsically Disordered Proteins (IDPs) in the eukaryotic proteome, and their ability to interact with each other, proteins, RNA, and DNA for functional purposes have made it important to quantitatively characterize their biophysical properties. Towards this end, we developed the transferable Self-Organized Polymer (SOP-IDP) model in order to calculate the properties of a number of IDPs. The calculated and measured radius of gyration values (Rgs) are in excellent agreement, with a correlation coefficient of 0.96. For AP180 and Epsin, the predicted and values obtained using Fluorescence Correlation Spectroscopy for the hydrodynamic radii (Rhs) are also in quantitative agreement. Strikingly, the calculated SAXS profiles for thirty six IDPs also nearly match the experiments. The dependence ofRg, the mean end-to-end distance (Re), andRhobey the Flory scaling law,Rα≈aαN0.59(α=g, e, andh), suggesting that globally IDPs behave as polymers in a good solvent. The values ofag,ae, andahare 0.21 nm, 0.53 nm, and 0.16 nm, respectively. Surprisingly, finite size corrections to scaling, expected on theoretical grounds, for all the three quantities are negligible. Sequence dependencies, masked in ensemble properties, emerge through a fine structure analyses of the conformational ensembles using a hierarchical clustering method. Typically, the ensemble of conformations partition into three distinct clusters, with differing extent of population and structural properties. The subpopulations could dictate phase separation tendencies and association with ligands. Without any adjustments to the three parameters in the SOP-IDP model, we obtained excellent agreement with paramagnetic relaxation enhancement (PRE) measurements forα-synuclein. The transferable SOP-IDP model sets the stage for a number of promising applications, including the study of phase separation in IDPs and interactions with nucleic acids.

The transition from a disordered to an assembly-competent monomeric state (N*) in amyloidogenic sequences is a crucial event in the aggregation cascade. Using a well-calibrated model for intrinsically disordered proteins (IDPs), we show that the N* states, which bear considerable resemblance to the polymorphic fibril structures found in experiments, not only appear as excitations in the free energy landscapes of Aβ40 and Aβ42, but also initiate the aggregation cascade. For Aβ42, the transitions to the different N* states are in accord with Ostwald’s rule of stages, with the least stable structures forming ahead of thermodynamically favored ones. The Aβ40 and Aβ42 monomer landscapes exhibit different extents of local frustration, which we show have profound implications in dictating subsequent self-assembly. Using kinetic transition networks, we illustrate that the most favored dimerization routes proceed via N* states. We argue that Ostwald’s rule also holds for the aggregation of fused in sarcoma and polyglutamine proteins.

The 99-residue C-terminal domain of amyloid precursor protein (APP-C99), precursor to amyloid beta (A β ), is a transmembrane (TM) protein containing intrinsically disordered N- and C-terminal extramembrane domains. Using molecular dynamics (MD) simulations, we show that the structural ensemble of the C99 monomer is best described in terms of thousands of states. The C99 monomer has a propensity to form β -strand in the C-terminal extramembrane domain, which explains the slow spin relaxation times observed in paramagnetic probe NMR experiments. Surprisingly, homodimerization of C99 not only narrows the conformational ensemble from thousands to a few states through the formation of metastable β -strands in extramembrane domains but also stabilizes extramembrane α -helices. The extramembrane domain structure is observed to dramatically impact the homodimerization motif, resulting in the modification of TM domain conformations. Our study provides an atomic-level structural basis for communication between the extramembrane domains of the C99 protein and TM homodimer formation. This finding could serve as a general model for understanding the influence of disordered extramembrane domains on TM protein structure.

The 99-residue C-terminal domain of amyloid precursor protein (APP-C99), precursor to amyloid beta (A

An approach for the efficient simulation of phase-separated lipid bilayers, for use in the calculation of equilibrium free energies of partitioning between lipid domains, is proposed. The methodology exploits restraint potentials and rectangular aspect ratios that enforce lipid phase separation, allowing for the simulation of smaller systems that approximately reproduce bulk behavior. The utility of this approach is demonstrated through the calculation of potentials of mean force for the translation of a transmembrane protein between lipid domains. The impact of the imposed restraints on lipid tail ordering and lipid packing are explored, providing insight into how restraints can best be employed to compute accurate free-energy surfaces. This approach should be useful in the accurate calculation of equilibrium partition coefficients for transmembrane protein partitioning in heterogeneous membranes, providing insight into the thermodynamic driving forces that control this fundamental biophysical phenomenon.

The transition from a disordered to an assembly-competent and sparsely populated monomeric state (N*) in amyloidogenic sequences is a crucial event in the aggregation cascade. Using a well-calibrated model for Intrinsically Disordered Proteins (IDPs), we show that the N* states, which bear considerable resemblance to distinct polymorphic fibril structures found in experiments, not only appear as excitations on the monomer free energy landscapes of Aβ40 and Aβ42 but also initiate the aggregation cascade. Interestingly, for Aβ42, the transitions to the different N* states are in accord with Ostwald’s rule of stages, with the least stable structures forming ahead of thermodynamically favored structures, which appear only on longer time-scales. Despite having similar topographies, the Aβ40 and Aβ42 monomer landscapes exhibit different extent of ruggedness, particularly in the vicinity of N* states, which we show have profound implications in dictating the intramolecular diffusion rates, and subsequent self-assembly into higher order structures. The network of connected kinetic states, which for Aβ42 is considerably more complex than for Aβ40, shows that the most favored dimerization routes proceed via the N* states. Direct transition between the disordered ground states within the monomer and dimer basins is less likely. The Ostwald’s rule of stages holds widely, qualitatively explaining the unusual features in other fibril forming IDPs, such as Fused in Sarcoma (FUS). Similarly, the N* theory accounts for dimer formation in small disordered polyglutamine peptides, implicated in the Huntington disease.Graphical TOC Entry

The MARTINI model is a widely used coarse-grained force field popular for its capacity to represent a diverse array of complex biomolecules. However, efforts to simulate increasingly realistic models of membranes, involving complex lipid mixtures and multiple proteins, suggest that membrane protein aggregates are overstabilized by the MARTINI v2.2 force field. In this study, we address this shortcoming of the MARTINI model. We determined the free energy of dimerization of four transmembrane protein systems using the nonpolarizable MARTINI model. Comparison with experimental FRET-based estimates of the dimerization free energy was used to quantify the significant overstabilization of each protein homodimer studied. To improve the agreement between simulation and experiment, a single uniform scaling factor, α, was used to enhance the protein–lipid Lennard–Jones interaction. A value of α = 1.04–1.045 was found to provide the best fit to the dimerization free energies for the proteins studied while maintaining the specificity of contacts at the dimer interface. To further validate the modified force field, we performed a multiprotein simulation using both MARTINI v2.2 and the reparameterized MARTINI model. While the original MARTINI model predicts oligomerization of protein into a single aggregate, the reparameterized MARTINI model maintains a dynamic equilibrium between monomers and dimers as predicted by experimental studies. The proposed reparameterization is an alternative to the standard MARTINI model for use in simulations of realistic models of a biological membrane containing diverse lipids and proteins.

Cholesterol is a ubiquitous component of mammalian cell membranes and affects membrane protein function. Although cholesterol-mediated formation of ordered membrane domains has been extensively studied, molecular-level structural information about cholesterol self-association has been absent. Here, we combine solid-state nuclear magnetic resonance (NMR) spectroscopy with all-atom molecular dynamics simulations to determine the oligomeric structure of cholesterol in phospholipid bilayers. Two-dimensional 13C-13C correlation spectra of differentially labeled cholesterol indicate that cholesterol self-associates in a face-to-face fashion at membrane concentrations from 17 to 44 mol %. 2D 13C and 19F spin-counting experiments allowed us to measure the average oligomeric number of these cholesterol clusters. At low cholesterol concentrations of ∼20%, the average cluster size is centered on dimers. At a high cholesterol concentration of 44%, which is representative of virus lipid envelopes and liquid-ordered domains of cell membranes, both dimers and tetramers are observed. The cholesterol dimers are found in both phase-separated membranes that contain sphingomyelin and in disordered and miscible membranes that are free of sphingomyelin. Molecular dynamics simulations support these experimental observations and moreover provide the lifetimes, stabilities, distributions, and structures of these nanoscopic cholesterol clusters. Taken together, these NMR and MD data strongly suggest that dimers are the basic structural unit of cholesterol in phospholipid bilayers. The direct observation of cholesterol dimers and tetramers provides a revised framework for studying cholesterol interactions with membrane proteins to regulate protein functions and for understanding the pathogenic role of cholesterol in diseases.

Protein association in lipid membranes is fundamental to membrane protein function and of great biomedical relevance. All-atom and coarse-grained models have been extensively used to understand the protein-protein interactions in the membrane and to compute equilibrium association constants. However, slow translational and rotational diffusion of protein in membrane presents challenges to the effective sampling of conformations defining the ensembles of free and bound states contributing to the association equilibrium and the free energy of dimerization. We revisit the homodimerization equilibrium of the TM region of glycophorin A. Conformational sampling is performed using umbrella sampling along previously proposed one-dimensional collective variables and compared with sampling over a two-dimensional collective variable space using the MARTINI v2.2 force field. We demonstrate that the one-dimensional collective variables suffer from restricted sampling of the native homodimer conformations leading to a biased free energy landscape. Conversely, simulations along the two-dimensional collective variable effectively characterize the thermodynamically relevant native and non-native interactions contributing to the association equilibrium. These results demonstrate the challenges associated with accurately characterizing binding equilibria when multiple poses contribute to the bound state ensemble.

Significance Alzheimer’s disease (AD) is caused by the accumulation of pathological aggregates, primarily composed of the A β 40 and A β 42 isoforms. Experiments have shown that A β 42 is more aggregation prone compared to A β 40. However, the molecular origin of this apparent anomaly remains elusive. Here, we provide a microscopic basis for the different aggregation rates in terms of the distinct populations of high free energy excited fibril-like states ( N * ) that are encoded in the monomer spectra. The N * theory explains the emergence of fibril polymorphs and predicts the relative kinetic stabilities of A β 42 fibrils using Ostwald’s rule of stages. Our work shows that sequence-specific conformational heterogeneity of the monomer ensembles provides important cues for understanding protein aggregation.

How the distinctive lipid composition of mammalian plasma membranes impacts membrane protein structure is largely unexplored, partly because of the dearth of isotropic model membrane systems that contain abundant sphingolipids and cholesterol. This gap is addressed by showing that sphingomyelin and cholesterol-rich (SCOR) lipid mixtures with phosphatidylcholine can be co-solubilized by n-dodecyl-β-melibioside to form bicelles. Small angle X-ray and neutron scattering, as well as cryo-electron microscopy demonstrate that these assemblies are stable over a wide range of conditions and exhibit the bilayered-disc morphology of ideal bicelles even at low lipid-to-detergent mole ratios. SCOR bicelles are shown to be compatible with a wide array of experimental techniques, as applied to the transmembrane human amyloid precursor C99 protein in this medium. These studies reveal an equilibrium between low order oligomer structures that differ significantly from previous experimental structures of C99, providing an example of how ordered membranes alter membrane protein structure.

Chemical exchange line broadening is an important phenomenon in nuclear magnetic resonance (NMR) spectroscopy, in which a nuclear spin experiences more than one magnetic environment as a result of chemical or conformational changes of a molecule. The dynamic process of chemical exchange strongly affects the sensitivity and resolution of NMR experiments and increasingly provides a powerful probe of the interconversion between chemical and conformational states of proteins, nucleic acids, and other biologic macromolecules. A simple and often used theoretic description of chemical exchange in NMR spectroscopy is based on an idealized 2-state jump model (the random phase or telegraph signal). However, chemical exchange can also be represented as a barrier crossing event that can be modeled by using chemical reaction rate theory. The timescale of crossing is determined by the barrier height, the temperature, and the dissipation modeled as collisional or frictional damping. This tutorial explores the connection between the NMR theory of chemical exchange line broadening and strong collision models for chemical kinetics in statistical mechanics. Theoretic modeling and numeric simulation are used to map the rate of barrier crossing dynamics of a particle on a potential energy surface to the chemical exchange relaxation rate constant. By developing explicit models for the exchange dynamics, the tutorial aims to elucidate the underlying dynamical processes that give rise to the rich phenomenology of chemical exchange observed in NMR spectroscopy. Software for generating and analyzing the numeric simulations is provided in the form of Python and Fortran source codes.

International audience; Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro, in vivo and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aβ, tau), α-synuclein, IAPP and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D) and amyotrophic lateral sclerosis (ALS) research, respectively for over many years.

Residues spanning distinct regions of the low-complexity domain of the RNA-binding protein, Fused in Sarcoma (FUS-LC), form fibril structures with different core morphologies. NMR experiments show that the 214 residue FUS-LC forms a fibril with an S-bend (core-1, residues 39-95), while the rest of the protein is disordered. In contrast, the fibrils of the C-terminal variant (FUS-LC-C; residues 111-214) has a U-bend topology (core-2, residues 112-150). Absence of the U-bend in FUS-LC implies that the two fibril cores do not coexist. Computer simulations show that these perplexing findings could be understood in terms of the population of sparsely-populated fibril-like excited states in the monomer. The propensity to form core-1 is higher compared to core-2. We predict that core-2 forms only in truncated variants that do not contain the core-1 sequence. At the monomer level, sequence-dependent enthalpic effects determine the relative stabilities of the core-1 and core-2 topologies.TOC graphic

Lipid-coated noble metal nanoparticles (L-NPs) combine the biomimetic surface properties of a self-assembled lipid membrane with the plasmonic properties of a nanoparticle (NP) core. In this work, we investigate derivatives of cholesterol, which can be found in high concentrations in biological membranes, and other terpenoids, as tunable, synthetic platforms to functionalize L-NPs. Side chains of different length and polarity, with a terminal alkyne group as Raman label, are introduced into cholesterol and betulin frameworks. The synthesized tags are shown to coexist in two conformations in the lipid layer of the L-NPs, identified as "head-out" and "head-in" orientations, whose relative ratio is determined by their interactions with the lipid-water hydrogen-bonding network. The orientational dimorphism of the tags introduces orthogonal functionalities into the NP surface for selective targeting and plasmon-enhanced Raman sensing, which is utilized for the identification and Raman imaging of epidermal growth factor receptor-overexpressing cancer cells.

Normal micelle aggregates of amphiphilic surfactant in aqueous solvent are formed by a process of entropically driven self-assembly. The self-assembly of reverse micelles from amphiphilic surfactant in non-polar solvent in the presence of water is considered to be an enthalpically driven process. While the formation of normal and reverse surfactant micelles has been well characterized in theory and experiment, the nature of dry micelle formation, from amphiphilic surfactant in non-polar solvent in the absence of water, is poorly understood. In this study, a theory of dry reverse micelle formation is developed. Variation in free energy during micelle assembly is derived for the specific case of AOT surfactant in isooctane solvent using atomistic molecular dynamics simulation analyzed using the energy representation method. The existence and thermodynamic stability of dry reverse micelles of limited size are confirmed. The abrupt occurence of monodisperse aggregates is a clear signature a critical micelle concentration, commonly observed in the formation of normal surfactant micelles. The morphology of large dry micelles provides insight into the nature of the thermodynamic driving forces stabilizing the formation of the surfactant aggregates. Overall, this study provides detailed insight into the structure and stability of dry reverse micelles assembly in non-polar solvent., Comment: 34 pages, 6 figure. In Ver2, GAP joined and re-plot figures, and we changed the title

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Computer simulations of complete viral particles can provide theoretical insights into large-scale viral processes including assembly, budding, egress, entry, and fusion. Detailed atomistic simulations, however, are constrained to shorter timescales and require billion-atom simulations for these processes. Here, we report the current status and on-going development of a largely “bottom-up” coarse-grained (CG) model of the SARS-CoV-2 virion. Structural data from a combination of cryo-electron microscopy (cryo-EM), x-ray crystallography, and computational predictions were used to build molecular models of structural SARS-CoV-2 proteins, which were then assembled into a complete virion model. We describe how CG molecular interactions can be derived from all-atom simulations, how viral behavior difficult to capture in atomistic simulations can be incorporated into the CG models, and how the CG models can be iteratively improved as new data becomes publicly available. Our initial CG model and the detailed methods presented are intended to serve as a resource for researchers working on COVID-19 who are interested in performing multiscale simulations of the SARS-CoV-2 virion.

The spontaneous formation of micelles in aqueous solutions is governed by the amphipathic nature of surfactants and is practically interesting due to the regular use of micelles as membrane mimics, for the characterization of protein structure, and for drug design and delivery. We performed a systematic characterization of the finite-size effect observed in single-component dodecylphosphocholine (DPC) micelles with the coarse-grained MARTINI model. Of multiple coarse-grained solvent models investigated using large system sizes, the non-polarizable solvent model was found to most-accurately reproduce SANS spectra of 100 mM DPC in aqueous solution. We systematically investigated the finite-size effect at constant 100 mM concentration in 23 systems of sizes 40 to 150 DPC, confirming the finite-size effect to manifest as an oscillation in the mean micelle aggregation number about the thermodynamic aggregation number as the system size increases, mostly diminishing once the system supports formation of three micelles. The accuracy of employing a multiscale simulation approach to avoid finite-size effects in the micelle size distribution and SANS spectra using MARTINI and CHARMM36 was explored using multiple long timescale 500-DPC coarse-grained simulations which were back-mapped to CHARMM36 all-atom systems. It was found that the MARTINI model generally occupies more volume than the all-atom model, leading to the formation of micelles that are of a reasonable radius of gyration, but are smaller in aggregation number. The systematic characterization of the finite-size effect and exploration of multiscale modeling presented in this work provides guidance for the accurate modeling of micelles in simulations., 19 pages, 5 figures, 2 tables, Corresponding Author: John E. Straub

The early events in the aggregation of the intrinsically disordered peptide, amyloid- [Formula: see text] (A [Formula: see text]), involve transitions from the disordered free energy ground state to assembly-competent states. Are the fingerprints of order found in the amyloid fibrils encoded in the conformations that the monomers access at equilibrium? If so, could the enhanced aggregation rate of A [Formula: see text] 42 compared to A [Formula: see text] 40 be rationalized from the sparsely populated high free energy states of the monomers? Here, we answer these questions in the affirmative using coarse-grained simulations of the self-organized polymer–intrinsically disordered protein (SOP-IDP) model of A [Formula: see text] 40 and A [Formula: see text] 42. Although both the peptides have practically identical ensemble-averaged properties, characteristic of random coils (RCs), the conformational ensembles of the two monomers exhibit sequence-specific heterogeneity. Hierarchical clustering of conformations reveals that both the peptides populate high free energy aggregation-prone ([Formula: see text]) states, which resemble the monomers in the fibril structure. The free energy gap between the ground (RC) and the [Formula: see text] states of A [Formula: see text] 42 peptide is smaller than that for A [Formula: see text] 40. By relating the populations of excited states of the two peptides to the fibril formation time scales using an empirical formula, we explain nearly quantitatively the faster aggregation rate of A [Formula: see text] 42 relative to A [Formula: see text] 40. The [Formula: see text] concept accounts for fibril polymorphs, leading to the prediction that the less stable [Formula: see text] state of A [Formula: see text] 42, encoding for the U-bend fibril, should form earlier than the structure with the S-bend topology, which is in accord with Ostwald’s rule rationalizing crystal polymorph formation.

© 2020 American Chemical Society. Vibrational spectroscopy is an essential tool in chemical analyses, biological assays, and studies of functional materials. Over the past decade, various coherent nonlinear vibrational spectroscopic techniques have been developed and enabled researchers to study time-correlations of the fluctuating frequencies that are directly related to solute-solvent dynamics, dynamical changes in molecular conformations and local electrostatic environments, chemical and biochemical reactions, protein structural dynamics and functions, characteristic processes of functional materials, and so on. In order to gain incisive and quantitative information on the local electrostatic environment, molecular conformation, protein structure and interprotein contacts, ligand binding kinetics, and electric and optical properties of functional materials, a variety of vibrational probes have been developed and site-specifically incorporated into molecular, biological, and material systems for time-resolved vibrational spectroscopic investigation. However, still, an all-encompassing theory that describes the vibrational solvatochromism, electrochromism, and dynamic fluctuation of vibrational frequencies has not been completely established mainly due to the intrinsic complexity of intermolecular interactions in condensed phases. In particular, the amount of data obtained from the linear and nonlinear vibrational spectroscopic experiments has been rapidly increasing, but the lack of a quantitative method to interpret these measurements has been one major obstacle in broadening the applications of these methods. Among various theoretical models, one of the most successful approaches is a semiempirical model generally referred to as the vibrational spectroscopic map that is based on a rigorous theory of intermolecular interactions. Recently, genetic algorithm, neural network, and machine learning approaches have been applied to the development of vibrational solvatochromism theory. In this review, we provide comprehensive descriptions of the theoretical foundation and various examples showing its extraordinary successes in the interpretations of experimental observations. In addition, a brief introduction to a newly created repository Web site (http://frequencymap.org) for vibrational spectroscopic maps is presented. We anticipate that a combination of the vibrational frequency map approach and state-of-the-art multidimensional vibrational spectroscopy will be one of the most fruitful ways to study the structure and dynamics of chemical, biological, and functional molecular systems in the future.

The early events in the aggregation of the intrinsically disordered peptide, Aβ, involve transitions from the disordered lowest free energy ground state to assembly-competent states. Are the finger-prints of order found in the amyloid fibrils encoded in the conformations that the monomers access at equilibrium? If so, could the enhanced aggregation rate of Aβ42 compared to Aβ40 be rationalized from the sparsely populated high free energy states of the monomers? Here, we answer these questions in the affirmative using coarse-grained simulations of the SOP-IDP model of Aβ40 and Aβ42. Although both the peptides have practically identical ensemble-averaged properties, characteristic of random coils (RCs), the conformational ensembles of the two monomers exhibit sequence-specific heterogeneity. Hierarchical clustering of conformations reveals that both the peptides populate high free energy aggregation-prone (N*) states, which resemble the monomers in the fibril structure. The free energy gap between the ground (RC) and the N* states of Aβ42 peptide is smaller than for Aβ40. By relating the populations of excited states of the two peptides to the fibril formation time scales using an empirical formula, we explain nearly quantitatively the faster aggregation rate of Aβ42 relative to Aβ40. The N* concept accounts for fibril polymorphs, leading to the prediction that the less stable N* state of Aβ42, encoding for the U-bend fibril, should form earlier than the structure with the S-bend topology, which is in accord with the Ostwald’s rule rationalizing crystal polymorph formation.Significance StatementAlzheimer’s disease (AD), a rampant neurodegenerative disorder, is caused by the accumulation of pathological aggregates, primarily composed of the two isoforms Aβ40 and Aβ42. Experiments have shown that Aβ42 is more aggregation-prone compared to Aβ40. However, the molecular origin of this apparent anomaly remains elusive. Here, we provide a microscopic basis for the different aggregation rates in terms of the distinct populations of high free energy excited fibril-like states (N*) that are encoded in the monomer spectra. The N* theory explains the emergence of fibril polymorphs, and predicts the relative kinetic stabilities of Aβ42 fibrils using Ostwald’s rule of stages. Our work shows that sequence-specific conformational heterogeneity of the monomer ensembles provides important cues for understanding protein aggregation.

Serum amyloid A (SAA) is a plasma protein that transports lipids during inflammation. To explore SAA solution conformations and lipid-binding mechanism, we used hydrogen-deuterium exchange mass spectrometry, lipoprotein reconstitution, amino acid sequence analysis, and molecular dynamics simulations. Solution conformations of lipid-bound and lipid-free mSAA1 at pH~7.4 agreed in details with the crystal structures but also showed important differences. The results revealed that amphipathic α-helices h1 and h3 comprise a lipid-binding site that is partially pre-formed in solution, is stabilized upon binding lipids, and shows lipid-induced folding of h3. This site sequesters apolar ligands via a concave hydrophobic surface in SAA oligomers. The largely disordered/dynamic C-terminal region is conjectured to mediate the promiscuous binding of other ligands. The h1-h2 linker region is predicted to form an unexpected β-hairpin that may represent an early amyloidogenic intermediate. The results help establish structural underpinnings for understanding SAA interactions with its key functional ligands, its evolutional conservation, and its transition to amyloid.

We propose a molecular simulation method using genetic algorithm (GA) for biomolecular systems to obtain ensemble averages efficiently. In this method, we incorporate the genetic crossover, which is one of the operations of GA, to any simulation method such as conventional molecular dynamics (MD), Monte Carlo, and other simulation methods. The genetic crossover proposes candidate conformations by exchanging parts of conformations of a target molecule between a pair of conformations during the simulation. If the candidate conformations are accepted, the simulation resumes from the accepted ones. While conventional simulations are based on local update of conformations, the genetic crossover introduces global update of conformations. As an example of the present approach, we incorporated genetic crossover to MD simulations. We tested the validity of the method by calculating ensemble averages and the sampling efficiency by using two kinds of peptides, ALA3 and (AAQAA)3 . The results show that for ALA3 system, the distribution probabilities of backbone dihedral angles are in good agreement with those of the conventional MD and replica-exchange MD simulations. In the case of (AAQAA)3 system, our method showed lower structural correlation of α-helix structures than the other two methods and more flexibility in the backbone ψ angles than the conventional MD simulation. These results suggest that our method gives more efficient conformational sampling than conventional simulation methods based on local update of conformations. © 2018 Wiley Periodicals, Inc.

Hereditary apolipoprotein A-I (apoA-I) amyloidosis is a life-threatening incurable genetic disorder whose molecular underpinnings are unclear. In this disease, variant apoA-I, the major structural and functional protein of high-density lipoprotein, is released in a free form, undergoes an α-helix to intermolecular cross-β-sheet conversion along with a proteolytic cleavage, and is deposited as amyloid fibrils in various organs, which can cause organ damage and death. Glu34Lys is the only known charge inversion mutation in apoA-I that causes human amyloidosis. To elucidate the structural underpinnings of the amyloidogenic behavior of Glu34Lys apoA-I, we generated its recombinant globular N-terminal domain (residues 1-184) and compared the conformation and dynamics of its lipid-free form with those of two other naturally occurring apoA-I variants, Phe71Tyr (amyloidogenic) and Leu159Arg (non-amyloidogenic). All variants showed reduced structural stability and altered aromatic residue packing. The greatest decrease in stability was observed in the non-amyloidogenic variant, suggesting that amyloid formation is driven by local structural perturbations at sensitive sites. Molecular dynamics simulations revealed local helical unfolding and suggested that transient opening of the Trp72 side chain induced mutation-dependent structural perturbations in a sensitive region, including the major amyloid hot spot residues Leu14-Leu22. We posit that a shift from the "closed" to the "open" orientation of the Trp72 side chain modulates structural protection of amyloid hot spots, suggesting a previously unknown early step in the protein misfolding pathway.

Sulphated polysaccharides play important roles in a number of physiological and patho-physiological processes including the coagulation cascade, viral transmission, and antioxidation. In nature, sulphated polysaccharides are highly diverse, possessing variations in the carbohydrate backbone, location of the sulphate group(s), and degree of sulphation. These compositional attributes lead to varied sulphated polymers with different negative charge densities and resultant structure–property–activity relationships. Sulphating naturally occurring polysaccharides and their synthetic analogs is challenging, and traditionally requires harsh conditions and long reaction times, often causing non-selective sulphation at different and/or multiple hydroxyl positions. In this Review, we begin with a discussion of both established and novel methods and reagents for sulphation of these polymers, along with the advantages and disadvantages of these various approaches. Next, we describe characterization methods to confirm sulphation. Finally, we provide examples of synthetically sulphated natural polysaccharides and sulphated synthetic polysaccharides, and discuss the utility of these novel polymers in various biomedical applications. This review provides a comprehensive analysis of synthetic sulphated polysaccharides, their current uses, and highlights biomedical opportunities.

For 40 years, the existence and possible functional importance of cholesterol dimer formation has been discussed. Due to challenges associated with structural studies of membrane lipids, there has as yet been no direct experimental verification of the existence and relevance of the cholesterol dimer. Building on recent advances in lipid force fields for molecular simulation, in this work the structure and stability of the cholesterol dimer is characterized in POPC bilayers in absence and presence of sphingomyelin. The cholesterol dimer structural ensemble is found to consist of sub-states that reflect, but also differ from, previously proposed dimer structures. While face-to-face dimer structures predominate, no evidence is found for the existence of tail-to-tail dimers in POPC lipid bilayers. Near stoichiometric complex formation of cholesterol with sphingomyelin is found to effect cholesterol dimer structure without impacting population. Comparison with NMR-derived order parameters provide validation for the simulation model employed and conclusions drawn related to the structure and stability of cholesterol dimers in multicomponent lipid bilayers. © 2016 Wiley Periodicals, Inc.

Simulated tempering (ST) is a generalized-ensemble algorithm that employs trajectories exploring a range of temperatures to effectively sample rugged energy landscapes. When implemented using the molecular dynamics method, ST can require the use of short time steps for ensuring the stability of trajectories at high temperatures. To address this shortcoming, a mass-scaling ST (MSST) method is presented in which the particle mass is scaled in proportion to the temperature. Mass scaling in the MSST method leads to velocity distributions that are independent of temperature and eliminates the need for velocity scaling after the accepted temperature updates that are required in conventional ST simulations. The homogeneity in time scales with changing temperature improves the stability of simulations and allows for the use of longer time steps at high temperatures. As a result, the MSST is found to be more efficient than the standard ST method, particularly for cases in which a large temperature range is employed. © 2016 Wiley Periodicals, Inc.

Poly-amido-saccharides (PAS) are carbohydrate-based, enantiopure synthetic polymers in which sugar repeat units are joined by amide linkages. This unique and relatively rigid pyranose backbone contributes to their defined helical secondary structure and remarkable chemical properties. Glucose- (glc-) and galactose- (gal-) PAS 10-mer structures are synthesized and investigated with molecular dynamics (MD) simulations and experimental measurements. Quantum mechanical DFT energy minimization calculations, as well as experimental observables including circular dichroism, (1)H,(13)C-HSQC, and (1)H,(1)H-NOESY 2D-NMR studies, validated the all-atom simulation models produced using a modified CHARMM force field. Water radial distribution functions show distinct differences in the glc- and gal-PAS systems that correlate well with observed differences in solubility between gal-PASs and glc-PASs. The computational analysis and MD simulations are in good agreement with experimental results, validating the proposed models as reliable representations of novel glc- and gal-PASs.

γ-Secretase (GS) is a multi-subunit membrane-embedded aspartyl protease that cleaves more than 80 integral membrane proteins, including the amyloid precursor protein (APP) to produce the amyloid-β (Aβ) peptide. Oligomerization and aggregation of the 42-amino acid length Aβ isoform in the brain has been associated with the development and progression of Alzheimer's disease (AD). Based on recent experimental structural studies and using multiscale computational modeling approaches, the conformational states and protein–membrane interactions of the GS complex embedded in six homogeneous and six heterogeneous lipid bilayers were characterized. In order to identify potential lipid and cholesterol binding sites, GS regions with high lipid/cholesterol occupancy values were analyzed using atomistic and coarse-grained simulations. Long lipid residence times were observed to be correlated with a large number of hydrogen bonds between the charged headgroups and key GS amino acids. This observation provides a plausible explanation for the inhibition of GS by charged lipids observed in previous experimental studies. Computed lateral pressure profiles suggest that higher transmembrane pressures favor active state conformations of the catalytic subunit. A probable mechanism for the regulation of the local stress response in cholesterol-rich multicomponent lipid bilayers is identified. Finally, it is demonstrated that interactions between the nicastrin extracellular domain and lipid headgroups leads to a compact structural conformation of the GS complex. Overall, this study provides valuable insight into the effect of bilayer lipid composition on the GS structural ensemble and its function.

Cholesterol is essential to the formation of phase separated lipid domains in membranes. Lipid domains can exist in different thermodynamic phases depending on the molecular composition, and play significant roles in determining structure and function of membrane proteins. We investigate the role of cholesterol in the structure and dynamics of ternary lipid mixtures displaying phase separation using Molecular Dynamics simulations, employing a physiologically-relevant span of cholesterol concentration. We find that cholesterol can induce formation of three regimes of phase behavior, I) miscible liquid disordered bulk, II) phase separated, domain registered coexistence of liquid disordered and liquid ordered and domains, and III) phase separated, domain-anti-registered coexistence of liquid-disordered and newly-identified nanoscopic gel domains composed of cholesterol threads we name “cholesterolic gel” domains. These findings are validated and discussed in the context of current experimental knowledge, models of cholesterol spatial distributions, and models of ternary lipid mixture phase separation.

Intrinsically disordered proteins (IDPs) lack well-defined three-dimensional structures, thus challenging the archetypal notion of structure-function relationships. Determining the ensemble of conformations that IDPs explore under physiological conditions is the first step towards understanding their diverse cellular functions. Here, we quantitatively characterize the structural features of IDPs as a function of sequence and length using coarse-grained simulations. For diverse IDP sequences, with the number of residues (NT) ranging from 24 to 441, our simulations not only reproduce the radii of gyration (Rg) obtained from experiments, but also predict the full scattering intensity profiles in very good agreement with Small Angle X-ray Scattering experiments. The Rg values are well-described by the standard Flory scaling law, , with v ≈ 0.588, making it tempting to assert that IDPs behave as polymers in a good solvent. However, clustering analysis reveals that the menagerie of structures explored by IDPs is diverse, with the extent of heterogeneity being highly sequence-dependent, even though ensemble-averaged properties, such as the dependence of Rg on chain length, may suggest synthetic polymer-like behavior in a good solvent. For example, we show that for the highly charged Prothymosin-α, a substantial fraction of conformations is highly compact. Even if the sequence compositions are similar, as is the case for α-Synuclein and a truncated construct from the Tau protein, there are substantial differences in the conformational heterogeneity. Taken together, these observations imply that metrics based on net charge or related quantities alone, cannot be used to anticipate the phases of IDPs, either in isolation or in complex with partner IDPs or RNA. Our work sets the stage for probing the interactions of IDPs with each other, with folded protein domains, or with partner RNAs, which are critical for describing the structures of stress granules and biomolecular condensates with important cellular functions.Graphical TOC Entry

Gold nanoparticles (NPs) wrapped in a membrane can be utilized as artificial virus nanoparticles (AVNs) that combine the large nonblinking or bleaching optical cross-section of the NP core with the biological surface properties and functionalities provided by a self-assembled lipid membrane. We used these hybrid nanomaterials to test the roles of monosialodihexosylganglioside (GM3) and phosphatidylserine (PS) for a lipid-mediated targeting of virus-containing compartments (VCCs) in macrophages. GM3-presenting AVNs bind to CD169 (Siglec-1)–expressing macrophages, but inclusion of PS in the GM3-containing AVN membrane decreases binding. Molecular dynamics simulations of the AVN membrane and experimental binding studies of CD169 to GM3-presenting AVNs reveal Na(+)-mediated interactions between GM3 and PS as a potential cause of the antagonistic action on binding by the two negatively charged lipids. GM3-functionalized AVNs with no or low PS content localize to tetherin(+), CD9(+) VCC in a membrane composition-depending fashion, but increasing amounts of PS in the AVN membrane redirect the NP to lysosomal compartments. Interestingly, this compartmentalization is highly GM3 specific. Even AVNs presenting the related monosialotetrahexosylganglioside (GM1) fail to achieve an accumulation in VCC. AVN localization to VCC was observed for AVN with gold NP core but not for liposomes, suggesting that NP sequestration into VCC has additional requirements beyond ligand (GM3)–receptor (CD169) recognition that are related to the physical properties of the NP core. Our results confirm AVN as a scalable platform for elucidating the mechanisms of lipid-mediated viral entry pathways and for selective intracellular targeting.

Hereditary apolipoprotein A-I (apoA-I) amyloidosis (AApoAI) is a life-threatening incurable genetic disorder whose molecular underpinnings and the full spectrum of afflicted organs are unclear. We report a new form of AApoAI with amyloid deposition in multiple organs, including an unprecedented retinal amyloidosis. Genetic and proteomic analyses identified Glu34Lys apoA-I as the fibrillar protein causing the clinical manifestations. A life-saving combined hepatorenal transplantation was performed for one Glu34Lys carrier. To elucidate structural underpinnings for amyloidogenic properties of Glu34Lys, we generated its recombinant globular domain and compared the conformation and dynamics of its lipid-free form with those of two other naturally occurring apoA-I variants, Phe71Tyr (amyloidogenic) and Leu159Arg (non-amyloidogenic). All variants showed reduced stability and altered aromatic residue packing. Molecular dynamics simulations revealed local helical unfolding and suggested that transient opening of Trp72 induced mutation-dependent structural perturbations in a sensitive region, including the major amyloid hotspot residues 14-22. We posit that a shift from the “closed” to an “open” orientation of Trp72 modulates structural protection of amyloid hotspots, suggesting a previously unknown early step in protein misfolding.

The 99 amino acid C-terminal fragment of Amyloid Precursor Protein APP-C99 (C99) is cleaved by γ-secretase to form Aβ peptide, which plays a critical role in the etiology of Alzheimer's Disease (AD). The structure of C99 consists of a single transmembrane domain flanked by intra and intercellular domains. While the structure of the transmembrane domain has been well characterized, little is known about the structure of the flanking domains and their role in C99 processing by γ-secretase. To gain insight into the structure of full-length C99, REMD simulations were performed for monomeric C99 in model membranes of varying thickness. We find equilibrium ensembles of C99 from simulation agree with experimentally-inferred residue insertion depths and protein backbone chemical shifts. In thin membranes, the transmembrane domain structure is correlated with extra-membrane structural states and the extra-membrane domain structural states become less correlated to each other. Mean and variance of the transmembrane and G37G38 hinge angles are found to increase with thinning membrane. The N-terminus of C99 forms β-strands that may seed aggregation of Aβ on the membrane surface, promoting amyloid formation. In thicker membranes the N-terminus forms α-helices that interact with the nicastrin domain of γ-secretase. The C-terminus of C99 becomes more α-helical as the membrane thickens, forming structures that may be suitable for binding by cytoplasmic proteins, while C-terminal residues essential to cytotoxic function become α-helical as the membrane thins. The heterogeneous but discrete extra-membrane domain states analyzed here open the path to new investigations of the role of C99 structure and membrane in amyloidogenesis. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.

The 99 amino acid C-terminal fragment of Amyloid Precursor Protein APP-C99 (C99) is cleaved by γ-secretase to form Aβ peptide, which plays a critical role in the etiology of Alzheimer’s Disease (AD). The structure of C99 consists of a single transmembrane domain flanked by intra and intercellular domains. While the structure of the transmembrane domain has been well characterized, little is known about the structure of the flanking domains and their role in C99 processing by γ-secretase. To gain insight into the structure of full-length C99, REMD simulations were performed for monomeric C99 in model membranes of varying thickness. We find equilibrium ensembles of C99 from simulation agree with experimentally-inferred residue insertion depths and protein backbone chemical shifts. In thin membranes, the transmembrane domain structure is correlated with extra-membrane structural states and the extra-membrane domain structural states become increasingly decorrelated to each other. Mean and variance of the transmembrane and G(37)G(38) hinge angles are found to increase with thinning membrane. The N-terminus of C99 forms β-strands that may seed aggregation of Aβ on the membrane surface, promoting amyloid formation. The N-terminus, which forms α-helices that interact with the nicastrin domain of γ-secretase. The C-terminus of C99 becomes more α-helical as the membrane thickens, forming structures that may be suitable for binding by cytoplasmic proteins, while C-terminal residues essential to cytotoxic function become α-helical as the membrane thins. The heterogeneous but discrete extra-membrane domain states analyzed here open the path to new investigations of the role of C99 structure and membrane in amyloidogenesis.

Novel adjustments are introduced to the docking algorithm, DOCK/PIERR, for the purpose of predicting structures of transmembrane protein complexes. Incorporating knowledge about the membrane environment is shown to significantly improve docking accuracy. The extended version of DOCK/PIERR is shown to perform comparably to other leading docking packages. This membrane version of DOCK/PIERR is applied to the prediction of coiled-coil homodimer structures of the transmembrane region of the C-terminal peptide of amyloid precursor protein (C99). Results from MD simulation of the C99 homodimer in POPC bilayer and docking are compared. Docking results are found to capture key aspects of the homodimer ensemble, including the existence of three topologically distinct conformers. Furthermore, the extended version of DOCK/PIERR is successful in capturing the effects of solvation in membrane and micelle. Specifically, DOCK/PIERR reproduces essential differences in the homodimer ensembles simulated in POPC bilayer and DPC micelle, where configurational entropy and surface curvature effects bias the handedness and topology of the homodimer ensemble.

Oligomeric forms of the amyloid-β (Aβ) peptide are thought to represent the primary synaptotoxic species underlying the neurodegenerative changes seen in Alzheimer's disease. It has been proposed that the cellular prion protein (PrPC) functions as a cell-surface receptor, which binds to Aβ oligomers and transduces their toxic effects. However, the molecular details of the PrPC–Aβ interaction remain uncertain. Here, we investigated the effect of PrPC on polymerization of Aβ under rigorously controlled conditions in which Aβ converts from a monomeric to a fibrillar state via a series of kinetically defined steps. We demonstrated that PrPC specifically inhibited elongation of Aβ fibrils, most likely by binding to the ends of growing fibrils. Surprisingly, this inhibitory effect required the globular C-terminal domain of PrPC, which has not been previously implicated in interactions with Aβ. Our results suggest that PrPC recognizes structural features common to both Aβ oligomers and fibril ends and that this interaction could contribute to the neurotoxic effect of Aβ aggregates. Additionally, our results identify the C terminus of PrPC as a new and potentially more druggable molecular target for treating Alzheimer's disease.

The propensity of peptides to form α-helices has been intensely studied using theory, computation, and experiment. Important model peptides for the study of the coil-to-helix transition have been alanine-lysine (AKA) peptides in which the lysine residues are placed on opposite sides of the helix avoiding charge repulsion while enhancing solubility. In this study, the effects of capped versus zwitterionic peptide termini on the secondary structure of alanine-rich peptides in reverse micelles are explored. The reverse micelles are found to undergo substantial shape fluctuations, a property observed in previous studies of AOT reverse micelles in the absence of solvated peptide. The peptides are observed to interact with water, as well as the AOT surfactant, including interactions between the nonpolar residues and the aliphatic surfactant tails. Computation of IR spectra for the amide I band of the peptide allows for direct comparison with experimental spectra. The results demonstrate that capped AKA2 peptides form more stable α helices than zwitterionic AKA2 peptides in reverse micelles. The rotational anisotropy decay of water is found to be distinctly different in the presence or absence of peptide within the reverse micelle, suggesting that the introduction of peptide significantly alters the number of free waters within the reverse micelle nanopool. However, neither the nature of the peptide termini (capped or charged) nor the degree of peptide helicity is found to significantly alter the balance of interactions between the peptides and the environment. Observed changes in the degree of helicity in AKA2 peptides in bulk solution and in reverse micelle environments result from changes in peptide confinement and hydration as well as direct nonpolar and polar interactions with the water-surfactant interface.

The 99 amino acid C-terminal fragment of amyloid precursor protein (C99), consisting of a single transmembrane (TM) helix, is known to form homodimers. Homodimers can be processed by γ-secretase to produce amyloid-β (Aβ) protein, which is implicated in Alzheimer's disease (AD). While knowledge of the structure of C99 homodimers is of great importance, experimental NMR studies and simulations have produced varying structural models, including right-handed and left-handed coiled-coils. In order to investigate the structure of this critical protein complex, simulations of the C99(15-55) homodimer in POPC membrane bilayer and DPC surfactant micelle environments were performed using a multiscale approach that blends atomistic and coarse-grained models. The C99(15-55) homodimer adopts a dominant right-handed coiled-coil topology consisting of three characteristic structural states in a bilayer, only one of which is dominant in the micelle. Our structural study, which provides a self-consistent framework for understanding a number of experiments, shows that the energy landscape of the C99 homodimer supports a variety of slowly interconverting structural states. The relative importance of any given state can be modulated through environmental selection realized by altering the membrane or micelle characteristics.

New sets of parameters (maps) for calculating amide I vibrational spectra for proteins through a vibrational exciton model are proposed. The maps are calculated as a function of electric field and van der Waals forces on the atoms of peptide bonds, taking into account the full interaction between peptide bonds and the surrounding environment. The maps are designed to be employed using data obtained from standard all-atom molecular simulations without any additional constraints on the system. Six proteins representing a wide range of sizes and secondary structure complexity were chosen as a test set. Spectra calculated for these proteins reproduce experimental data both qualitatively and quantitatively. The proposed maps lead to spectra that capture the weak second peak observed in proteins containing β-sheets, allowing for clear distinction between α-helical and β-sheet proteins. While the parametrization is specific to the CHARMM force field, the methodology presented can be readily applied to any empirical force field.

The amyloid β (Aβ) peptide associated with Alzheimer’s Disease results from processing of the amyloid precursor protein (APP) by secretases. Following cleavage of APP by β-secretase, a 99 amino acid C-terminal fragment of APP (C99) is produced consisting of a single transmembrane (TM) helix. Simulation studies of C99 congeners and structural studies of C99 in surfactant micelles and lipid vesicles have shown that a key peptide structural motif is a prominent “GG kink,” centered at two glycines and dividing the TM helix. The flexibility of the GG kink is important in the processing of C99 by γ-secretase. We performed multiscale simulations of C9915–55 in a DPC surfactant micelle and POPC lipid bilayer in order to elucidate the role of membrane surface curvature in modulating the peptide structure. C9915–55 in a DPC surfactant micelle possesses a “GG kink” in the TM domain near the dynamic hinge located at G37/G38, while such a kink is not observed in C9915–55 in a POPC lipid bilayer. Intramolecular interaction between the extracellular and TM domains of C9915–55 is enhanced in the micelle environment, influencing helical stability, TM helix extension, exposure to water, and depth of insertion in the lipophilic region. Our results point to critical differences in C9915–55 structures in micelle and bilayer environments, and show that the fluctuations of the structural ensemble of APP are strongly influenced by membrane surface curvature.

The separation of lipid mixtures into thermodynamically stable phase-separated domains is dependent on lipid composition, temperature, and system size. Using molecular dynamics simulations, the line tension between thermodynamically stable lipid domains formed from ternary mixtures of di-C16:0 PC:di-C18:2 PC:cholesterol at 40:40:20 mol. % ratio was investigated via two theoretical approaches. The line tension was found to be 3.1 ± 0.2 pN by capillary wave theory and 4.7 ± 3.7 pN by pressure tensor anisotropy approaches for coarse-grained models based on the Martini force field. Using an all-atom model of the lipid membrane based on the CHARMM36 force field, the line tension was found to be 3.6 ± 0.9 pN using capillary wave theory and 1.8 ± 2.2 pN using pressure anisotropy approaches. The discrepancy between estimates of the line tension based on capillary wave theory and pressure tensor anisotropy methods is discussed. Inclusion of protein in Martini membrane lipid mixtures was found to reduce the line tension by 25%–35% as calculated by the capillary wave theory approach. To further understand and predict the behavior of proteins in phase-separated membranes, we have formulated an analytical Flory-Huggins model and parameterized it against the simulation results. Taken together these results suggest a general role for proteins in reducing the thermodynamic cost associated with domain formation in lipid mixtures and quantifies the thermodynamic driving force promoting the association of proteins to domain interfaces.

Recent NMR chemical shift measurements of the 99 residue C-terminal fragment of amyloid precursor protein (APP-C99) in the presence of cholesterol provide evidence of binary complex formation between C99 and cholesterol in membrane mimetic environments. It has also been observed that the production of Aβ protein is enhanced under conditions of high cholesterol concentration. In this study, we investigated the impact of the charge state of C99 on the structure and stability of the C99-cholesterol complex. We observed that the binding of C99 to cholesterol depends critically on the charge state of Glu 693 (E22) and Asp 694 (D23). Evaluation of the pKa values of the Asp and Glu side chains suggests that these residues may be predominantly neutral in existing experimental observations of a stable C99-cholesterol complex at lower pH (characteristic of the endosomal environment), while binding is destabilized near neutral pH (characteristic of the cytoplasm). These observations suggest that specific binding of cholesterol to C99 is a sensitive function of the pH encountered in vivo, with key E22 and D23 residues serving as a "pH switch" controlling C99-cholesterol binding.