Unraveling the Role of Hydrogen Bonds in Thrombin via Two Machine Learning Methods.

Hydrogen bonds play a critical role in the folding and stability of proteins, such as proteins and nucleic acids, by providing strong and directional interactions. They help to maintain the secondary and 3D structure of proteins, and structural changes in these molecules often result from the formation or breaking of hydrogen bonds. To gain insights into these hydrogen bonding networks, we applied two machine learning models - a logistic regression model and a decision tree model - to study four variants of thrombin: wild-type, ΔK9, E8K, and R4A. Our results showed that both models have their unique advantages. The logistic regression model highlighted potential key residues (GLU295) in thrombin's allosteric pathways, while the decision tree model identified important hydrogen bonding motifs. This information can aid in understanding the mechanisms of folding in proteins and has potential applications in drug design and other therapies. The use of these two models highlights their usefulness in studying hydrogen bonding networks in proteins.

Impact of A2T and D23N mutations on C99 homodimer conformations.

The proteolytic cleavage of C99 by γ-secretase is the last step in the production of amyloid-ß (Aß) peptides. Previous studies have shown that membrane lipid composition, cholesterol concentration, and mutation in the transmembrane helix modified the structures and fluctuations of C99. In this study, we performed atomistic molecular dynamics simulations of the homodimer of the 55-residue congener of the C-terminal domain of the amyloid protein precursor, C99(1-55), in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine-cholesterol lipid bilayer and compared the conformational ensemble of wild-type (WT) sequence to those of the A2T and D23N variants. These mutations are particularly interesting as the protective Alzheimer's disease (AD) A2T mutation is known to decrease Aß production, whereas the early onset AD D23N mutation does not affect Aß production. We found noticeable differences in the structural ensembles of the three sequences. In particular, A2T varies from both WT and D23N by having long-range effects on the population of the extracellular juxtamembrane helix, the interface between the G29xxx-G33xxx-G37 motifs, and the fluctuations of the transmembrane helical topologies.

Light Chain Mutation Effects on the Dynamics of Thrombin.

Thrombin plays an important role in the process of hemostasis and blood coagulation. Studies in thrombin can help us find ways to treat cancer because thrombin is able to reduce the characteristic hypercoagulability of cancer. Thrombin is composed of two chains, the light chain and the heavy chain. The function of the heavy chain has been largely explored, while the function of the light chain was obscured until several disease-associated mutations in the light chain come to light. In this study, we want to explore the dynamic and conformation effects of mutations on the light chain further to determine possible associations between mutation, conformational changes, and disease. The study, which is a follow-up for our studies on apo thrombin and the mutant, ΔK9, mainly focuses on the mutants E8K and R4A. E8K is a disease-associated mutation, and R4A is used to study the role of Arg4, which is suggested experimentally to play a critical role for thrombin's catalytic activities. We performed five all-atom one microsecond-scale molecular dynamics (MD) simulations for both E8K and R4A, and quantified the changes in the conformational ensemble of the mutants. From the root-mean-square fluctuations (RMSF) for the α-carbons, we find that the atomic fluctuations change in the mutants in the 60s loop and γ loop. The correlation coefficients for the α-carbons indicate that the correlation relation for atom-pairs in the protein is also impacted. The clustering analysis and the principal component analysis (PCA) consistently tell us that the catalytic pocket and the regulatory loops are destabilized by the mutations. We also find that there are two binding modes for Na+ by clustering the vector difference between the Na+ ions and the 220s loop. After further analysis, we find that there is a relation between the Na+ binding and the rigidification of the γ loop, which may shed light on the mysterious role of the γ loop in thrombin.

Na+-binding modes involved in thrombin's allosteric response as revealed by molecular dynamics simulations, correlation networks and Markov modeling.

The monovalent sodium ion (Na+) is a critical modulator of thrombin. However, the mechanism of thrombin's activation by Na+ has been widely debated for more than twenty years. Details of the linkage between thrombin and Na+ remain vague due to limited temporal and spatial resolution in experiments. In this work, we combine microsecond scale atomic-detailed molecular dynamics simulations with correlation network analyses and hidden Markov modeling to probe the detailed thermodynamic and kinetic picture of Na+-binding events and their resulting allosteric responses in thrombin. We reveal that ASP189 and ALA190 comprise a stable Na+-binding site (referred as "inner" Na+-binding site) along with the previously known one (referred as "outer" Na+-binding site). The corresponding newly identified Na+-binding mode introduces significant allosteric responses in thrombin's regulatory regions by stabilizing selected torsion angles of residues responsive to Na+-binding. Our Markov model indicates that the bound Na+ prefers to transfer between the two Na+-binding sites when an unbinding event takes place. These results suggest a testable hypothesis of a substrate-driven Na+ migration (ΔG ∼ 1.7 kcal mol-1) from the "inner" Na+-binding site to the "outer" one during thrombin's catalytic activities. The binding of a Na+ ion at the "inner" Na+-binding site should be inferred as a prerequisite for thrombin's efficient recognition to the substrate, which opens a new angle for our understanding of Na+-binding's allosteric activation on thrombin and sheds light on detailed processes in thrombin's activation.

Influence of electric field on the amyloid-ß(29-42) peptides embedded in a membrane bilayer.

Alzheimer's disease is linked to various types of aggregates of amyloid-ß (Aß) peptide and their interactions with protein receptors and neuronal cell membranes. Little is known on the impact of the electric field on membrane-embedded Aß. Here we use atomistic molecular dynamics simulations to study the effects of a constant electric field on the conformations of Aß29-42 dimer inside a membrane, where the electric field has a strength of 20 mV/nm which exists across the membrane of a human neuron. Starting from α-helix peptides, the transmembrane electric field (TMEF) accelerates the conversion from the Gly-out substate to the Gly-side and Gly-in substates. Starting from ß-sheet peptides, TMEF induces changes of the kink and tilt angles at Gly33 and Gly37. Overall, in the simulations totaling 10 µs, TMEF establishes new ground states for the dimer, similar to induced-fit in ligand binding. Our findings indicate that TMEF can stabilize rare conformations of amyloid peptides, and this could influence the cleavage of the amyloid precursor protein and the formation of ß-sheet oligomers in membrane bilayers.

Binding Site Configurations Probe the Structure and Dynamics of the Zinc Finger of NEMO (NF-κB Essential Modulator).

Zinc-finger proteins are regulators of critical signaling pathways for various cellular functions, including apoptosis and oncogenesis. Here, we investigate how binding site protonation states and zinc coordination influence protein structure, dynamics, and ultimately function, as these pivotal regulatory proteins are increasingly important for protein engineering and therapeutic discovery. To better understand the thermodynamics and dynamics of the zinc finger of NEMO (NF-κB essential modulator), as well as the role of zinc, we present results of 20 µs molecular dynamics trajectories, 5 µs for each of four active site configurations. Consistent with experimental evidence, the zinc ion is essential for mechanical stabilization of the functional, folded conformation. Hydrogen bond motifs are unique for deprotonated configurations yet overlap in protonated cases. Correlated motions and principal component analysis corroborate the similarity of the protonated configurations and highlight unique relationships of the zinc-bound configuration. We hypothesize a potential mechanism for zinc binding from results of the thiol configurations. The deprotonated, zinc-bound configuration alone predominantly maintains its tertiary structure throughout all 5 µs and alludes rare conformations potentially important for (im)proper zinc-finger-related protein-protein or protein-DNA interactions.

Mechanistic insights into thrombin's switch between "slow" and "fast" forms.

Thrombin is a multifunctional enzyme that plays an important role in blood coagulation, cell growth, and metastasis. Depending upon the binding of sodium ions, thrombin presents significantly different enzymatic activities. In the environment with sodium ions, thrombin is highly active in cleaving the coagulated substrates and this is referred to as the "fast" form; in the environment without sodium ions, thrombin turns catalytically less active and is in the "slow" form. Although many experimental studies over the last two decades have attempted to reveal the structural and kinetic differences between these two forms, it remains vague and disputed how the functional switch between the "fast" and "slow" forms is mediated by Na+ cations. In this work, we employ microsecond-scale all-atom molecular dynamics simulations to investigate the differences in the structural ensembles in sodium-bound/unbound and potassium-bound/unbound thrombin. Our calculations indicate that the regulatory regions, including the 60s, γ loops, and exosite I and II, are primarily affected by both the bound and unbound cations. Conformational free energy surfaces, estimated from principal component analysis, further reveal the existence of multiple conformational states. The binding of a cation introduces changes in the distribution of these states. Through comparisons with potassium-binding, the binding of sodium ions appears to shift the population toward conformational states that might be catalytically favorable. Our study of thrombin in the presence of sodium/potassium ions suggests Na+-mediated generalized allostery is the mechanism of thrombin's functional switch between the "fast" and "slow" forms.

All-atom MD indicates ion-dependent behavior of therapeutic DNA polymer.

Understanding the efficacy of and creating delivery mechanisms for therapeutic nucleic acids requires understanding structural and kinetic properties which allow these polymers to promote the death of cancerous cells. One molecule of interest is a 10 mer of FdUMP (5-fluoro-2'-deoxyuridine-5'-O-monophosphate) - also called F10. Here we investigate the structural and kinetic behavior of F10 in intracellular and extracellular solvent conditions along with non-biological conditions that may be efficacious in in vitro preparations of F10 delivery systems. From our all-atom molecular dynamics simulations totaling 80 microseconds, we predict that F10's phosphate groups form close-range interactions with calcium and zinc ions, with calcium having the highest affinity of the five ions investigated. We also predict that F10's interactions with magnesium, potassium and sodium are almost exclusively long-range interactions. In terms of intramolecular interactions, we find that F10 is least structured (in terms of hydrogen bonds among bases) in the 150 mM NaCl (extracellular-like solvent conditions) and most structured in 150 mM ZnCl2. Kinetically, we see that F10 is unstable in the presence of magnesium, sodium or potassium, finding stable kinetic traps in the presence of calcium or zinc.

Small static electric field strength promotes aggregation-prone structures in amyloid-ß(29-42).

The formation of senile plaques in central neural system resulting from the aggregation of the amyloid ß (Aß) of 40 and 42 residues is one of the two hallmarks of Alzheimer's disease. Numerous experiments and computational studies have shown that the aggregation of Aß peptides in vitro is very complex and depends on many factors such as pH, agitation, temperature, and peptide concentration. The impact of a static electric field (EF) on amyloid peptide aggregation has been much less studied, although EFs may have some applications to treat Parkinson's disease symptoms. Here, we study the influence of an EF strength of 20 mV/nm, present in the human brains, on the conformation of the Aß29-42 dimer. Our 7 µs non-equilibrium atomistic simulations in aqueous solution show that this field-strength promotes substantially the formation of ß-hairpins, believed to be a very important intermediate state during aggregation. This work also suggests that structural biology experiments conducted under appropriate EF strengths may help reduce the conformational heterogeneity of Aß1-40/Aß1-42 dimers and provide significant insights into their structures that may be disease-causing.

Site-specific DNA-doxorubicin conjugates display enhanced cytotoxicity to breast cancer cells.

Doxorubicin (Dox) is widely used for breast cancer treatment but causes serious side effects including cardiotoxicity that may adversely impact patient lifespan even if treatment is successful. Herein, we describe selective conjugation of Dox to a single site in a DNA hairpin resulting in a highly stable complex that enables Dox to be used more effectively. Selective conjugation of Dox to G15 in the hairpin loop was verified using site-specific labeling with [2-(15)N]-2'-deoxyguanosine in conjunction with [(1)H-(15)N] 2D NMR, while 1:1 stoichiometry for the conjugate was validated by ESI-QTOF mass spectrometry and UV spectroscopy. Molecular modeling indicated covalently bound Dox also intercalated into the stem of the hairpin and stability studies demonstrated the resulting Dox-conjugated hairpin (DCH) complex had a half-life >30 h, considerably longer than alternative covalent and noncovalent complexes. Secondary conjugation of DCH with folic acid (FA) resulted in increased internalization into breast cancer cells. The dual conjugate, DCH-FA, can be used for safer and more effective chemotherapy with Dox and this conjugation strategy can be expanded to include additional anticancer drugs.

Allosteric Modulation of Thrombin by Thrombomodulin: Insights from Logistic Regression and Statistical Analysis of Molecular Dynamics Simulations.

Thrombomodulin (TM), a transmembrane receptor integral to the anticoagulant pathway, governs thrombin's substrate specificity via interaction with thrombin's anion-binding exosite I. Despite its established role, the precise mechanisms underlying this regulatory function are yet to be fully unraveled. In this study, we deepen the understanding of these mechanisms through eight independent 1 µs all-atom simulations, analyzing thrombin both in its free form and when bound to TM fragments TM456 and TM56. Our investigations revealed distinct and significant conformational changes in thrombin mediated by the binding of TM56 and TM456. While TM56 predominantly influences motions within exosite I, TM456 orchestrates coordinated alterations across various loop regions, thereby unveiling a multifaceted modulatory role that extends beyond that of TM56. A highlight of our study is the identification of critical hydrogen bonds that undergo transformations during TM56 and TM456 binding, shedding light on the pivotal allosteric influence exerted by TM4 on thrombin's structural dynamics. This work offers a nuanced appreciation of TM's regulatory role in blood coagulation, paving the way for innovative approaches in the development of anticoagulant therapies and expanding the horizons in oncology therapeutics through a deeper understanding of molecular interactions in the coagulation pathway.

Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif.

We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.

Thrombin - A Molecular Dynamics Perspective.

Thrombin is a crucial enzyme involved in blood coagulation, essential for maintaining circulatory system integrity and preventing excessive bleeding. However, thrombin is also implicated in pathological conditions such as thrombosis and cancer. Despite the application of various experimental techniques, including X-ray crystallography, NMR spectroscopy, and HDXMS, none of these methods can precisely detect thrombin's dynamics and conformational ensembles at high spatial and temporal resolution. Fortunately, molecular dynamics (MD) simulation, a computational technique that allows the investigation of molecular functions and dynamics in atomic detail, can be used to explore thrombin behavior. This review summarizes recent MD simulation studies on thrombin and its interactions with other biomolecules. Specifically, the 17 studies discussed here provide insights into thrombin's switch between 'slow' and 'fast' forms, active and inactive forms, the role of Na+ binding, the effects of light chain mutation, and thrombin's interactions with other biomolecules. The findings of these studies have significant implications for developing new therapies for thrombosis and cancer. By understanding thrombin's complex behavior, researchers can design more effective drugs and treatments that target thrombin.

Electrostatics of cysteine residues in proteins: parameterization and validation of a simple model.

One of the most popular and simple models for the calculation of pK(a) s from a protein structure is the semi-macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pK(a) s. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pK(a) s; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pK(a) s. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pK(a) values (where the calculation should reproduce the pK(a) within experimental error). Both the general behavior of cysteines in proteins and the perturbed pK(a) in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pK(a) should be shifted, and validation of force field parameters for cysteine residues.

Simulations suggest double sodium binding induces unexpected conformational changes in thrombin.

Thrombin is a Na[Formula: see text]-activated serine protease existing in two forms targeted to procoagulant and anticoagulant activities, respectively. There is one Na[Formula: see text]-binding site that has been the focus of the study of the thrombin. However, molecular dynamics (MD) simulations suggest that there might be actually two Na[Formula: see text]-binding sites in thrombin and that Na[Formula: see text] ions can even bind to two sites simultaneously. In this study, we performed 12 independent 2-µs all-atom MD simulations for the wild-type (WT) thrombin and we studied the effects of the different Na[Formula: see text] binding modes on thrombin. From the root-mean-square fluctuations (RMSF) for the [Formula: see text]-carbons, we see that the atomic fluctuations mainly change in the 60s, 170s, and 220s loops, and the connection (residue 167 to 170). The correlation matrices for different binding modes suggest regions that may play an important role in thrombin's allosteric response and provide us a possible allosteric pathway for the sodium binding. Amorim-Hennig (AH) clustering tells us how the structure of the regions of interest changes on sodium binding. Principal component analysis (PCA) shows us how the different regions of thrombin change conformation together with sodium binding. Solvent-accessible surface area (SASA) exposes the conformational change in exosite I and catalytic triad. Finally, we argue that the double binding mode might be an inactive mode and that the kinetic scheme for the Na[Formula: see text] binding to thrombin might be a multiple-step mechanism rather than a 2-step mechanism.

Compound C Inhibits Ovarian Cancer Progression via PI3K-AKT-mTOR-NFκB Pathway.

Epithelial Ovarian cancer (OvCa) is the leading cause of death from gynecologic malignancies in the United States, with most patients diagnosed at late stages. High-grade serous cancer (HGSC) is the most common and lethal subtype. Despite aggressive surgical debulking and chemotherapy, recurrence of chemo-resistant disease occurs in ~80% of patients. Thus, developing therapeutics that not only targets OvCa cell survival, but also target their interactions within their unique peritoneal tumor microenvironment (TME) is warranted. Herein, we report therapeutic efficacy of compound C (also known as dorsomorphin) with a novel mechanism of action in OvCa. We found that CC not only inhibited OvCa growth and invasiveness, but also blunted their reciprocal crosstalk with macrophages, and mesothelial cells. Mechanistic studies indicated that compound C exerts its effects on OvCa cells through inhibition of PI3K-AKT-NFκB pathways, whereas in macrophages and mesothelial cells, CC inhibited cancer-cell-induced canonical NFκB activation. We further validated the specificity of the PI3K-AKT-NFκB as targets of compound C by overexpression of constitutively active subunits as well as computational modeling. In addition, real-time monitoring of OvCa cellular bioenergetics revealed that compound C inhibits ATP production, mitochondrial respiration, and non-mitochondrial oxygen consumption. Importantly, compound C significantly decreased tumor burden of OvCa xenografts in nude mice and increased their sensitivity to cisplatin-treatment. Moreover, compound C re-sensitized patient-derived resistant cells to cisplatin. Together, our findings highlight compound C as a potent multi-faceted therapeutic in OvCa.

TREX1 as a Novel Immunotherapeutic Target.

Mutations in the TREX1 3' → 5' exonuclease are associated with a spectrum of autoimmune disease phenotypes in humans and mice. Failure to degrade DNA activates the cGAS-STING DNA-sensing pathway signaling a type-I interferon (IFN) response that ultimately drives immune system activation. TREX1 and the cGAS-STING DNA-sensing pathway have also been implicated in the tumor microenvironment, where TREX1 is proposed to degrade tumor-derived DNA that would otherwise activate cGAS-STING. If tumor-derived DNA were not degraded, the cGAS-STING pathway would be activated to promote IFN-dependent antitumor immunity. Thus, we hypothesize TREX1 exonuclease inhibition as a novel immunotherapeutic strategy. We present data demonstrating antitumor immunity in the TREX1 D18N mouse model and discuss theory surrounding the best strategy for TREX1 inhibition. Potential complications of TREX1 inhibition as a therapeutic strategy are also discussed.

Small molecule induction of MSH2-dependent cell death suggests a vital role of mismatch repair proteins in cell death.

Avoidance of apoptosis is one of the hallmarks of cancer development and progression. Chemotherapeutic agents aim to initiate an apoptotic response, but often fail due to dysregulation. MSH proteins are capable of recognizing cisplatin damage in DNA and participate in the initiation of cell death. We have exploited this recognition and computationally simulated a MutS homolog (MSH) "death conformation". Screening and docking experiments based on this model determined that the MSH2-dependent cell-death pathway can be induced by a small molecule without DNA damage, reserpine. Reserpine was identified via virtual screening on structures obtained from molecular dynamics as a small molecule that selectively binds a protein "death" conformation. The virtual screening predicts that this small molecule binds in the absence of DNA. Cell biology confirmed that reserpine triggers the MSH2-dependent cell-death pathway. This result supports the hypothesis that the MSH2-dependent pathway is initiated by specific protein conformational changes triggered by binding to either DNA damage or small compound molecules. These findings have multiple implications for drug discovery and cell biology. Computational modeling may be used to identify and eventually design small molecules that selectively activate particular pathways through conformational control. Molecular dynamics simulations can be used to model the biologically relevant conformations and virtual screening can then be used to select for small molecules that bind specific conformations. The ability of a small molecule to induce the cell-death pathway suggests a broader role for MMR proteins in cellular events, such as cell-death pathways, than previously suspected.

Probing light chain mutation effects on thrombin via molecular dynamics simulations and machine learning.

Thrombin is a key component for chemotherapeutic and antithrombotic therapy development. As the physiologic and pathologic roles of the light chain still remain vague, here, we continue previous efforts to understand the impacts of the disease-associated single deletion of LYS9 in the light chain. By combining supervised and unsupervised machine learning methodologies and more traditional structural analyses on data from 10 µs molecular dynamics simulations, we show that the conformational ensemble of the ΔK9 mutant is significantly perturbed. Our analyses consistently indicate that LYS9 deletion destabilizes both the catalytic cleft and regulatory functional regions and result in some conformational changes that occur in tens to hundreds of nanosecond scaled motions. We also reveal that the two forms of thrombin each prefer a distinct binding mode of a Na+ ion. We expand our understanding of previous experimental observations and shed light on the mechanisms of the LYS9 deletion associated bleeding disorder by providing consistent but more quantitative and detailed structural analyses than early studies in literature. With a novel application of supervised learning, i.e. the decision tree learning on the hydrogen bonding features in the wild-type and ΔK9 mutant forms of thrombin, we predict that seven pairs of critical hydrogen bonding interactions are significant for establishing distinct behaviors of wild-type thrombin and its ΔK9 mutant form. Our calculations indicate the LYS9 in the light chain has both localized and long-range allosteric effects on thrombin, supporting the opinion that light chain has an important role as an allosteric effector.

Amyloid-ß(29-42) Dimeric Conformations in Membranes Rich in Omega-3 and Omega-6 Polyunsaturated Fatty Acids.

The omega-3 and omega-6 polyunsaturated fatty acids are two important components of cell membranes in human brains. When incorporated into phospholipids, omega-3 slows the progression of Alzheimer's disease (AD), whereas omega-6 is linked to increased risk of AD. Little is known on the amyloid-ß (Aß) conformations in membranes rich in omega-3 and omega-6 phospholipids. Herein, the structural properties of the Aß29-42 dimer embedded in both fatty acid membranes were comparatively studied to a 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) bilayer using all-atom molecular dynamics (MD) simulations. Starting from α-helix, both omega-6 and omega-3 membranes promote new orientations and conformations of the dimer, in agreement with the observed dependence of Aß production upon addition of these two fatty acids. This conformational result is corroborated by atomistic MD simulations of the dimer of the 99 amino acid C-terminal fragment of amyloid precursor protein spanning the residues 15-55. Starting from ß-sheet, omega-6 membrane promotes helical and disordered structures of Aß29-42 dimer, whereas omega-3 membrane preserves the ß-sheet structures differing however from those observed in POPC. Remarkably, the mixture of the two fatty acids and POPC depicts another conformational ensemble of the Aß29-42 dimer. This finding demonstrates that variation in the abundance of the molecular phospholipids, which changes with age, modulates membrane-embedded Aß oligomerization.