Quantum Chemical Calculations of Amide-¹⁵N Chemical Shift Anisotropy Tensors for a Membrane-Bound Cytochrome‑<i>b</i>₅

There is considerable interest in determining amide-¹⁵N chemical shift anisotropy (CSA) tensors from biomolecules and understanding their variation for structural and dynamics studies using solution and solid-state NMR spectroscopy and also by quantum chemical calculations. Due to the difficulties associated with the measurement of CSA tensors from membrane proteins, NMR-based structural studies heavily relied on the CSA tensors determined from model systems, typically single crystals of model peptides. In the present study, the principal components of backbone amide-¹⁵N CSA tensors have been determined using density functional theory for a 16.7 kDa membrane-bound paramagnetic heme containing protein, cytochrome-<i>b</i>₅ (cytb₅). All the calculations were performed by taking residues within 5 Å distance from the backbone amide-¹⁵N nucleus of interest. The calculated amide-¹⁵N CSA spans agree less well with our solution NMR data determined for an effective internuclear distance <i>r</i>_N–H = 1.023 Å and a constant angle β = 18° that the least shielded component (δ₁₁) makes with the N–H bond. The variation of amide-¹⁵N CSA span obtained using quantum chemical calculations is found to be smaller than that obtained from solution NMR measurements, whereas the trends of the variations are found to be in close agreement. We believe that the results reported in this study will be useful in studying the structure and dynamics of membrane proteins and heme-containing proteins, and also membrane-bound protein–protein complexes such as cytochromes-b5-P450

Phosphatidylethanolamine Enhances Amyloid Fiber-Dependent Membrane Fragmentation

The toxicity of amyloid-forming peptides has been hypothesized to reside in the ability of protein oligomers to interact with and disrupt the cell membrane. Much of the evidence for this hypothesis comes from in vitro experiments using model membranes. However, the accuracy of this approach depends on the ability of the model membrane to accurately mimic the cell membrane. The effect of membrane composition has been overlooked in many studies of amyloid toxicity in model systems. By combining measurements of membrane binding, membrane permeabilization, and fiber formation, we show that lipids with the phosphatidylethanolamine (PE) headgroup strongly modulate the membrane disruption induced by IAPP (islet amyloid polypeptide protein), an amyloidogenic protein involved in type II diabetes. Our results suggest that PE lipids hamper the interaction of prefibrillar IAPP with membranes but enhance the membrane disruption correlated with the growth of fibers on the membrane surface via a detergent-like mechanism. These findings provide insights into the mechanism of membrane disruption induced by IAPP, suggesting a possible role of PE and other amyloids involved in other pathologies

Detergent-Type Membrane Fragmentation by MSI-78, MSI-367, MSI-594, and MSI-843 Antimicrobial Peptides and Inhibition by Cholesterol: A Solid-State Nuclear Magnetic Resonance Study

Multidrug resistance against the existing antibiotics is becoming a global threat, and any potential drug that can be designed using cationic antimicrobial peptides (AMP) could be an alternate solution to alleviate this existing problem. The mechanism of action of killing bacteria by an AMP differs drastically in comparison to that of small molecule antibiotics. The main target of AMPs is to interact with the lipid bilayer of the cell membrane and disrupt it to kill bacteria. Consequently, the modes of membrane interaction that lead to the selectivity of an AMP are very important to understand. Here, we have used different membrane compositions, such as negatively charged, zwitterionic, or mixed large unilamellar vesicles (LUVs), to study the interaction of four different synthetically designed cationic, linear antimicrobial peptides: MSI-78 (commercially known as pexiganan), MSI-367, MSI-594, and MSI-843. Our solid-state nuclear magnetic resonance (NMR) experiments confirmed that the MSI peptides fragmented LUVs through a detergent-like carpet mechanism depending on the amino acid sequence of the MSI peptide and/or the membrane composition of LUVs. Interestingly, the fragmented lipid aggregates such as SUVs or micelles are sufficiently small to produce an isotropic peak in the ³¹P NMR spectrum. These fragmented lipid aggregates contain only MSI peptides bestowed with lipid molecules as confirmed by NMR in conjunction with circular dichroism spectroscopy. Our results also demonstrate that cholesterol, which is present only in the eukaryotic cell membrane, inhibits the MSI-induced fragmentation of LUVs, suggesting that the MSI peptides can discriminate the bacteria and the eukaryotic cell membranes, and this selectivity could be used for further development of novel antibiotics

pH Tunable and Divalent Metal Ion Tolerant Polymer Lipid Nanodiscs

The development and applications of detergent-free membrane mimetics have been the focus for the high-resolution structural and functional studies on membrane proteins. The introduction of lipid nanodiscs has attracted new attention toward the structural biology of membrane proteins and also enabled biomedical applications. Lipid nanodiscs provide a native lipid bilayer environment similar to the cell membrane surrounded by a belt made up of proteins or peptides. Recent studies have shown that the hydrolyzed form of styrene maleic anhydride copolymer (SMA) has the ability to form lipid nanodiscs and has several advantages over protein or peptide based nanodiscs. SMA polymer lipid nanodiscs have become very important for structural biology and nanobiotechnological applications. However, applications of the presently available polymer nanodiscs are limited by their instability toward divalent metal ions and acidic conditions. To overcome the limitations of SMA nanodiscs and to broaden the potential applications of polymer nanodiscs, the present study investigates the tunability of SMA polymer nanodiscs by systematically modifying the maleic acid functional group. The two newly developed polymers and subsequent lipid nanodiscs were characterized using solid-state NMR, FT-IR, TEM, and DLS experiments. The pH dependence and metal ion stability of these nanodiscs were studied using static light scattering and FTIR. The reported polymer nanodiscs exhibit unique pH dependent stability based on the modified functional group and show a high tolerance toward divalent metal ions. We also show these tunable nanodiscs can be used to encapsulate and stabilize a polyphenolic natural product curcumin

Side-Chain Dynamics Reveals Transient Association of Aβ_1–40 Monomers with Amyloid Fibers

Low-lying excited states that correspond to rare conformations or transiently bound species have been hypothesized to play an important role for amyloid nucleation. Despite their hypothesized importance in amyloid formation, transiently occupied states have proved difficult to detect directly. To experimentally characterize these invisible states, we performed a series of Carr–Purcell–Meiboom–Gill (CPMG)-based relaxation dispersion NMR experiments for the amyloidogenic Aβ_1–40 peptide implicated in Alzheimer’s disease. Significant relaxation dispersion of the resonances corresponding to the side-chain amides of Q15 and N27 was detected before the onset of aggregation. The resonances corresponding to the peptide backbone did not show detectable relaxation dispersion, suggesting an exchange rate that is not within the practical limit of detection. This finding is consistent with the proposed “dock and lock” mechanism based on molecular dynamics simulations in which the Aβ_1–40 monomer transiently binds to the Aβ_1–40 oligomer by non-native contacts with the side chains before being incorporated into the fiber through native contacts with the peptide backbone

Alternative Pathways of Human Islet Amyloid Polypeptide Aggregation Distinguished by ¹⁹F Nuclear Magnetic Resonance-Detected Kinetics of Monomer Consumption

Amyloid formation, a complex process involving many intermediate states, is proposed to be the driving force for amyloid-related toxicity in common degenerative diseases. Unfortunately, the details of this process have been obscured by the limitations in the methods that can follow this reaction in real time. We show that alternative pathways of aggregation can be distinguished by using ¹⁹F nuclear magnetic resonance (NMR) to monitor monomer consumption along with complementary measurements of fibrillogenesis. The utility of this technique is demonstrated by tracking amyloid formation in the diabetes-related islet amyloid polypeptide (IAPP). Using this technique, we show IAPP fibrillizes without an appreciable buildup of nonfibrillar intermediates, in contrast to the well-studied Aβ and α-synuclein proteins. To further develop the usage of ¹⁹F NMR, we have tracked the influence of the polyphenolic amyloid inhibitor epigallocatechin gallate (EGCG) on the aggregation pathway. Polyphenols have been shown to strongly inhibit amyloid formation in many systems. However, spectroscopic measurements of amyloid inhibition by these compounds can be severely compromised by background signals and competitive binding with extrinsic probes. Using ¹⁹F NMR, we show that thioflavin T strongly competes with EGCG for binding sites on IAPP fibers. By comparing the rates of monomer consumption and fiber formation, we are able to show that EGCG stabilizes nonfibrillar large aggregates during fibrillogenesis

Pseudonegative Thermal Expansion and the State of Water in Graphene Oxide Layered Assemblies

Unraveling the complex interplay between thermal properties and hydration is a part of understanding the fundamental properties of many soft materials and very essential for many applications. Here we show that graphene oxide (GO) demonstrates a highly negative thermal expansion (NTE) coefficient owing to unique thermohydration processes related with fast transport of water between the GO sheets, the amphiphilic nature of nanochannels, and close-to-zero intrinsic thermal expansion of GO. The humidity-dependent NTE of GO layered assemblies, or “pseudonegative thermal expansion” (PNTE), differs from that of other hygroscopic materials due to its relatively fast and highly reversible expansion/contraction cycles and occurrence at low humidity levels while bearing similarities to classic NTE. Thermal expansion of polyvinyl alcohol/GO composites is easily tunable with additional intricacy of thermohydration effects. PNTE combined with isotropy, nontoxicity, and mechanical robustness is an asset for applications of actuators, sensors, MEMS devices, and memory materials and crucial for developing methods of thermal/photopatterning of GO devices

Real-Time Monitoring of Lipid Exchange via Fusion of Peptide Based Lipid-Nanodiscs

Real-Time Monitoring of Lipid Exchange via Fusion of Peptide Based Lipid-Nanodisc

Real-Time Monitoring of Lipid Exchange via Fusion of Peptide Based Lipid-Nanodiscs

Real-Time Monitoring of Lipid Exchange via Fusion of Peptide Based Lipid-Nanodisc

Lipid Composition-Dependent Membrane Fragmentation and Pore-Forming Mechanisms of Membrane Disruption by Pexiganan (MSI-78)

The potency and selectivity of many antimicrobial peptides (AMPs) are correlated with their ability to interact with and disrupt the bacterial cell membrane. <i>In vitro</i> experiments using model membranes have been used to determine the mechanism of membrane disruption of AMPs. Because the mechanism of action of an AMP depends on the ability of the model membrane to accurately mimic the cell membrane, it is important to understand the effect of membrane composition. Anionic lipids that are present in the outer membrane of prokaryotes but are less common in eukaryotic membranes are usually thought to be key for the bacterial selectivity of AMPs. We show by fluorescence measurements of peptide-induced membrane permeabilization that the presence of anionic lipids at high concentrations can actually inhibit membrane disruption by the AMP MSI-78 (pexiganan), a representative of a large class of highly cationic AMPs. Paramagnetic quenching studies suggest MSI-78 is in a surface-associated inactive mode in anionic sodium dodecyl sulfate micelles but is in a deeply buried and presumably more active mode in zwitterionic dodecylphosphocholine micelles. Furthermore, a switch in mechanism occurs with lipid composition. Membrane fragmentation with MSI-78 can be observed in mixed vesicles containing both anionic and zwitterionic lipids but not in vesicles composed of a single lipid of either type. These findings suggest membrane affinity and membrane permeabilization are not always correlated, and additional effects that may be more reflective of the actual cellular environment can be seen as the complexity of the model membranes is increased