Neutron scattering has significant benefits for examining the structure of protein-lipid complexes. Cold neutrons are non-damaging and predominantly interact with the atomic nucleus, meaning that neutron beams can penetrate deeply into samples, which allows for flexibility in the design of samples studied. Components within a complex can be individually resolved by leveraging the strong difference in neutron scattering length between protium ( H 1 , 99.984% natural abundance) and deuterium ( H 2 or D, 0.016%) namely through the mixing of H2O and D2O in the samples or by the deuterium labelling of the biomolecules. Thus, the relative distribution of components within a membrane can be elucidated. Using neutron scattering techniques lipid-protein complexes are most commonly studied using neutron reflectometry (NR) and small-angle neutron scattering (SANS). In this review, the methodologies to produce and examine a variety of model biological membrane systems using SANS and NR are detailed. These systems include supported lipid bilayers derived from vesicle dispersions or Langmuir-Blodgett deposition, tethered and floating bilayer systems, membrane protein-lipid complexes, and polymer wrapped lipid nanodiscs. The three key stages of any SANS/NR study on model membrane systems-sample preparation, data collection, and analysis-are described together with some background on the techniques themselves.

Abstract Cell-membrane signaling and trafficking rely on proteins that associate with lipid bilayers through dynamic, low-affinity interactions. Defining how these proteins dock onto membrane surfaces is therefore essential for understanding their function. While Neutron Reflectometry (NR) combined with molecular dynamics (MD) simulations is frequently used, complementary approaches that do not require access to large-scale neutron facilities are needed. Here, we establish membrane Paramagnetic Relaxation Enhancement (mPRE) Nuclear Magnetic Resonance (NMR), combined with nanodiscs as membrane mimics and optimized acquisition strategies, as an accessible solution for extracting membrane–protein distance constraints even for weakly bound systems. Using the PI(4,5)P₂-binding ASAP1 Pleckstrin Homology (PH) domain as a model, we show that both conventional mPRE and a new dynamic-exchange mPRE (EX-mPRE) method reproduce the membrane orientation obtained by NR. In addition, we show that increasing PI(4,5)P₂ levels to mimic nanoscale membrane clustering broadened the orientational distribution of ASAP1 PH. EX-mPRE, which transfers PREs from transient bound states to the observable free state, further enables studies of temperature-sensitive or rapidly exchanging membrane interactions. Together, these results provide the formalism and establish mPRE and EX-mPRE NMR as a powerful alternative for resolving the membrane orientation of peripheral proteins and for probing how lipid composition affects their behavior.

Abstract Antimicrobial resistance poses a major global health challenge, necessitating efficient strategies to discover potent antimicrobial peptides (AMPs). While recent generative models can produce many candidate sequences, experimentally validating all generated peptides in wet labs is impractical due to the high costs and time involved in such measurements. As a result, there is a strong demand for accurate predictions of peptide efficacy, typically measured as the minimum inhibitory concentration (MIC). We introduce STAMP, a framework for Species- and Topic-aware Representation Learning in AMP Discovery. This unified machine learning framework allows for cross-species predictions of AMP activity. STAMP integrates protein language model embeddings with species conditioning and topic-aware representations that capture sequence-level patterns, enabling generalizable predictions across multiple bacterial species within a single model. We evaluated STAMP on three benchmark datasets, which include two previously published datasets and a newly curated dataset derived from DBAASP, addressing duplicates and inconsistencies systematically. STAMP achieved strong predictive performance across these datasets, demonstrating a Pearson correlation coefficient (PCC) of 0.837 and an R 2 of 0.70, outperforming several baseline models. Importantly, we further validated our prediction model using peptides that were experimentally tested for their antimicrobial activity against E . coli . and S . epidermidis bacteria, demonstrating its real-world applicability. Furthermore, residue-level importance analyses provide insights into the sequence determinants governing antimicrobial activity. Together, these results establish STAMP as a scalable framework for MIC prediction and an effective computational tool for accelerating AMP discovery and optimization.

Membrane-active peptides (MAPs) have garnered significant attention as potential alternatives to conventional cancer therapies, which are frequently limited by severe side effects. Among them, antimicrobial peptides (AMPs) that leverage differences between the plasma membranes of cancer cells and healthy cells are particularly attractive. While several AMPs have demonstrated anticancer potency, structure-function relationship studies are lacking to explain the molecular basis of their selectivity and to help design improved analogs. Here, we contribute to filling this gap by investigating Nile tilapia piscidin 4 (TP4), an AMP with demonstrated activity against several solid organ cancers. First, we discover through biological assays that the anticancer activity of the peptide, which underscores a promising therapeutic window, is associated with increased plasma membrane permeability in cancer cells compared to normal cells and positively (negatively) correlated with enzymes that enrich (deplete) anionic PS in the outer leaflet. Next, we utilize a suite of complementary techniques on model membranes to investigate the interactions of TP4 with membranes, uncovering behaviors not previously observed in related AMPs. Circular dichroism experiments reveal that TP4 preferentially binds to zwitterionic phosphatidylcholine (PC) membranes enriched in anionic PS, while cholesterol markedly impairs binding. X-ray diffraction demonstrates that TP4 disrupts PC-PS membranes by inducing lipid segregation. Covering a range of biologically relevant peptide concentrations with neutron diffraction and reflectometry measurements in fluid bilayers and MD simulations, we unveil how TP4 and associated water gradually insert into the hydrocarbon region and cause convoluted membrane deformations to breach the membrane barriers. These studies highlight the pivotal role of the TP4 polyarginine tail in driving selective membrane binding and disruption on membranes enriched with the anionic lipid PS. Together, our results elucidate the molecular determinants underpinning the selective anticancer effects of TP4, providing a strategic framework for the rational design of advanced membrane-active therapeutics.

Sequence motifs or patterns found in natural antimicrobial peptides (AMPs) have a great impact on their microbicidal activities. Here, through database inquiries and biological assays, we explore the enhanced antibacterial function associated with poly arginine (poly-R) motifs that typically occur as 3-5 concatenated R residues in many natural AMPs. Using a suite of biophysical techniques, we explore the structural consequences of a C-terminal poly-R motif at membranes and correlate our findings with the functional assays. We use natural peptides, such as Tilapia piscidin 4 (TP4), as an example of how various segments in an AMP play separate and synergistic roles to achieve unmatched bactericidal effects. The function of the poly-R segment is highly consequential since the simple addition of a five-arginine (R5) tail to an otherwise inert and weakly binding helical peptide creates a potent AMP. We investigate interactions of AMPs with lipid bilayers mimicking bacterial membrane compositions, including lipopolysaccharides, to show that the poly-R tail has a key role in initiating membrane destabilization through lipid segregation and water sequestration effects, all of which facilitate insertion and translocation of the amphipathic, α-helical N-terminal segment through the membrane. We compiled a large set of natural AMP sequences and MIC values to show that, statistically, the poly-R sequence motifs have, in average, a greater impact on the overall antimicrobial efficacy than equivalent sequences with poly-K motifs and similar charge densities. We discuss our observations in light of the unique structural and hydration properties of arginine residues.

The effect of cholesterol on the bending modulus KC of DOPC lipid bilayers has been controversial. Previous analysis of dynamic neutron spin echo (NSE) data reported that 50% cholesterol increased KC by a factor of three in contrast to earlier studies using four different static methods that reported essentially no increase. We reanalyzed the previous NSE data using new developments in NSE analysis. We find that the same NSE data require non-zero viscosity in pure DOPC and they are consistent with no increases in KC with cholesterol. Instead, we find more than a five-fold increase in the membrane viscosity ηm. We have further added diffusional softening dynamical theory to the basic phenomenological model. This generally decreases the 5-fold increase in viscosity, but the NSE data are not sufficient to determine by how much.

ASAP1 is a multidomain Arf GTPase-activating protein (ArfGAP) that catalyzes GTP hydrolysis on the small GTPase Arf1 and is implicated in cancer progression. The PH domain of ASAP1 enhances its activity greater than 7 orders of magnitude but the underlying mechanisms remain poorly understood. Here, we combined Nuclear Magnetic Resonance (NMR), Molecular Dynamic (MD) simulations and mathematical modeling of functional data to build a comprehensive structural-mechanistic model of the complex of Arf1 and the ASAP1 PH domain on a membrane surface. Our results support a new conceptual model in which the PH domain contributes to efficient catalysis not only by membrane recruitment but by acting as a critical component of the catalytic interface, binding Arf·GTP and allosterically driving it towards the catalytic transition state. We discuss the biological implications of these results and how they may apply more broadly to poorly understood membrane-dependent regulatory mechanisms controlling catalysis of the ArfGAP superfamily as well as other peripheral membrane enzymes.

One promising strategy to combat worldwide antimicrobial resistance involves using cyclic peptides as antibacterial agents. Cyclization of peptides can confer several advantages, including enhanced stability to proteolysis, decreased toxicity and increased bactericidal efficacy. This paper examines two cyclic peptides CE-03 (12 AAs) and CE-05 (16 AAs) and evaluates their effectiveness in combating bacterial infections, their stability and toxicity. We compare them to their linear versions. Circular dichroism (CD) reveals that CE-03 and CE-05 both adopt random coil and β-sheet structures in lipid model membranes (LMMs) mimicking G(-) and G(+) bacteria, where they are both bactericidal. Using X-ray diffuse scattering (XDS), their effects on lipid model membranes show a deep penetration of both peptides into eukaryotic LMMs where they are nontoxic, while a headgroup location in bacterial LMMs correlates with bacterial killing. Neutron reflectometry (NR) confirms the AMP locations determined using XDS. Further, solution small-angle X-ray scattering demonstrates that both peptides induce vesicle fusion in bacterial LMMs without affecting eukaryotic LMMs. Proteolytic degradation studies show that both CE-05 and CE-03 do not lose activity when incubated with the elastase enzyme, while the helical E2-35 AMP becomes inactive upon proteolysis.

Antibiotics have been developed to effectively target and eliminate bacteria, but the rise in antimicrobial resistance (AR) complicates the treatment of certain infections. To address this issue, researchers have explored antimicrobial peptides (AMPs) that disrupt bacterial membranes. A promising method for this exploration is motif-based analysis, which identifies hidden patterns in AMPs to better understand their mechanism of action. While existing methods rely on expert knowledge, incorporating topic models can enhance analysis by revealing the contextual relationships between sequence elements. This is complemented by a data analytics tool designed to analyze AMP motifs and their biochemical properties. Such integration allows for the extraction of valuable motifs and the development of a robust data analytics module for predicting membrane activity. Additionally, we evaluated the biological relevance of motifs by extracting biochemical features, making structural predictions via Evolutionary Scale Modeling (ESM). Our results indicate that topic model-derived motifs are strongly associated with antimicrobial activity and demonstrate lower minimum inhibitory concentration values and capture contextual information more effectively than traditional frequency-based motifs. We also performed a comparative analysis between the two approaches regarding motif evolution, sequence-level attributes, and entropy measures, ultimately contributing to ongoing efforts to combat AR.

As amphiphiles, proteins adsorb at hydrophobic air-liquid interfaces, often forming a viscoelastic film. This surface viscoelasticity is of interest in many fields of science and technology, such as foamability and coalescence in food and emulsion science as well as stability in biopharmaceutical formulations. For example, the long-term stability of monoclonal antibody (mAb) formulations correlates with surface aggregation and interfacial shear elasticity. To investigate this viscoelastic interface, a new interfacial rheometer is used to probe the dilatational and shear rheology of an adsorbed mAb interfacial film as a function of coverage, including "jammed" interfacial states that have not been explored in previous studies. This interfacial film is primarily elastic (solid-like), and the calculated 2D Poisson ratios ν2D decrease from 0.9 to 0.4 with increasing compression. X-ray reflectivity (XRR) resolves the out-of-plane structure, and Brewster angle microscopy (BAM) measurements provide in-plane structural information on the film. BAM images confirm the homogeneity of the film upon compression, while XRR reveals a thin, high-protein concentration region at the air interface and enables accurate determination of the true surface excess. Thus, the scaling of interfacial rheology with the actual, measured surface coverage is determined. Using the Naïve mode-coupling theory, a cage-localization length rloc between 2.5 and 4.0 Å is calculated, further confirming the hypothesis that the high interfacial elasticity is due to the beta-sheet structure forming from localized, partial unfolding of mAb at the interface. These results provide a fundamental understanding of the structure-rheological property of the adsorbed mAb interfacial layer with scientific importance and technological application.

GTPase-activating proteins are important regulators of small GTPases; among these, ASAP1 stimulates GTP hydrolysis on Arf1 and is implicated in cancer progression. ASAP1 contains a Pleckstrin Homology (PH) domain essential for maximum Arf·GTP hydrolysis. The prevailing view of PH domains is that they regulate proteins through passive mechanisms like membrane recruitment. In sharp contrast, we show that the PH domain of ASAP1 actively contributes to Arf1 GTP hydrolysis. By combining NMR, molecular dynamics simulations, kinetic assays, and mutational analysis, we find that the PH domain binds Arf·GTP at the membrane, to establish an active state primed for GTP hydrolysis. We identify key residues on the PH domain and Arf that drive this allosteric mechanism, which mathematical modeling shows contributes as much to GTPase activation as membrane recruitment. The finding that PH domains directly modulate small GTPases has broad implications for the Ras and Rho oncoprotein families.

Monoclonal antibodies (mAbs), due to their amphiphilicity, adsorb strongly at the air-water (a/w) interface, such that interfacial stresses can lead to the formation of visible and subvisible particles in solution that adversely affect the therapeutic efficacy and immunogenicity of protein formulations. Furthermore, prior work in our group shows that the interfacial shear elastic modulus of mAbs adsorbed at the a/w interface correlates strongly with the long-term (3 year) stability in solution, providing a possible means to accelerate the testing of formulations. To mitigate interface-induced instability in formulation and delivery, the biopharmaceutical industry employs excipients, such as nonionic surfactants, to rapidly adsorb to and protect against mAb adsorption at interfaces. However, the molecular interactions at the a/w interface during mechanical deformation are not established despite their technological importance. To address this need, the adsorption of a mAb (studied previously) at the a/w interface in the presence and absence of the nonionic surfactant Poloxamer 188 (P188) is examined through measurements of interfacial pressure and rheology, Brewster angle microscopy (BAM), and neutron reflectometry (NR). We find that mAb adsorbs rapidly to the air-water interface, but the evolution of the surface pressure and interfacial shear moduli continues long after the surface excess has reached a plateau. NR measurements quantify changes in the molecular layer when undergoing dilatational stresses, which may occur during the storage and transport of mAbs. The injection of P188 into the subphase below an established mAb interface increases the surface pressure and decreases interfacial shear moduli, but mAbs remain at the interface with a modified surface topology. Comparisons to adsorption from a premixed mAb-P188 solution highlight the importance of the order of addition in therapeutic formulations, and the results in totality provide molecular evidence for the effect of the a/w interface on mAb interactions and stability.