A Targetable N-Terminal Motif Orchestrates α-Synuclein Oligomer-to-Fibril Conversion.

Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson's disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. Phenol-soluble modulin α3 (PSMα3) is a nanomolar peptide binder of α-synuclein oligomers that inhibits aggregation by blocking oligomer-to-fibril conversion. Here, we investigate the binding of PSMα3 to α-synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSMα3 selectively targets an α-synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the mono- and oligomeric states. This α-synuclein region plays a pivotal role in oligomer-to-fibril conversion as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early onset Parkinson's disease, causes a conformational fluctuation in this region, leading to delayed oligomer-to-fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in α-synuclein oligomers, advance our comprehension of oligomer-to-amyloid fibril conversion, and reveal a new facet of α-synuclein pathogenic mutations.

Structural basis for kinase inhibition in the tripartite E. coli HipBST toxin-antitoxin system.

Many bacteria encode multiple toxin-antitoxin (TA) systems targeting separate, but closely related, cellular functions. The toxin of the Escherichia coli hipBA system, HipA, is a kinase that inhibits translation via phosphorylation of glutamyl-tRNA synthetase. Enteropathogenic E. coli O127:H6 encodes the hipBA-like, tripartite TA system; hipBST, in which the HipT toxin specifically targets the tryptophanyl-tRNA synthetase, TrpS. Notably, in the tripartite system, the function as antitoxin has been taken over by the third protein, HipS, but the molecular details of how activity of HipT is inhibited remain poorly understood. Here, we show that HipBST is structurally different from E. coli HipBA and that the unique HipS protein, which is homologous to the N-terminal subdomain of HipA, inhibits the kinase through insertion of a conserved Trp residue into the active site. We also show how auto-phosphorylation at two conserved sites in the kinase toxin serve different roles and affect the ability of HipS to neutralize HipT. Finally, solution structural studies show how phosphorylation affects overall TA complex flexibility.

Periostin C-Terminal Is Intrinsically Disordered and Interacts with 143 Proteins in an In Vitro Epidermal Model of Atopic Dermatitis.

Human periostin is a 78-91 kDa matricellular protein implicated in extracellular matrix remodeling, tumor development, metastasis, and inflammatory diseases like atopic dermatitis, psoriasis, and asthma. The protein consists of six domains, including an N-terminal Cys-rich CROPT domain, four fasciclin-1 domains, and a C-terminal domain. The exons encoding the C-terminal domain may be alternatively spliced by shuffling four exons, generating ten variants of unknown function. Here, we investigate the structure and interactome of the full-length variant of the C-terminal domain with no exons spliced out. The structural analysis showed that the C-terminal domain lacked a tertiary structure and was intrinsically disordered. In addition, we show that the motif responsible for heparin-binding is in the conserved very C-terminal part of periostin. Pull-down confirmed three known interaction partners and identified an additional 140 proteins, among which nine previously have been implicated in atopic dermatitis. Based on our findings, we suggest that the C-terminal domain of periostin facilitates interactions between connective tissue components in concert with the four fasciclin domains.

The evolution of equilibrium poly(styrene sulfonate) and dodecyl trimethylammonium bromide supramolecular structure in dilute aqueous solution with increasing surfactant binding.

HYPOTHESIS: In the last 20 years, it has been demonstrated that oppositely charged polyelectrolyte-surfactant (PE-S) mixtures are prone to forming kinetically arrested non-equilibrium aggregates, which are present in the prepared mixtures from rather low surfactant-to-polymer-repeat-unit ratios. Practically, this means that the PE-S mixtures used for the structural investigations of the formed PE-S complexes are typically a mixture of the primary PE-S complexes and large non-equilibrium aggregates of close to charge-neutral complexes. EXPERIMENTS: In this work, we present a unique approach that allows the preparation of PE-S mixtures in the equilibrium one-phase region (surfactant binding ß, is typically below 80%) without forming non-equilibrium aggregates. We used this method to prepare equilibrium, non-aggregated complexes of sodium poly(styrene sulfonate) (NaPSS, Mw = 17 kDa) and dodecyltrimethylammonium bromide (DTAB) (ß = 10 - 70%) both in water and in an inert electrolyte (100 mM NaCl). The evolution of the complex structure was monitored by small-angle X-ray scattering (SAXS) as a function of increasing surfactant binding (ß), and the measured scattering data were fitted by suitable structural models on an absolute scale where concentrations, compositions, and scattering contrasts calculated from molecular properties are used as restraints. FINDINGS: We could show that at low binding (ß < 30%), the system is a mixture of bare polyelectrolyte coils and NaPSS-DTAB complexes containing a closed surfactant associates of low aggregation number wrapped by the polyelectrolyte chain. Once all polymer chains are occupied by a micelle-like surfactant aggregate, the aggregation number increases linearly with increasing surfactant chemical potential. Using the structural insight provided by the SAXS measurements, we could fit the experimental binding isotherm data with a physically coherent, simple thermodynamic model. Finally, we also compared the stoichiometric NaPSS-DTAB precipitate's structure with the equilibrium complexes' structure.

Tau Fibrillation Induced by Heparin or a Lysophospholipid Show Different Initial Oligomer Formation.

The protein tau is involved in several neurogenerative diseases such as Alzheimer's Disease, where tau content and fibrillation have been linked to disease progression. Tau colocalizes with phospholipids and glycosaminoglycans in vivo. We investigated if and how tau fibrillation can be induced by two lysophospholipids, namely the zwitterionic 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC) and the anionic 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (LPG) as well as the glycosaminoglycan heparin. We used a range of biophysical techniques including small-angle X-ray scattering, Thioflavin T fluorescence, and SDS-PAGE, collecting data at various time points to obtain structural information on each phase of the fibrillation. We find that LPC does not induce fibrillation or interact with tau. Low concentrations of LPG induce fibrillation by formation of small hydrophobic clusters with monomeric tau. At higher LPG concentrations, a core-shell complex is formed where tau wraps around LPG micelles with regions extending away from the micelles. For heparin, loosely associated oligomers are formed rapidly with around ten tau molecules. Fibrils formed with either LPG or heparin show similar final cross-section dimensions. Furthermore, SDS-resistant oligomers are observed for both LPG and heparin. Our study demonstrates that tau fibrillation can be induced by two different biologically relevant cofactors leading to structurally different initial states but similar cross-sectional dimensions for the fibrils. Structural information about initial states prior to fibril formation is important both to gain a better understanding of the onset of fibrillation in vivo, and for the development of targeted drugs that can reduce or abolish tau fibrillation.

Structural and regulatory insights into the glideosome-associated connector from Toxoplasma gondii.

The phylum of Apicomplexa groups intracellular parasites that employ substrate-dependent gliding motility to invade host cells, egress from the infected cells, and cross biological barriers. The glideosome-associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the solution properties together with membrane and F-actin binding interfaces suggests that GAC adopts several conformations from closed to open and extended. A multi-conformational model for assembly and regulation of GAC within the glideosome is proposed.

Structure, folding and flexibility of co-transcriptional RNA origami.

RNA origami is a method for designing RNA nanostructures that can self-assemble through co-transcriptional folding with applications in nanomedicine and synthetic biology. However, to advance the method further, an improved understanding of RNA structural properties and folding principles is required. Here we use cryogenic electron microscopy to study RNA origami sheets and bundles at sub-nanometre resolution revealing structural parameters of kissing-loop and crossover motifs, which are used to improve designs. In RNA bundle designs, we discover a kinetic folding trap that forms during folding and is only released after 10 h. Exploration of the conformational landscape of several RNA designs reveal the flexibility of helices and structural motifs. Finally, sheets and bundles are combined to construct a multidomain satellite shape, which is characterized by individual-particle cryo-electron tomography to reveal the domain flexibility. Together, the study provides a structural basis for future improvements to the design cycle of genetically encoded RNA nanodevices.

FapA is an Intrinsically Disordered Chaperone for Pseudomonas Functional Amyloid FapC.

Bacterial functional amyloids contribute to biofilm development by bacteria and provide protection from the immune system and prevent antibiotic treatment. Strategies to target amyloid formation and interrupt biofilm formation have attracted recent interest due to their antimicrobial potential. Functional amyloid in Pseudomonas (Fap) includes FapC as the major component of the fibril while FapB is a minor component suggested to function as a nucleator of FapC. The system also includes the small periplasmic protein FapA, which has been shown to regulate fibril composition and morphology. The interplay between these three components is central in Fap fibril biogenesis. Here we present a comprehensive biophysical and spectroscopy analysis of FapA, FapB and FapC and provide insight into their molecular interactions. We show that all three proteins are primarily disordered with some regions with structural propensities for α-helix and ß-sheet. FapA inhibits FapC fibrillation by targeting the nucleation step, whereas for FapB the elongation step is modulated. Furthermore, FapA alters the morphology of FapC (more than FapB) fibrils. Complex formation is observed between FapA and FapC, but not between FapA and FapB, and likely involves the N-terminus of FapA. We conclude that FapA is an intrinsically disordered chaperone for FapC that guards against fibrillation within the periplasm. This new understanding of a natural protective mechanism of Pseudomonas against amyloid formations can serve as inspiration for strategies blocking biofilm formation in infections.

Stable nanovesicles formed by intrinsically planar bilayers.

HYPOTHESIS: Quatsome nanovesicles, formed through the self-assembly of cholesterol (CHOL) and cetyltrimethylammonium bromide (CTAB) in water, have shown long-term stability in terms of size and morphology, while at the same time exhibiting high CHOL-CTAB intermolecular binding energies. We hypothesize that CHOL/CTAB quatsomes are indeed thermodynamically stable nanovesicles, and investigate the mechanism underlying their formation. EXPERIMENTS: A systematic study was performed to determine whether CHOL/CTAB quatsomes satisfy the experimental requisites of thermodynamically stable vesicles. Coarse-grain molecular dynamics simulations were used to investigate the molecular organization in the vesicle membrane, and the characteristics of the simulated vesicle were corroborated with experimental data obtained by cryo-electron microscopy, small- and wide-angle X-ray scattering, and multi-angle static light scattering. FINDINGS: CHOL/CTAB quatsomes fulfill the requisites of thermodynamically stable nanovesicles, but they do not exhibit the classical membrane curvature induced by a composition asymmetry between the bilayer leaflets, like catanionic nanovesicles. Instead, CHOL/CTAB quatsomes are formed through the association of intrinsically planar bilayers in a faceted vesicle with defects, indicating that distortions in the organization and orientation of molecules can play a major role in the formation of thermodynamically stable nanovesicles.

A model on an absolute scale for the small-angle X-ray scattering from bovine casein micelles.

Casein micelles extracted from milk are 100-400 nm-sized particles, made up of proteins and calcium phosphates, with the latter as colloidal calcium phosphate particles (CCPs) in a size range of 2-4 nm embedded in a protein network. The hierarchical structures give rise to a variation of scattering intensity over many orders of magnitude, which can be measured by small-angle X-ray scattering and static light scattering. Expressions for the scattering intensity of a general simple model for composite particles with polydispersities of overall size and subparticles are derived, and some approximations are checked by generating scattering data for systems generated by Monte Carlo simulations. Based on the simpler models, a new model has been developed for casein micelles, where the scattering is expressed on an absolute scale and where the concentrations of, respectively, protein and CCPs are used as constraints, providing a consistent model. The CCPs are modelled as oblate ellipsoids and the protein as star structures. Correlations between the substructures of CCPs and protein structures are taken into account in terms of partial structure factors. The overall structure as well as some heterogeneities at intermediate length scale are modelled as polydisperse spheres. The model fits the data very well on all length scales and demonstrates that both the scattering from CCPs and protein is important. Thus, the model provides a detailed description of the casein structure, which is consistent with the information available in the literature.

How do surfactants unfold and refold proteins?

Although the anionic surfactant sodium dodecyl sulfate, SDS, has been used for more than half a century as a versatile and efficient protein denaturant for protein separation and size estimation, there is still controversy about its mode of interaction with proteins. The term "rod-like" structures for the complexes that form between SDS and protein, originally introduced by Tanford, is not sufficiently descriptive and does not distinguish between the two current vying models, namely protein-decorated micelles a.k.a. the core-shell model (in which denatured protein covers the surface of micelles) versus beads-on-a-string model (where unfolded proteins are surrounded by surfactant micelles). Thanks to a combination of structural, kinetic and computational work particularly within the last 5-10 years, it is now possible to rule decisively in favor of the core-shell model. This is supported unambiguously by a combination of calorimetric and small-angle X-ray scattering (SAXS) techniques and confirmed by increasingly sophisticated molecular dynamics simulations. Depending on the SDS:protein ratio and the protein molecular mass, the formed structures can range from multiple partly unfolded protein molecules surrounding a single shared micelle to a single polypeptide chain decorating multiple micelles. We also have much new insight into how this species forms. It is preceded by the binding of small numbers of SDS molecules which subsequently grow by accretion. Time-resolved SAXS analysis reveals an asymmetric attack by SDS micelles followed by distribution of the increasingly unfolded protein around the micelle. The compactness of the protein chain continues to evolve at higher SDS concentrations according to single-molecule studies, though the protein remains completely denatured on the tertiary structural level. SDS denaturation can be reversed by addition of nonionic surfactants that absorb SDS forming mixed micelles, leaving the protein free to refold. Refolding can occur in parallel tracks if only a fraction of the protein is initially stripped of SDS. SDS unfolding is nearly always reversible unless carried out at low pH, where charge neutralization can lead to superclusters of protein-surfactant complexes. With the general mechanism of SDS denaturation now firmly established, it largely remains to explore how other ionic surfactants (including biosurfactants) may diverge from this path.

Superanionic Solvent-Free Liquid Enzymes Exhibit Enhanced Structures and Activities.

The surface of a carboxylate-enriched octuple mutant of Bacillus subtilis lipase A (8M) is chemically anionized to produce core (8M)-shell (cationic polymer surfactants) bionanoconjugates in protein liquid form, which are termed anion-type biofluids. The resultant lipase biofluids exhibit a 2.5-fold increase in hydrolytic activity when compared with analogous lipase biofluids based on anionic polymer surfactants. In addition, the applicability of the anion-type biofluid using Myoglobin (Mb) that is well studied in anion-type solvent-free liquid proteins is evaluated. Although anionization resulted in the complete unfolding of Mb, the active α-helix level is partially recovered in the anion-type biofluids, and the effect is accentuated in the cation-type Mb biofluids. These highly active anion-type solvent-free liquid enzymes exhibit increased thermal stability and provide a new direction in solvent-free liquid protein research.

Human myelin proteolipid protein structure and lipid bilayer stacking.

The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure-function differences, as well as fundamental protein-lipid interplay in CNS compact myelin.

Universal effective interactions of globular proteins close to liquid-liquid phase separation: Corresponding-states behavior reflected in the structure factor.

Intermolecular interactions in protein solutions, in general, contain many contributions. If short-range attractions dominate, the state diagram exhibits liquid-liquid phase separation (LLPS) that is metastable with respect to crystallization. In this case, the extended law of corresponding states (ELCS) suggests that thermodynamic properties are insensitive to details of the underlying interaction potential. Using lysozyme solutions, we investigate the applicability of the ELCS to the static structure factor and how far effective colloidal interaction models can help to rationalize the phase behavior and interactions of protein solutions in the vicinity of the LLPS binodal. The (effective) structure factor has been determined by small-angle x-ray scattering. It can be described by Baxter's adhesive hard-sphere model, which implies a single fit parameter from which the normalized second virial coefficient b2 is inferred and found to quantitatively agree with previous results from static light scattering. The b2 values are independent of protein concentration but systematically vary with temperature and solution composition, i.e., salt and additive content. If plotted as a function of temperature normalized by the critical temperature, the values of b2 follow a universal behavior. These findings validate the applicability of the ELCS to globular protein solutions and indicate that the ELCS can also be reflected in the structure factor.

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization.

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized ß-sheet structures that accumulate in plaques in brains of Parkinson's disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.

Structure and Orientation of the SARS-Coronavirus-2 Spike Protein at Air-Water Interfaces.

The SARS coronavirus 2 (SARS-CoV-2) spike protein is located at the outermost perimeter of the viral envelope and is the first component of the virus to make contact with surrounding interfaces. The stability of the spike protein when in contact with surfaces plays a deciding role for infection pathways and for the viability of the virus after surface contact. While cryo-EM structures of the spike protein have been solved with high resolution and structural studies in solution have provided information about the secondary and tertiary structures, only little is known about the folding when adsorbed to surfaces. We here report on the secondary structure and orientation of the S1 segment of the spike protein, which is often used as a model protein for in vitro studies of SARS-CoV-2, at the air-water interface using surface-sensitive vibrational sum-frequency generation (SFG) spectroscopy. The air-water interface plays an important role for SARS-CoV-2 when suspended in aerosol droplets, and it serves as a model system for hydrophobic surfaces in general. The SFG experiments show that the S1 segment of the spike protein remains folded at the air-water interface and predominantly binds in its monomeric state, while the combination of small-angle X-ray scattering and two-dimensional infrared spectroscopy measurements indicate that it forms hexamers with the same secondary structure in aqueous solution.

An Atomistic Model Describing the Structure and Morphology of Cu-Doped C-S-H Hardening Accelerator Nanoparticles.

Calcium silicate hydrate (C-S-H) is the main binding phase in Portland cement. The addition of C-S-H nanoparticles as nucleation seeds has successfully been used to accelerate the hydration process and the precipitation of binding phases either in conventional Portland cement or in alternative binders. Indeed, the modulation of the hydration kinetics during the early-stage dissolution-precipitation reactions, by acting on the nucleation and growth of binding phases, improves the early strength development. The fine-tuning of concrete properties in terms of compressive strength and durability by designed structural modifications can be achieved through the detailed description of the reaction products at the atomic scale. The nano-sized, chemically complex and structurally disordered nature of these phases hamper their thorough structural characterization. To this aim, we implement a novel multi-scale approach by combining forefront small-angle X-ray scattering (SAXS) and synchrotron wide-angle X-ray total scattering (WAXTS) analyses for the characterization of Cu-doped C-S-H nanoparticles dispersed in a colloidal suspension, used as hardening accelerator. SAXS and WAXTS data were analyzed under a unified modeling approach by developing suitable atomistic models for C-S-H nanoparticles to be used to simulate the experimental X-ray scattering pattern through the Debye scattering equation. The optimization of atomistic models against the experimental pattern, together with complementary information on the structural local order from 29Si solid-state nuclear magnetic resonance and X-ray absorption spectroscopy, provided a comprehensive description of the structure, size and morphology of C-S-H nanoparticles from the atomic to the nanometer scale. C-S-H nanoparticles were modeled as an assembly of layers composed of 7-fold coordinated Ca atoms and decorated by silicate dimers and chains. The structural layers are a few tens of nanometers in length and width, with a crystal structure resembling that of a defective tobermorite, but lacking any ordering between stacking layers.

Advancement of Fluorescent and Structural Properties of Bovine Serum Albumin-Gold Bioconjugates in Normal and Heavy Water with pH Conditioning and Ageing.

The red-emitting fluorescent properties of bovine serum albumin (BSA)-gold conjugates are commonly attributed to gold nanoclusters formed by metallic and ionized gold atoms, stabilized by the protein. Others argue that red fluorescence originates from gold cation-protein complexes instead, not gold nanoclusters. Our fluorescence and infrared spectroscopy, neutron, and X-ray small-angle scattering measurements show that the fluorescence and structural behavior of BSA-Au conjugates are different in normal and heavy water, strengthening the argument for the existence of loose ionic gold-protein complexes. The quantum yield for red-emitting luminescence is higher in heavy water (3.5%) than normal water (2.4%), emphasizing the impact of hydration effects. Changes in red luminescence are associated with the perturbations of BSA conformations and alterations to interatomic gold-sulfur and gold-oxygen interactions. The relative alignment of domains I and II, II and III, III and IV of BSA, determined from small-angle scattering measurements, indicate a loose ("expanded-like") structure at pH 12 (pD ~12); by contrast, at pH 7 (pD ~7), a more regular formation appears with an increased distance between the I and II domains, suggesting the localization of gold atoms in these regions.

The C-terminal tail of α-synuclein protects against aggregate replication but is critical for oligomerization.

Aggregation of the 140-residue protein α-synuclein (αSN) is a key factor in the etiology of Parkinson's disease. Although the intensely anionic C-terminal domain (CTD) of αSN does not form part of the amyloid core region or affect membrane binding ability, truncation or reduction of charges in the CTD promotes fibrillation through as yet unknown mechanisms. Here, we study stepwise truncated CTDs and identify a threshold region around residue 121; constructs shorter than this dramatically increase their fibrillation tendency. Remarkably, these effects persist even when as little as 10% of the truncated variant is mixed with the full-length protein. Increased fibrillation can be explained by a substantial increase in self-replication, most likely via fragmentation. Paradoxically, truncation also suppresses toxic oligomer formation, and oligomers that can be formed by chemical modification show reduced membrane affinity and cytotoxicity. These remarkable changes correlate to the loss of negative electrostatic potential in the CTD and highlight a double-edged electrostatic safety guard.

The changing face of SDS denaturation: Complexes of Thermomyces lanuginosus lipase with SDS at pH 4.0, 6.0 and 8.0.

HYPOTHESIS: Lipases are widely used in the detergent industry and must withstand harsh conditions involving both anionic and zwitterionic surfactants at alkaline pH. Thermomyces lanuginosus lipase (TlL) is often used and stays active at high concentrations of the anionic surfactant sodium dodecyl sulfate (SDS) at pH 8.0, but is sensitive to SDS at pH 6.0 and below. We propose that enhanced stability at pH 8.0 results from a structurally distinct complex formation with SDS. EXPERIMENTS: We use small-angle X-ray scattering (SAXS) to elucidate structures of TlL:SDS at pH 4.0, 6.0, and 8.0 and further investigate the complexes at pH 8.0 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). FINDINGS: At pH 4.0, large dense aggregates are formed at low [SDS], which become gradually less dense at higher [SDS], resulting in a core-shell structure. At pH 6.0, SDS induces a TlL dimer and forms a hemi-micelle along the side of the dimer. At higher [SDS], TlL adopts a core-shell structure. At pH 8.0, TlL forms a dimer with a SDS hemi-micelle but avoids a core-shell structure and maintains activity. Three helices are identified as SDS anchor points. This study provides important structural insight into the stability of TlL towards SDS under alkaline conditions.