Biophysical Chemistry

Mutations in MAPT, the tau gene, give rise to forms of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T), with abundant filamentous tau inclusions in brain cells. Some mutations that encode missense and deletion variants can give rise to a clinical picture of Picks disease and filaments made of three-repeat tau. Here we report the electron cryo-microscopy (cryo-EM) structures of tau filaments from individuals with MAPT mutations D252V, G272V, S320F and {Delta}G389-I392. The two-layered Pick fold was present in the individuals with mutations D252V and {Delta}G389-I392. By contrast, mutations G272V and S320F gave rise to a more open variant of the Pick fold, with residues 272-341 rotated by 20-25{degrees} with respect to the rest of the structure. These findings show that missense mutations within the filament core can modify the Pick fold, generating closely related structural variants. In addition, we were able to reconstitute the Pick fold and some of its variants using seeded assembly with recombinant 0N3R tau carrying 12 serine or threonine to aspartate substitutions (PAD12) and missense mutations D252V, G272V or S320F. This work provides a foundation for the development of structure-based diagnostic and therapeutic approaches.

Formation of the {beta}-amyloid (A{beta}) plaques is a pathological hallmark of Alzheimers disease (AD), and is believed to be a primary cause of dementia in elderly individuals. In the present work, we simulated the conformational evolution of A{beta}42 dimers in solution and in membrane-like environment to explore the folding of A{beta}42 along fibrillation. The molecular dynamics (MD) simulation was steered by experimental internuclear distance restraints obtained using solid-state nuclear magnetic resonance (ssNMR) spectroscopy. Our results revealed that several hydrophobic and polar motifs within the A{beta}42 sequence played key roles in the early-stage nucleation process of fibrillation and those motifs are also the stabilizing agents in the mature fibrils judged by the energy contribution. Our results also indicated that the peptide association with membrane bilayers could modulate the structural evolution pathways towards fibrillation. These findings contributed to a better understanding of the molecular level structural polymorphisms inherent to A{beta}42 fibrils. Further, the current work demonstrated that the combination of MD simulations with ssNMR-based experimental restraints provided a reliable method for studying structural changes of A{beta}. HighlightO_LIUsing solid-state NMR restraints guided molecular dynamic simulation, {beta}-amyloid dimers displayed consistent {beta}-strand-prone regions, which are major stabilizing segments for mature fibrils. C_LIO_LI{beta}-amyloid dimers evolved differently with or without interacting with the lipid bilayers. C_LIO_LIExperimental restraints guided simulation provided molecular level insights about early-stage interactions along the progress of {beta}-amyloid fibrillation C_LI

The accumulation of amyloid-beta (A{beta}) plaques is a hallmark of Alzheimers disease (AD), with A{beta}42 representing the predominant and most aggregation-prone isoform. Reliable preparation of monomeric A{beta}42 is essential for investigating the kinetics and mechanisms of its aggregation into oligomers and fibrils. This study provides a direct comparison of two monomerization protocols for recombinantly expressed A{beta}42: one incorporating size-exclusion chromatography (SEC) and the other relying solely on chemical denaturation, using agents such as NaOH and NH4OH. A{beta}42 was produced in E. coli, purified through urea solubilization followed by HPLC, and subjected to monomerization via the respective methods. Monomeric preparations were evaluated using Thioflavin T (ThT) fluorescence to assess aggregation kinetics, TEM to detect fibrils and preformed aggregates, and NMR spectroscopy. SEC-isolated monomers displayed sigmoidal aggregation profiles in ThT assays, featuring distinct lag, growth, and plateau phases consistent with secondary nucleation-dominated models as determined by AmyloFit analysis. Increasing the initial peptide concentration resulted in higher fibril yields, which was further supported by TEM images showing extensive fibrillization following incubation. In contrast, non-SEC preparations containing pre-existing aggregates detectable by TEM and showed attenuated NMR signals, leading to impaired aggregation behavior. NaOH-denatured samples predominantly exhibited flat ThT curves, whereas NH4OH-denatured samples displayed extended lag phases. NH4OH performance better than NaOH, likely because its gradual pH neutralization reduced peptide structural perturbation. Overall, these findings demonstrate that SEC is critical for obtaining highly pure monomeric A{beta}42 and improving the reproducibility of aggregation assays, highlighting the importance of standardized monomer preparation protocols in AD research. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=49 SRC="FIGDIR/small/724608v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a3b9caorg.highwire.dtl.DTLVardef@1fa85d2org.highwire.dtl.DTLVardef@67a83dorg.highwire.dtl.DTLVardef@1564f77_HPS_FORMAT_FIGEXP M_FIG C_FIG

Intramolecular diffusion is an important, but often overlooked, property of intrinsically disordered proteins and plays an important role in folding, assembly and aggregation. Fluorescence resonance energy transfer (FRET) is used to observe reconfiguration over nanometer length scales while close range quenching over Angstrom length scales provides a complimentary view with different dynamics. There are several probe/quencher pairs that have been employed with varying levels of quantification of the quenching rate. Here we measure the electron transfer quenching parameters of the fluorophore Atto-655 by tryptophan using fluorescence correlation spectroscopy. Measurements with varying concentrations of quencher with low diffusion yield a distance dependent quenching rate. These parameters provide for a more quantitative analysis of measurements of intramolecular diffusion, particularly in crowded environments.

The importance of human aldehyde oxidase (hAOX1) has increased over the last decades due to its involvement in drug metabolism. Inhibition studies concerning hAOX1 are extensive and a common reducing agent, dithiothreitol (DTT), was recently found to inactivate the enzyme. However, in previous crystallographic studies of hAOX1, DTT was found to be essential for crystallization. To surpass this concern another reducing agent used in crystallization trials. Using tris(2-carboxyethyl)phosphine (TCEP), a sulphur-free reducing agent, it was possible to obtain well-ordered crystals from hAOX1 wild type and variant, hAOX1_6A, which diffracted beyond 2.3 [A]. Instead of the typical star-shaped crystals of hAOX1, at pH 4.7, plates are obtained in the orthorhombic space group (P22121) with two molecules in the asymmetric unit. Activity assays with the enzyme incubated with both reducing agents show that contrary to DTT, TCEP does not lead to irreversible inactivation of the enzyme. The replacement of DTT with TCEP in crystallization of hAOX1 provides a strategy to circumvent enzyme inactivation during crystallographic studies, allowing future applications of new assays, such as time-resolved crystallography.

Proteins with intrinsically disordered regions (IDRs) migrate at a higher apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) complicating their analysis and identification. Here, we investigate the sequence determinants of the hypomobility of IDRs using a series of synthetic low complexity domains. We find that negative charge increases the apparent molecular weight, but neutral polar tracts also have abnormally slow migration. Positive charge and hydrophobic residues decrease the apparent molecular weight, although lysine residues show a biphasic effect with decreased migration at high fractional contents. Combinations of residues show that different sequence contributions to the apparent molecular weight are not additive. The results can be rationalized by the protein-decorated micelle model by considering both SDS binding and the compaction of protein SDS-complexes.

Magic-angle spinning (MAS) solid-state NMR (SSNMR) has been widely used to determine amyloid fibril structures at atomic resolution. Such studies typically rely on homogeneous fibril preparations that produce narrow linewidths and high spectral resolution, enabling reliable resonance assignment and structural analysis. However, many biologically relevant amyloid aggregates are structurally heterogeneous, resulting in spectral broadening and reduced sensitivity that hinder atomic-resolution characterization. Lipids are known to modulate amyloid aggregation pathways and promote the formation of toxic species that are often less homogeneous, further complicating NMR-based investigations. Here, we evaluate the feasibility of utilizing the benefits associated with high-field (1.1 GHz) SSNMR for studying ganglioside GD3-catalyzed A{beta}42 aggregates. Uniformly-13C,15N-labeled A{beta}42 was incubated with GD3 to generate lipid-associated aggregates and analyzed under MAS conditions. 13C cross-polarization magic-angle spinning (CPMAS) spectra and 2D 13C-13C chemical shift correlation experiments using CORD (COmbined R2nv-Driven) mixing were acquired and compared with data collected at 600 MHz. Despite the heterogeneous nature of the GM1-associated assemblies, the 1.1 GHz spectra exhibit enhanced sensitivity and improved spectral resolution. Better resolved resonances corresponding to selectively structured regions of A{beta}42 are observed, indicating the presence of an ordered core within the lipid-associated aggregates. These results demonstrate that ultrahigh-field SSNMR significantly improves the characterization of heterogeneous amyloid assemblies and provides a promising approach for atomic-level investigation of biologically relevant, lipid-modulated A{beta} aggregates.

This study investigates the interaction between the cationic antimicrobial peptide protamine and bacterial porin OmpF (E. coli) at the single-molecule level. Using high-resolution conductance measurements in planar lipid bilayers, strong voltage- and concentration-dependent ion current blockages with OmpF, indicating significant protamine binding were observed. Further analysis revealed that peptide length influences binding kinetics, with longer peptides showing reduced affinity and slower exchange rates. These findings demonstrate that OmpF is a tractable model for studying cationic peptide-channel interactions and translocation mechanisms relevant to antimicrobial action.

The NAD+ dependent deacetylase sirtuin-1 (SIRT1) is known to elicit cellular defenses against aging, cancer, and other aberrant pathologies. Previous studies have identified an intrinsically disordered region of SIRT1 comprised of N-terminal residues 1-52, herein referred to as motif A, which activates SIRT1 activity, likely through intramolecular interactions. Additionally, phosphorylation of N-terminal residues Ser27 and Ser47 has been shown to be important for regulating SIRT1 activity and stability. The lack of in vitro characterization of these effects hampers our further understanding of the role of motif A in SIRT1 regulation. In this study, we elucidate the role phosphorylation plays in motif As structure as well as its regulatory effects on SIRT1 activity against Ac-p65. We find that phosphomimetic mutation at Ser27 significantly increases the activation effect of motif A towards SIRT1. This result is supported by molecular dynamics simulations of the phosphomimetics, which reveal stabilization of different transient structures for motif A depending on whether Ser27 and Ser47 have been modified. A key finding suggested by this study is that phosphorylation of S27 appears to activate SIRT1 by causing motif A, which is intrinsically disordered in the WT, to fold into an ordered structure. This conclusion is based on both the experimental findings and simulation results. These findings contribute to our understanding of SIRT1 regulation, specifically the role played by phosphorylation within the N-terminal disordered region.

Single-crystal X-ray diffraction remains one of the most direct and reliable techniques for clarifying the three-dimensional structures of small molecules; however, its wider use in developing research settings has historically been limited by access to advanced instrumentation. Here, we consider the performance of the in-house diffractometer, Turkish Light Source, for small-molecule structure determination using three rhodanine-derivative compounds. Diffraction data were collected, processed, and followed by full-matrix least-squares refinement as a user-friendly pipeline. The compounds were successfully resolved in the triclinic space group P-1 and refined to chemically reasonable models, although notable differences in data quality and refinement parameters were observed. Compounds 1 and 2 produced the most robust and internally coherent structure, whereas compound 3 displayed refinement tribulations. These might be attributed to the intrinsic structural disorder of c-5b, analogous to polymorphic perversity in higher Z' phase, likely due to the presence of dissymmetric molecules within the asymmetric unit (Z' = 2), rather than empirical limitations. Anisotropic displacement parameters were systematically computed by atom-resolved Ueq factors and anisotropy index. The combined analyses reveal that structural ambiguity of c-5b is largely governed by localized maxima in atomic displacement (up to 0.29 [A]2 in Ueq with 6.67 anisotropy) rather than by global disorder, caused by the fluorinated aryl moiety of c-5b. These findings indicate that the in-house SCXRD system, when coupled with our user-friendly downstream pipeline, can yield reliable structural data for small molecules. Brief video tutorials and detailed SOPs have been provided in the Tutorials folder, including CrysAlisPro and Olex2 tutorials, as well as are easily accessible for users.

Amyloid beta (A{beta}), one of the hallmark proteins of Alzheimers Disease (AD), aggregates into plaques that are strongly linked to cognitive decline and neuronal death. Reducing its aggregation propensity may provide a strategy to slow the progression of AD. While chirality modulation has emerged as an innovative approach to disrupt this process, research has primarily focused on alterations at the C position, often overlooking the impact of the second chiral center, such as the C{beta} atom of Threonine. Furthermore, the underlying mechanisms governing these chiral effects remain elusive. Given the intrinsically disordered nature of the A{beta} peptide, we employed temperature-replica exchange molecular dynamics (T-REMD) simulations to explore its rugged conformational landscape. We considered sequence mutations (A2T, A2V), N-terminal chirality inversion of the first six residues (A2V1-6D and WT1-6D), and alteration of the second chiral center (C{beta}) of Threonine (A2TC{beta}). By analyzing the effect size and population change induced by these mutations and chiral modulation, we concluded that the modulation at the N-termini is not confined locally but also exerts specific effects on the central hydrophobic core (CHC) region. Inspection of their free energy landscape and representative structures reveals that the protective or pathogenic effects of these variants correlate with their similarity to the wild type (WT) ensemble. Beyond these static thermodynamics analyses, a direct connection to phase transitions was made by estimating heat capacity as a function of temperature. Both analyses predict that A2TC{beta} may exert a pathogenic effect, in contrast to the protective nature of A2T. These findings offer a deeper understanding of the effects of site-specific mutations and chirality and shed light on the development of advanced therapeutic strategies for AD.

We have conducted all atom molecular dynamics simulations of POPC and DPPC lipid bilayers using AMBER Lipid21 force field with eight different water models, including SPC/E, TIP3P, TIP3P-FB, TIP4P-FB, TIP4P-Ew, TIP4P/2005, TIP4P-D, and OPC, to identify the most compatible one without any modification. A number of parameters have been computed in order to understand the structure of the lipid bilayer: Area per lipid, Isothermal compressibility modulus, average Volume per lipid, electron density profile, bilayer thickness, X-ray and neutron scattering form factors, deuterium order parameter, and radial distribution function. The estimated Area per lipid, Isothermal compressibility factor, volume per lipid and bilayer thickness are highly consistent with experimental results for the SPC/E water model, indicating its suitability with the AMBER Lipid21 force field, insted of any modification. The bilayer electron density profiles of both the lipid bilayers demonstrate a little augmentation of water penetration with respect to the membrane surface for TIP4P-D water model. However, the experimental X-ray and neutron scattering form factors are aligning well with the simulated results for all studied water models, and TIP4P-D shows better for X-ray data. The deuterium order parameter for lipid acyl chains value less than 0.25 for all observed water models, depicting their disorderness for both the lipid bilayers. The lateral diffusion and reorientation autocorrelation function of the lipid molecules in both the bilayers are computed to reveal their dynamics across all water models. In comparison to other water models, the simulated trajectories predict better structure and reasonably fair dynamic properties for the SPC/E water model. The TIP4P-Ew water model reproduces the lateral diffusion co-efficient in close agreement with experiment. Reorientational dynamics for both the lipids in the bilayers for eight different water models are observed; the presence of slow and slowest time components corresponds to the lipid axial motion (wobble motion) and Twist/Splay motions. So, in view of the overall performance of the different water models with the AMBER Lipid21 all atom force field in reproducing membrane physical properties, the SPC/E water model appears to be an optimal choice.

The molten globule (MG) state is an intermediate in the unfolding pathway of proteins, typically triggered by denaturing agents such as urea, extreme pH, high pressure, or heat. The microscopic details of such states are far from understood. Here we study the MG states in protein Hen Egg-White Lysozyme (PDB ID: 1AKI) using microscopic constant pH molecular dynamics (CpHMD) simulations and experiments across a wide pH range. We observe that the titratable residues act as key drivers of conformational fluctuations, promoting the emergence of MG states at extreme pH. These states display partial unfolding, and small global structural changes (< 7% deviation). Hydration around the fluctuating acidic residues shows reduced water density and weakened hydrogen bonding at low pH. At high pH, hydration around acidic residues increases relative to pH = 7, whereas hydration around basic residues decreases. The translational and rotational dynamics of hydration water also exhibit pronounced pH dependence: the translational diffusion coefficient (Dtrans) increases linearly with decrease in pH in acidic medium and increases linearly with increasing pH in the basic regime. The rotational diffusion (Drot) shows similar dependencies on pH except a break at pH {approx} 4 corresponding to acidic residue pKa values. Our results may be useful to identify ligand binding of lysozyme in extreme pH conditions.

Intercalation of small molecules between DNA base pairs affects DNA conformation, disrupting essential cellular processes including replication, transcription, and repair. We investigated conformational changes in 18-mer DNA upon intercalation of doxorubicin, SYBR Gold and YOYO-1 using extensive MD simulations. Two main patterns for the intercalation were identified: RISE-type intercalation occurs between adjacent base pairs and extends the DNA helix with decreased twist angles, while OPEN-type intercalation proceeds through base-pair opening without significant DNA extension. Kinetic analysis revealed that association rates for intercalation followed the order: first YO-moiety (mono-intercalation) > SYBR Gold > doxorubicin > YOYO-1 (bis-intercalation). Free energy landscape showed that forces at DNA termini reached up to 117 pN during stretching. Notably, base pairs adjacent to intercalators were protected from strand separation, accompanied by additional helical unwinding. MM-PBSA/GBSA analysis revealed that the driving force for intercalation is the stacking energy, and the binding affinity was highest for minor groove binding. Persistence length decreased with single molecule binding but recovered with two molecules due to their electrostatic repulsion. Mechanical properties of intercalated DNA showed position-dependence, demonstrating that multiple intercalation modes coexist in solution. The heterogeneous nature of intercalation explains why experimental measurements reflect ensemble averages rather than single binding configurations.

Ligand binding has been shown to induce significant alterations in the conformational landscape of proteins. Traditional crystallography approaches have provided valuable input about the end states in ligand-binding reactions. However, dynamical relationships between ligand binding and backbone rearrangement often remain obscured by crystallographic structures. In the present study, we use time-resolved serial synchrotron crystallography (TR-SSX) to directly visualize indole binding in the cavity of T4 lysozyme L99A in microcrystals under controlled environmental conditions. By integrating fixed target crystallography with LAMA-based ligand delivery, we have been able to track the progression of ligand binding and backbone rearrangement. By utilizing an occupancy refinement protocol, we have been able to quantify structural populations. Our studies reveal that ligand binding for this protein cavity follows a diffusion-limited process that progressively rearranges the F -helix of the protein towards a dominant conformational state. These findings establish an observable link between ligand diffusion, occupancy evolution and conformational adaptation within a crystalline environment. More broadly, our work shows how TR-SSX can quantify ligand and conformational populations during binding, providing a framework to interpret structural adaptation in real time.

Single molecule methods have become prevalent tools in elucidating molecular processes across various life science fields over the past three decades, driving breakthroughs in understanding their underlying molecular mechanisms. In our study, we employed two single-molecule methods, Forster Resonance Energy Transfer (smFRET) and high-resolution optical tweezers, to investigate the transcription of Mycobacterium tuberculosis RNA polymerase (MtbRNAP) from initiation through to termination. We aim to provide a set of comprehensive biophysical tools to deepen our current understanding of MtbRNAP and its transcription factors. These experimental assays represent an important step towards unraveling the molecular dynamics and interactions that support transcription in Mycobacterium tuberculosis.

G-quadruplexes (G4s) are critical nucleic acid secondary structures that play pivotal roles in regulating gene expression. In this study, we conducted a proteome-wide in silico analysis across multiple viruses causing hemorrhagic fevers to identify candidate proteins containing a conserved G4-binding motif. Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. We selected the NS3 protease domain of Yellow Fever virus for further validation. Biochemical assays demonstrated that the NS3 protease domain binds G4 structures with high specificity and affinity, particularly favoring the parallel conformation. Molecular docking and simulations further revealed that the NS3 protease domain interacts with the terminal G-tetrads and loop regions of G4 via key residues, including PHE40, adopting an insertion and stacking composite binding mode. These findings expand our understanding of virus - G4 interactions and offer novel potential targets for G4-based antiviral strategies. Bullet points- We screened viruses causing hemorrhagic fevers for potential G4-binding peptides. - Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. - Biochemical assays demonstrated that the NS3 protease domain of YFV binds G4 structures with high specificity and affinity.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

Saturated fatty acids such as stearic acid (SA) can exhibit both antimicrobial and growth-promoting effects on bacteria, depending on their concentration and chemical structure. However, the physical properties of the bacterial cell envelope in response to such molecules remain under-explored compared to their biochemical pathways. In this study, a comprehensive investigation is presented on the interaction of SA with the Gram-positive bacterium, Staphylococcus epider-midis (S. epi). SA alters bacterial growth, reflected in a higher maximum specific growth rate, a shorter lag phase, and an extended exponential phase, consistent with a prebiotic effect. Using fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy, we show that SA incorporation leads to significant fluidization of the lipid membrane, characterized by enhanced lateral diffusion and reduced membrane viscosity. Coarse-grained molecular dynamics (CG-MD) simulations demonstrate spontaneous insertion of SA into the membrane and a significant increase in mean-square displacement after insertion, supporting our experimental observations. Importantly, atomic force microscopy measurements show an increase in cell-envelope stiffness, reflected by a higher Youngs modulus which can be attributed to modulations in the glycan-peptide linkage density based on earlier studies that correlate stiffness changes to peptidoglycan (PG) crosslinking in Gram-positive strains [1]. These results provide direct evidence linking membrane fluidization induced by SA and increased cell wall stiffness due to transport modifications in the membrane mediated PG synthesis pathways to enhance bacterial cell viability.

The intracellular transport system is pivotal for cellular function and integrity, facilitated by cytoskeletal motor proteins such as dynein, which traverse along microtubules (MTs). The heterogeneity of the tubulin isotypes composing MTs introduces functional diversity, potentially affecting cytoskeletal motor proteins interactions with the MT. This in silico study investigated the influence of amino acid sequence variations in the C-terminal tails (CTTs) of six different Homo sapiens tubulin isotypes, TUBB2A, TUBB2B, TUBB2C, TUBB3, TUBB4A, and TUBB5, highly expressed in human brain tumors, and assessed the isotypes effect on the binding of motor protein dynein to MT. Among these isotypes, TUBB2A, TUBB2B, and TUBB2C were found to affect conformational motions of the dyneins microtubule-binding domain (MTBD) and stalk domain. The investigation highlighted the novel role of isotype-specific variations in lateral interactions between tubulin protofilaments (PFs) in determining the proximity of the {beta}-CTT of the adjacent PF to the MTBD, potentially affecting dyneins motility and suggesting how changes in isotype expression directly influence dyneins velocity and processivity and contribute to transport defects associated with neurological disorders and cancers. Isolating specific tubulin isotypes experimentally is challenging due to their high sequence similarity and complex interactions with other microtubule-associated proteins. This makes it challenging to distinguish between different tubulin isotypes and their effects, particularly in tissues where multiple isotypes are co-expressed. Additionally, these isotypes are heavily modified in vivo by post-translational modifications, which further complicate the isolation of a single, unmodified tubulin isotype. As a result, computational approaches have been necessary in this study for exploring these effects in a controlled, isotype-specific manner.