Biology subjects

Akbey, U.

Publications and source records attributed to Akbey, U..

11 recordsLinked to original sources

bioRxiv2026

Structural and Mechanistic Basis of FapB Accessory Functional Amyloid Assembly in Pseudomonas

Functional amyloid formation in Pseudomonas biofilms is coordinated by the six-gene Fap operon (fapA-fapF), which governs the production, secretion, and assembly of extracellular fibrils that reinforce the biofilm matrix. FapC, FapB, and FapE constitute the extracellular amyloid. FapC is the major fibrillar component, whereas FapB and FapE occur at lower abundance and other accessory Fap proteins regulate fibril composition, processing, and secretion. We recently determined the structure of FapC, a cross-b-solenoid major Pseudomonas extracellular fibril. The assembly mechanism and architecture of FapB, however, have remained undefined. Here, we elucidate the structural and mechanistic basis of FapB accessory amyloid, from an intrinsically disordered monomer to a cross-b-solenoid fibril. Using well-defined monomeric preparations, controlled fibrillation assays, NMR spectroscopy, cryo-EM, and structure prediction, we show that FapB efficiently self-assembles into structurally homogeneous helical fibrils. FapB fibrillation is highly sensitive to its initial oligomeric state and purity, as well as to environmental factors such as pH. Monomeric FapC does not measurably perturb the disordered conformational ensemble of FapB, whereas preformed FapB and FapC fibrils reciprocally cross-seed each other, revealing direct kinetic coupling between the two amyloid components. Finally, we present a 3.4A resolution cryo-EM structure of the FapB accessory fibril, revealing a compact cross-b-solenoid architecture closely related to that of FapC. Together, these findings establish FapB as an independently amyloidogenic, structurally defined component of the Fap system, provide structural basis for the first of accessory cross-b-solenoid fibril, and reveal a mechanistic framework for the post-secretion assembly of multicomponent extracellular functional amyloids in Pseudomonas biofilms.

microbiology↗

bioRxiv2026

Structural Insights into Native Intact Mycobacterium abscessus by Conventional and Ultrahigh-field solid-state NMR at 1.2 GHz

We present an ultrahigh-field magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize intact nontuberculous mycobacteria (NTM) at the molecular level. Hydrated and dried whole-cell Mycobacterium abscessus samples were investigated by combining conventional high-field ssNMR at 750 MHz with ultrahigh-field ssNMR at 1.2 GHz and ultrafast MAS at 100 kHz. To improve sensitivity and enable multidimensional experiments, 13C/15N isotope labeling was performed after growth in synthetic cystic fibrosis medium (SCFM). We utilized 1D 13C and multidimensional 1H-13C and 13C-13C ssNMR experiments to characterize the chemical composition, dynamics, and structural organization of the M. abscessus cell envelope. The isotope-labeling efficiency was found to be non-uniform across different molecular classes, with high incorporation into polysaccharides and lower incorporation into lipid and peptide-associated signals. INEPT- and CP-based experiments selectively probed flexible and rigid fractions of the samples, revealing substantial differences in linewidth, dynamics, and sensitivity between hydrated and dried preparations. Conventional 750 MHz experiments provided high-resolution multidimensional spectra and enabled identification of distinct chemical environments associated with peptidoglycan, arabinogalactan, mycolic acids, lipids, and peptide-associated components. Ultrahigh-field ssNMR at 1.2 GHz combined with ultrafast MAS and 1H detection substantially improved spectral resolution and sensitivity in particular per mg of sample amount, allowing detection of weak and previously unresolved resonances, including polysaccharide and possible nucleic-acid-associated signals. Together, these results demonstrate that ultra-high-field and ultrafast-MAS ssNMR enables detailed characterization of intact NTM cell envelopes under near-native conditions and provides a framework for future molecular investigations of antimicrobial interactions.

microbiology↗

bioRxiv2026

Fibrillation of the Pseudomonas Extracellular Functional Amyloid FapC is Controlled by Oligomeric State, Hierarchical Architecture and the C-Terminus

Functional amyloids enable bacteria to harness the exceptional stability of the amyloid fold while preventing uncontrolled aggregation. However, the molecular mechanisms that encode this balance between robustness and regulation remain incompletely understood. Here, we define how the Pseudomonas functional amyloid FapC, a secreted extracellular functional amyloid in bacterial biofilm, achieves controlled fibril assembly through a modular architecture and a short C-terminal regulatory element. Using controlled purification, modular truncation constructs, and quantitative kinetic assays, we reveal that full-length FapC fibrillation, comprising three irregular layers, is highly sensitive to its initial oligomeric state. Non-monomeric species exert opposing effects: high-molecular-weight assemblies accelerate aggregation via heterogeneous nucleation, while low-molecular-weight species potently inhibit productive assembly. In contrast, isolated single Layer 3, a structurally defined {beta}-solenoid segment, fibrillates rapidly with minimal lag and is largely insensitive to oligomeric heterogeneity, identifying it as a dominant amyloidogenic unit. Yet, despite its minimal architecture, Layer 3 still benefits from the presence of the C-terminal region, which enhances fibrillation rate and yield. Removal of this C-terminal segment delays nucleation, slows elongation, and reduces final fibril formation in both full-length and Layer 3 contexts, highlighting its role as a non-structural regulator of productive folding. Comparative studies show that hierarchical layering imposes kinetic checkpoints and expands regulatory potential, tuning the timing and efficiency of assembly. Together, our results establish that FapC encodes a fibrillation mechanism built on hierarchical architectural design and localized sequence-specific regulation. This suggests that precise control, rather than mere aggregation propensity, maybe the defining hallmark of functional amyloids, distinguishing them from their pathological counterparts.

biophysics↗

bioRxiv2025

Elucidation of Radical Degradation in Native Biofilms by EPR Sheds Light on Bacterial Resistance and Efficient DNP Solid-state NMR

Bacterial biofilms exhibit enhanced antimicrobial resistance, yet the mechanisms determining molecular transport and reactivity within these complex communities remain poorly understood. Here, we combine electron paramagnetic resonance (EPR), solid-state NMR (ssNMR), and dynamic nuclear polarization (DNP) ssNMR to determine how native Pseudomonas fluorescens Pf0-1 colony biofilms regulate nitroxide radicals. EPR measurements reveal that radical reduction depends on biofilm morphology, hydration, and composition of extracellular matrix (ECM). Wild-type biofilms exhibit slower nitroxide reduction than planktonic cells, while isolated ECM and dehydrated biofilms show no radical reduction, indicating that bacterial cells primarily drive radical reduction. Complementary ssNMR measurements identify polysaccharides and lipids within the ECM as primary interaction sites for nitroxide radicals. DNP-enhanced ssNMR further reveals compositional differences across biofilm morphologies, with increasingly polysaccharide-rich ECM environments correlating with slower reduction kinetics. These findings support a mechanism in which the ECM acts as a diffusion barrier and selective interaction region for nitroxide radicals to regulate cellular penetration. This work showcases an integrated magnetic resonance approach that provides molecular insight into how biofilm structure determines the fate of toxic redox-active small molecules. We also set the stage for high-sensitivity measurements of structure-function relationships in these medically relevant assemblies.

biophysics↗

bioRxiv2025

Structural Basis of Pseudomonas FapC Biofilm-Forming Functional Amyloid Formation

Biofilm-protected Pseudomonas aeruginosa causes chronic infections that are difficult to treat. FapC, the major biofilm forming functional-amyloid in Pseudomonas, is essential for biofilm integrity, yet its structural details remain unresolved. Using an integrative structural biology approach, we combine solution NMR-based structural ensemble of unfolded monomeric FapC, a [~]3.3 [A] resolution CryoEM density map of FapC fibril, and all-atom MD simulations to capture transition from unfolded to folded monomer to fibrillar fold, providing a complete structural view of FapC biogenesis. CryoEM reveals a unique triple-layer {beta}-solenoid cross-{beta} fibril composed of a single protofilament. MD simulations initiated from monomeric and fibrillar FapC mapped structural transitions, offering mechanistic insights into amyloid assembly and disassembly. Understanding FapC reveals how Pseudomonas exploits functional amyloids for biofilm formation and establishes a structural and mechanistic foundation for developing therapeutics targeting biofilm-related infection and antimicrobial resistance.

microbiology↗

bioRxiv2024

High-Sensitivity Analysis of Native Bacterial Biofilms Using Dynamic Nuclear Polarization-Enhanced Solid-State NMR

Bacterial biofilms cause persistent infections that are difficult to treat and contribute greatly to antimicrobial resistance. However, high-resolution structural information on native bacterial biofilms remain very limited. This limitation is primarily due to methodological constraints associated with analyzing complex native samples. Although solid-state NMR (ssNMR) is a promising method in this regard, its conventional applications typically suffer from sensitivity limitations, particularly for unlabeled native samples. Through the use of Dynamic Nuclear Polarization (DNP), we applied sensitivity enhanced ssNMR to characterize native Pseudomonas fluorescens colony biofilms. The increased ssNMR sensitivity by DNP enabled ultrafast structural characterization of the biofilm samples without isotope-labelling, and chemical or physical modification. We collected 1D 13C and 15N, and 2D 1H-13C, 1H-15N and 13C-13C ssNMR spectra within seconds/minutes or hours, respectively which enabled us to identify biofilm components as polysaccharides, proteins, and eDNA effectively. This study represents the first application of ultrasensitive DNP ssNMR to characterize a native bacterial biofilm and expands the technical scope of ssNMR towards obtaining insights into the composition and structure of a wide array of in vitro and ex vivo biofilm applications. Such versatility should greatly boost efforts to develop structure-guided approaches for combating infections caused by biofilm-forming microbes.

microbiology↗

bioRxiv2024

High-resolution 2D Solid-State NMR provides insights into Nontuberculous Mycobacteria

We present a high-resolution magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize native nontuberculous mycobacteria (NTM). We studied two different NTM strains, Mycobacterium smegmatis, a model, non-pathogenic strain, and Mycobacterium abscessus, an emerging and important human pathogen. Native hydrated NTM samples were studied at natural abundance without isotope-labelling and any chemical or physical modification. We utilized 1D 13C and 2D 1H-13C ssNMR spectra and peak deconvolution to identify NTM cell-wall chemical sites. More than [~]100 distinct 13C signals were identified in the ssNMR spectra. The signals originating from both the flexible and rigid fractions of the native bacteria samples were selectively analyzed by utilizing either CP or INEPT based 13C ssNMR spectra. CP buildup curves provide insights into the dynamical similarity of the cell-wall components for NTM strains. Signals from peptidoglycan, arabinogalactan and mycolic acid were identified. We also provide tentative assignments for [~]30 polysaccharides by using well resolved 1H/13C chemical shifts from the 2D INEPT-based 1H-13C ssNMR spectrum. As an orthogonal way of characterizing the bacteria, electron microscopy (EM) was used to provide spatial characterization. ssNMR and EM data suggest that M. abscessus cell-wall is composed of a smaller peptidoglycan layer which is more flexible compared to M. smegmatis, which may be related to its higher pathogenicity. Here in this work, we used high-resolution 2D ssNMR first time to characterize native NTM strains and identified chemical sites. These results will aid the development of structure-based approaches to combat NTM infections.

microbiology↗

bioRxiv2023

Tapping into the native Pseudomonas Bacterial Biofilm Structure by High-Resolution 1D and 2D MAS solid-state NMR

We present a high-resolution 1D and 2D magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize native Pseudomonas fluorescens colony biofilms at natural abundance without isotope-labelling. By using a high-resolution INEPT-based 2D 1H-13C ssNMR spectrum and thorough peak deconvolution approach at the 1D ssNMR spectra, approximately 80/134 (in 1D/2D) distinct biofilm chemical sites were identified. We compared CP and INEPT 13C ssNMR spectra to different signals originating from the mobile and rigid fractions of the biofilm, and qualitative determined dynamical changes by comparing CP buildup behaviors. Protein and polysaccharide signals were differentiated and identified by utilizing FapC signals as a template, a biofilm forming functional amyloid from Pseudomonas. We also attempted to identify biofilm polysaccharide species by using 1H/13C chemical shifts obtained from the 2D spectrum. This study marks the first demonstration of high-resolution 2D ssNMR spectroscopy for characterizing native bacterial biofilms and expands the scope of ssNMR in studying biofilms. Our experimental pipeline can be readily applied to other in vitro biofilm model systems and natural biofilms and holds the promise of making a substantial impact on biofilm research, fostering new ideas and breakthroughs to aid in the development of strategic approaches to combat infections caused by biofilm-forming bacteria.

microbiology↗

bioRxiv2023

Solution-state NMR Assignment and Secondary Structure Analysis of the Monomeric Pseudomonas Biofilm-forming Functional Amyloid Accessory Protein FapA

FapA is an accessory protein within the biofilm forming functional bacterial amyloid related fap-operon in Pseudomonas. We present a complete sequential assignment of 1Hamide, 13C, 13C{beta}, and 15N NMR resonances for the functional form of the monomeric soluble FapA protein, comprising amino acids between 29-152. From these observed chemical shifts, the secondary structure propensities (SSPs) were determined. FapA predominantly adopts a random coil conformation, however, we also identified small propensities for -helical and {beta}-sheet conformations. Notably, these observed SSPs are smaller compared to the ones we recently observed for the monomeric soluble FapC protein. These NMR results will provide valuable insights into the activity of FapA in functional amyloid formation and regulation, that will also aid developing strategies targeting amyloid formation within biofilms and addressing chronic infections.

microbiology↗

bioRxiv2023

Initial Steps of Chaperone-Aided Fibrillation of Pseudomonas aeruginosa Biofilm Forming Functional Amyloid FapC

Functional bacterial amyloids (FuBA) play a crucial role in the formation of biofilms, which are mediating chronic infections and contribute to antimicrobial resistance. This study focuses on the FapC protein from Pseudomonas, a major contributor to biofilm formation. We investigate the initial steps of FapC amyloid formation and the impact of the chaperone-like protein FapA on this process. Using solution NMR spectroscopy, we show that both FapC and FapA, which are part of the same biofilm-forming protein operon, are intrinsically disordered proteins (IDPs) in their soluble monomeric state. These SSPs were determined and compared to the Alphafold models. We further demonstrate that the IDP chaperone FapA interacts with FapC and significantly slows down the formation of FapC fibrils, while maintaining the fibril morphology unchanged. Our NMR titration experiments reveal that [~]18% of the resonances show FapA induced chemical shift perturbations (CPSs) which has not been previously observed, the largest being for A82, N201, C237, C240, A241 and G245 residues. These sites may suggest a specific interaction site and/or hotspots of fibrillation inhibition/control interface at the R1/L2 and L2/R3 transition areas and at the C-terminus of FapC. Remarkably, [~]90% of FapA NMR signals exhibit substantial CSPs upon titration with FapC. A temperature dependent effect of FapA was observed on FapC by ThT and NMR experiments. This study provides a detailed understanding of the interaction between the chaperone/chaperone-like FapA and the functional amyloid protein FapC, shedding light on the regulation and slowing down of amyloid formation. Our findings have important implications for the development of therapeutic strategies targeting biofilms and associated infections, leveraging these structural and mechanistic insights.

microbiology↗

bioRxiv2023

Solution-state NMR Assignment and Secondary Structural Propensities of the Full-Length and Minimalistic-Truncated Prefibrillar Monomeric Form of Biofilm-Forming Functional-Amyloid FapC from Pseudomonas aeruginosa

Functional bacterial amyloids provide structural scaffolding to bacterial biofilms. In contrast to the pathological amyloids, they have a role in vivo and are tightly regulated. Their presence is essential to the integrity of the bacterial communities surviving in biofilms and may cause serious health complications. Targeting amyloids in biofilms could be a novel approach to prevent chronic infections. However, structural information is very scarce on them in both soluble monomeric and insoluble fibrillar forms, hindering our molecular understanding and strategies to fight biofilm related diseases. Here, we present solution-state NMR assignment of 250 amino acid long biofilm-forming functional-amyloid FapC from Pseudomonas aeruginosa. We studied the full-length and shorter minimalistic-truncated FapC constructs without signal-sequence that is required for secretion. 91% and 100% backbone NH resonance assignment for FL and short constructs, respectively, indicates that soluble monomeric FapC is predominantly disordered, with sizeable secondary structural propensities mostly as PP2 helices, but also as -helices and {beta}-sheets highlighting hotspots for fibrillation initiation interface. Shorter construct showing almost identical NMR chemical shifts highlights the promise of utilizing it for more demanding solid-state NMR studies that requires methods to alleviate signal redundancy due to almost identical repeat units. This study provides key NMR resonance assignment for future structural studies of soluble, pre-fibrillar and fibrillar forms of FapC.

microbiology↗