Biology subjects

Akbey, U.

Publications and source records attributed to Akbey, U..

4 recordsLinked to original sources

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↗