NMR-based Structural Biology of Dynamic Biomolecular Complexes DOI Open Access

Agnes Adler

Опубликована: Авг. 22, 2023

Cells rely on various biomolecular interactions and dynamics to perform their functions. The interacting building blocks range from small molecules complex dynamic molecular networks such as membrane-less organelles or the cell’s cytoskeleton. These systems play a significant role in biological function development of diseases. X-ray crystallography cryogenic-electron microscopy (cryo-EM) enable atomic investigation these complexes, by largely probing static states. As result, insight into dynamics, subspecies heterogeneity is often limited. Additionally, structural biology techniques in-vitro sample preparations. Nuclear magnetic resonance (NMR) spectroscopy, however, can probe at under psychological conditions. utilizing solid-state NMR (ssNMR), it becomes possible study complexes. This thesis presents means NMR. We focused system microtubules (MTs) with microtubule-associated proteins (MAPs) they are essential processes cell division, intracellular transport structure. Moreover, we studied gel-like phase mRNA processing bodies (PBs) S.Pombe which involved homeostasis. Chapter 2 characterization microtubule-binding domain (MTBD) protein 7 (MAP7). assignment for this were obtained solution its secondary structure was characterized an alpha-helical fold. In 3 provided protocol isotopically label MTs ssNMR experiments. A novel through-bond hCCH pulse sequence discussed 4. applicable High-resolution Magic-Angle Spinning uses proton detection. applied labelled Tau K32 fungal wall S. commune. Building results, examined 5 interaction MAP7s MTBD MTs. Furthermore, combined approach cryo-EM confirmed our observations fluorescence anisotropy solution-state Combination results allowed us obtain information binding MAP7, suggests that forms elongated alpha-helix upon By designing peptides compromising tubulin carboxy-terminal tails (CTTs), also MAP7 similar employed Tau-MTs 6. SsNMR used rigid flexible regions complex. Thus, gained about register extend model interacts investigated CTTs protein, led identification residues both partners. 7, explored another system. conducted experiments investigate PBs. Our data shows state, overall conformations enhancer decapping (Edc3) (Dcp2) conserved. able changes state found Dcp2 might persist equilibrium conformations.

Язык: Английский

Molecular Distinction of Cell Wall and Capsular Polysaccharides in Encapsulated Pathogens by In Situ Magic-Angle Spinning NMR Techniques DOI
Alons Lends, Gaëlle Lamon,

Loic Delcourte

и другие.

Journal of the American Chemical Society, Год журнала: 2025, Номер unknown

Опубликована: Фев. 16, 2025

Pathogenic fungal and bacterial cells are enveloped within a cell wall, molecular barrier at their surface, critical architecture that constantly evolves during pathogenesis. Understanding the composition, structural organization, mobility of polysaccharides constituting this envelope is crucial to correlate wall organization with its role in pathogenicity identify potential antifungal targets. For pathogen Cryptococcus neoformans, characterization has been complexified by presence an additional external polysaccharide capsular shell. Here, we investigate how magic-angle spinning (MAS) solid-state NMR techniques increase analytical capabilities characterize structure dynamics encapsulated pathogen. The versatility proton detection experiments, dynamic-based filters, relaxation measurements facilitate discrimination highly mobile from internal rigid C. neoformans. In addition, report situ triglyceride molecules lipid droplets based on dynamic filters. Together, demonstrate nondestructive technique study microbes using neoformans as model, airborne opportunistic infects mainly immunocompromised but also competent hosts.

Язык: Английский

Процитировано

5
Structural adaptation of fungal cell wall in hypersaline environment DOI Creative Commons

Liyanage D. Fernando,

Yordanis Pérez‐Llano, Malitha C. Dickwella Widanage

и другие.

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Ноя. 4, 2023

Abstract Halophilic fungi thrive in hypersaline habitats and face a range of extreme conditions. These fungal species have gained considerable attention due to their potential applications harsh industrial processes, such as bioremediation fermentation under unfavorable conditions hypersalinity, low water activity, pH. However, the role cell wall surviving these environmental remains unclear. Here we employ solid-state NMR spectroscopy compare architecture Aspergillus sydowii across salinity gradients. Analyses intact cells reveal that A. walls contain rigid core comprising chitin, β-glucan, chitosan, shielded by surface shell composed galactomannan galactosaminogalactan. When exposed conditions, enhances chitin biosynthesis incorporates α-glucan create thick, stiff, hydrophobic walls. Such structural rearrangements enable fungus adapt both salt-deprived providing robust mechanism for withstanding external stress. molecular principles can aid optimization halophilic strains biotechnology applications.

Язык: Английский

Процитировано

36
Molecular architecture of chitin and chitosan-dominated cell walls in zygomycetous fungal pathogens by solid-state NMR DOI Creative Commons

Qinghui Cheng,

Malitha C. Dickwella Widanage, Jayasubba Reddy Yarava

и другие.

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Сен. 27, 2024

Язык: Английский

Процитировано

12
MAS NMR experiments of corynebacterial cell walls: Complementary 1H- and CPMAS CryoProbe-enhanced 13C-detected experiments DOI Creative Commons
Alicia Vallet, Isabel Ayala,

Barbara Perrone

и другие.

Journal of Magnetic Resonance, Год журнала: 2024, Номер 364, С. 107708 - 107708

Опубликована: Июнь 6, 2024

Bacterial cell walls are gigadalton-large cross-linked polymers with a wide range of motional amplitudes, including rather rigid as well highly flexible parts. Magic-angle spinning NMR is powerful method to obtain atomic-level information about intact walls. Here we investigate sensitivity and content different homonuclear

Язык: Английский

Процитировано

10
New Vision of Cell Walls in Aspergillus fumigatus from Solid-State NMR Spectroscopy DOI Creative Commons
Isha Gautam,

Kalpana Singh,

Malitha C. Dickwella Widanage

и другие.

Journal of Fungi, Год журнала: 2024, Номер 10(3), С. 219 - 219

Опубликована: Март 16, 2024

The fungal cell wall plays a critical role in regulating cellular integrity and communication, serves as frontline defense against stress. It is also prime target for the development of antifungal agents. comprised diverse polysaccharides proteins poses challenging high-resolution structural characterization. Recently, solid-state nuclear magnetic resonance (ssNMR) analysis intact Aspergillus fumigatus cells has provided atomic-level insights into polymorphism functional assembly principles carbohydrate components within wall. This physical perspective, alongside information from biochemical assays, offers renewed understanding highly complex dynamic organelle. Here, we summarize key conceptual advancements elucidation A. mycelial conidial walls their responses to stressors. We highlight underexplored areas discuss opportunities facilitated by technical ssNMR spectroscopy.

Язык: Английский

Процитировано

7
Comparative analysis of polysaccharide and cell wall structure in Aspergillus nidulans and Aspergillus fumigatus by solid-state NMR DOI
Isha Gautam, Jayasubba Reddy Yarava, Yifan Xu

и другие.

Carbohydrate Polymers, Год журнала: 2024, Номер 348, С. 122907 - 122907

Опубликована: Окт. 28, 2024

Язык: Английский

Процитировано

6
Polysaccharide assemblies in fungal and plant cell walls explored by solid-state NMR DOI Creative Commons

Liyanage D. Fernando,

Wancheng Zhao, Isha Gautam

и другие.

Structure, Год журнала: 2023, Номер 31(11), С. 1375 - 1385

Опубликована: Авг. 18, 2023

Язык: Английский

Процитировано

10
A structural and dynamic visualization of the interaction between MAP7 and microtubules DOI Creative Commons

Agnes Adler,

Mamata Bangera, J. Wouter Beugelink

и другие.

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Март 2, 2024

Abstract Microtubules (MTs) are key components of the eukaryotic cytoskeleton and essential for intracellular organization, organelle trafficking mitosis. MT tasks depend on binding interactions with MT-associated proteins (MAPs). protein 7 (MAP7) has unusual ability both activating kinesin-1-mediated cargo transport along MTs. Additionally, is reported to stabilize MTs its 112 amino-acid long MT-binding domain (MTBD). Here we investigate structural basis interaction MAP7 MTBD lattice. Using a combination solid solution-state nuclear magnetic resonance (NMR) spectroscopy electron microscopy, fluorescence anisotropy isothermal titration calorimetry, shed light mode at an atomic level. Our results show that between lattice extending beyond single tubulin dimer including C-terminal tails contribute formation MAP7-MT complex.

Язык: Английский

Процитировано

4
Solid-State NMR Analysis of Schizosaccharomyces pombe Reveals Role of α-Amylase Family Enzymes in Cell Wall Structure and Function DOI Creative Commons

Anand Jacob,

Alaina H. Willet, Maya G. Igarashi

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Март 11, 2025

ABSTRACT The fission yeast Schizosaccharomyces pombe is a widely employed model organism for studying the eukaryotic cell cycle. Like plants and bacteria, S. must build wall in concert with its cycle, but how wall-synthesizing remodeling enzymes mediate this process remains unclear. Here we characterize functions of Aah1 Aah3, two related α-amylases that are putative members evolutionarily conserved family wall-modifying proteins. We found unlike rod-shaped wildtype cells, aah1 Δ aah3 cells nearly spherical, grow slowly, have thickened walls, severe defects separation following cytokinesis. Solid-state NMR spectroscopy analyses intact revealed walls rigidified significant reduction α-glucan matrix, characterized by reduced amounts major α-1,3-glucan minor α-1,4-glucan within rigid mobile phases; was compensated two-fold increase β-glucan content. Indeed, viability depended on upregulation integrity pathway mediates it. While resemble impaired function transglycosylation domain synthase 1 (Ags1), increased expression Aah3 does not compensate Ags1 or vice-versa. Overall, our data suggest required addition to Ags1, likely downstream, chains generate fibers appropriate dimensions support proper morphology, growth, division. Significance Statement This study utilized range imaging techniques high-resolution solid-state refine understanding composition. also determined GPI-anchored α-amylase proteins, act as transglycosylases non-redundantly an synthesis content size polarized growth Our results highlight anti-fungal therapeutic potential acting glucan synthases.

Язык: Английский

Процитировано

0
Proton-Detected Solid-State NMR for Deciphering Structural Polymorphism and Dynamic Heterogeneity of Cellular Carbohydrates in Pathogenic Fungi DOI Creative Commons
Jayasubba Reddy Yarava, Isha Gautam,

Anand Jacob

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Март 13, 2025

Carbohydrate polymers in their cellular context display highly polymorphic structures and dynamics essential to diverse functions, yet they are challenging analyze biochemically. Proton-detection solid-state NMR spectroscopy offers high isotopic abundance sensitivity, enabling rapid high-resolution structural characterization of biomolecules. Here, an array 2D/3D 1 H-detection techniques tailored investigate polysaccharides fully protonated or partially deuterated cells three prevalent pathogenic fungi: Rhizopus delemar , Aspergillus fumigatus Candida albicans representing filamentous species yeast forms. Selective detection acetylated carbohydrates reveals fifteen forms N-acetylglucosamine units R. chitin, which coexists with chitosan as separate domains associates proteins only at limited sites. This is supported by distinct order parameters effective correlation times motions, analyzed through relaxation measurements model-free analysis. Five α-1,3-glucan origins were identified A. important for this buffering polysaccharide perform roles supporting wall mechanics regenerating soft matrix under antifungal stress. Eight α-1,2-mannan sidechain variants C. resolved, highlighting the crucial role mannan sidechains maintaining interactions other cell preserve integrity. These methodologies provide novel insights into functional key fungal create new opportunities exploring carbohydrate biosynthesis modifications across organisms.

Язык: Английский

Процитировано

0