scholarly journals Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability

2020 ◽  
Vol 295 (52) ◽  
pp. 18474-18484
Author(s):  
Tejaswini Pradhan ◽  
Karthikeyan Annamalai ◽  
Riddhiman Sarkar ◽  
Stefanie Huhn ◽  
Ute Hegenbart ◽  
...  
Keyword(s):  
Solid State ◽  
Light Chain ◽  
Solid State Nmr ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Chemical Shifts ◽  
Single Point ◽  
Single Point Mutation ◽  
Al Amyloidosis ◽  
Fibril Structure

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.

scholarly journals Amyloidogenic core of a human λ-III immunoglobulin light chain fibril and their germline variants probed by MAS solid state NMR

2020 ◽  
Author(s):  
Tejaswini Pradhan ◽  
Karthikeyan Annamalai ◽  
Riddhiman Sarkar ◽  
Stephanie Huhn ◽  
Ute Hegenbart ◽  
...  
Keyword(s):  
Solid State ◽  
Light Chain ◽  
Solid State Nmr ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Chemical Shifts ◽  
Single Point ◽  
Single Point Mutation ◽  
Al Amyloidosis ◽  
Fibril Structure

AbstractSystemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we study the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that reflect the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify glycine-49 that is mutated with respect to the germline sequence into arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity for the C-terminal residues 80-102 which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra show 13Cα chemical shifts that are highly similar to patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C-terminus in the fibril state, while the overall fibril topology is retained.

scholarly journals Solid state NMR assignments of a human λ-III immunoglobulin light chain amyloid fibril

2020 ◽  
Author(s):  
Tejaswini Pradhan ◽  
Karthikeyan Annamalai ◽  
Riddhiman Sarkar ◽  
Ute Hegenbart ◽  
Stefan Schönland ◽  
...  
Keyword(s):  
Solid State ◽  
Light Chain ◽  
Solid State Nmr ◽  
Cardiac Involvement ◽  
Gene Segment ◽  
Al Amyloidosis ◽  
Immunoglobulin Light Chain ◽  
Fibril Structure ◽  
Amyloid Deposits ◽  
Complementarity Determining Regions

Abstract The aggregation of antibody light chains is linked to systemic light chain (AL) amyloidosis, a disease where amyloid deposits frequently affect the heart and the kidney. We here investigate fibrils from the λ-III FOR005 light chain (LC), which is derived from an AL-patient with severe cardiac involvement. In FOR005, five residues are mutated with respect to its closest germline gene segment IGLV3-19 and IGLJ3. All mutations are located close to the complementarity determining regions (CDRs). The sequence segments responsible for the fibril formation are not yet known. We use fibrils extracted from the heart of this particular amyloidosis patient as seeds to prepare fibrils for solid-state NMR. We show that the seeds induce the formation of a specific fibril structure from the biochemically produced protein. We have assigned the fibril core region of the FOR005-derived fibrils and characterized the secondary structure propensity of the observed amino acids. As the primary structure of the aggregated patient protein is different for every AL patient, it is important to study, analyze and report a greater number of light chain sequences associated with AL amyloidosis.

scholarly journals Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

2016 ◽  
Vol 113 (6) ◽  
pp. 1546-1551 ◽  
Author(s):  
Cody L. Hoop ◽  
Hsiang-Kai Lin ◽  
Karunakar Kar ◽  
Gábor Magyarfalvi ◽  
Jonathan M. Lamley ◽  
...  
Keyword(s):  
Solid State ◽  
Solid State Nmr ◽  
Protein Misfolding ◽  
Amyloid Fibrils ◽  
Chemical Shifts ◽  
Magic Angle Spinning ◽  
Cag Repeat ◽  
Magic Angle ◽  
Polyglutamine Expansion ◽  
Angle Spinning

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington’s disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin–containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand–based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of “intrinsic” polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.

scholarly journals Different Dynamics in 6aJL2 Proteins Associated with AL Amyloidosis, a Conformational Disease

2019 ◽  
Vol 20 (17) ◽  
pp. 4078
Author(s):  
Roberto Maya-Martinez ◽  
Leidys French-Pacheco ◽  
Gilberto Valdés-García ◽  
Nina Pastor ◽  
Carlos Amero
Keyword(s):  
Light Chain ◽  
Amyloid Fibrils ◽  
Tertiary Structure ◽  
Germ Line ◽  
Single Point ◽  
Fiber Formation ◽  
Resonance Spectroscopy ◽  
Al Amyloidosis ◽  
Multiple Organs ◽  
Dynamics Simulations

Light-chain amyloidosis (AL) is the most common systemic amyloidosis and is caused by the deposition of mainly insoluble immunoglobulin light chain amyloid fibrils in multiple organs, causing organ failure and eventually death. The germ-line λ6a has been implicated in AL, where a single point mutant at amino acid 24 (6aJL2-R24G) has been observed in around 25% of patient samples. Structural analysis has shown only subtle differences between both proteins; nevertheless, 6aJL2-R24G is more prone to form amyloid fibrils. To improve our understanding of the role of protein flexibility in amyloid fibril formation, we have used a combination of solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations to complement the structural insight with dynamic knowledge. Fast timescale dynamics (ps–ns) were equivalent for both proteins, but suggested exchange events for some residues. Even though most of the intermediate dynamics (μs–ms) occurred at a similar region for both proteins, the specific characteristics are very different. A minor population detected in the dispersion experiments could be associated with the formation of an off-pathway intermediate that protects from fiber formation more efficiently in the germ-line protein. Moreover, we found that the hydrogen bond patterns for both proteins are similar, but the lifetime for the mutant is significantly reduced; as a consequence, there is a decrease in the stability of the tertiary structure that extends throughout the protein and leads to an increase in the propensity to form amyloid fibers.

Scarcity of λ1 B cells in mice with a single point mutation in Cλ1 is due to a low BCR signal caused by misfolded lambda1 light chain

2007 ◽  
Vol 44 (6) ◽  
pp. 1417-1428 ◽  
Author(s):  
Veronica V. Volgina ◽  
Tianhe Sun ◽  
Grazyna Bozek ◽  
Terence E. Martin ◽  
Ursula Storb
Keyword(s):  
B Cells ◽  
Point Mutation ◽  
Light Chain ◽  
Single Point ◽  
Single Point Mutation

scholarly journals Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Simon Erlendsson ◽  
Kamil Gotfryd ◽  
Flemming Hofmann Larsen ◽  
Jonas Sigurd Mortensen ◽  
Michel-Andreas Geiger ◽  
...  
Keyword(s):  
Solid State ◽  
Binding Site ◽  
Solid State Nmr ◽  
Chemical Shifts ◽  
Substrate Binding ◽  
Experimental Conditions ◽  
Specific Chemical ◽  
Sodium Dependent ◽  
Number Of Binding Sites ◽  
Neurotransmitter:Sodium Symporter

The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

scholarly journals Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Thomas Heerde ◽  
Matthies Rennegarbe ◽  
Alexander Biedermann ◽  
Dilan Savran ◽  
Peter B. Pfeiffer ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Proteinase K ◽  
Seed Structure ◽  
In Vitro Proliferation ◽  
Amyloid A ◽  
Fibril Structure ◽  
Proteolytic Stability ◽  
Fibril Morphology

AbstractSeveral studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection.

scholarly journals 14N1H HMQC Solid-State NMR as a Powerful Tool to Study Amorphous Formulations – an Exemplary Study of Paclitaxel Loaded Polymer Micelles

2020 ◽  
Author(s):  
Marvin Grüne ◽  
Robert Luxenhofer ◽  
Dinu Iuga ◽  
Steven P. Brown ◽  
Ann-Christin Pöppler
Keyword(s):  
Solid State ◽  
Solid State Nmr ◽  
Chemical Shifts ◽  
Mas Nmr ◽  
Cancer Drug ◽  
Polymer Micelles ◽  
Promising Tool ◽  
Nmr Characterization

We present <sup>14</sup>N-<sup>1</sup>H HMQC MAS NMR experiments in the solid state as a promising tool to study amorphous formulations. Poly(2-oxazoline) based polymer micelles loaded with different amounts of the cancer drug paclitaxel serve to highlight the possibilities offered by these experiments: While the very similar <sup>15</sup>N chemical shifts hamper a solid-state NMR characterization based on this nucleus, <sup>14</sup>N is a very versatile alternative. <sup>14</sup>N-<sup>1</sup>H HMQC experiments yield well-separated signals, which are spread over a large ppm range, provide information on the symmetry of the nitrogen environment and probe <sup>14</sup>N-<sup>1</sup>H through-space proximities.

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