Structural Changes Associated with Transthyretin Misfolding and Amyloid Formation Revealed by Solution and Solid-State NMR

Lim, Kwang Hun; Dasari, Anvesh K. R.; Hung, Ivan; Gan, Zhehong; Wemmer, David E.

doi:10.1021/acs.biochem.6b00164

Cited by 41 publications

(65 citation statements)

References 26 publications

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“…19,20 In particular, the native CBEF β-sheet remains unchanged in the amyloidogenic intermediate state of TTR, while the other β-sheet (DAGH) undergoes conformational fluctuations in millisecond time scales. The structural feature of the aggregation-prone states of TTR was investigated with CD spectroscopy (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…However, our recent solid-state NMR experiments showed that TTR amyloid used in this study is a uniform protein assembly consisting of a single monomeric core conformer. 20 …”

Section: Resultsmentioning

confidence: 99%

“…The TTR amyloid was examined by transmission electron microscopy (TEM) and thioflavin T binding assay in our previous studies. 20 …”

Section: Experimental Methodsmentioning

confidence: 99%

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Solid-State NMR Studies Reveal Native-like β-Sheet Structures in Transthyretin Amyloid

et al. 2016

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Structural characterization of amyloid rich in cross-β structures is crucial for unraveling molecular basis of protein misfolding and amyloid formation associated with a wide range of human disorders. Elucidation of the β-sheet structure in non-crystalline amyloid has, however, remained an enormous challenge. Here we report structural analyses of the β-sheet structure in full-length transthyretin amyloid using solid-state NMR spectroscopy. Magic-angle-spinning (MAS) solid-state NMR was employed to investigate native-like β-sheet structures in amyloid state using selective labeling schemes for more efficient solid-state NMR studies. Analyses of extensive long-range 13C-13C correlation MAS spectra obtained with selectively 13CO- and 13Cα-labeled TTR reveal that the two main β-structures in the native state, the CBEF and DAGH β-sheets, remain intact after amyloid formation. The tertiary structural information would be of great use for examining quaternary structure of TTR amyloid.

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Section: Resultsmentioning

confidence: 99%

“…However, our recent solid-state NMR experiments showed that TTR amyloid used in this study is a uniform protein assembly consisting of a single monomeric core conformer. 20 …”

Section: Resultsmentioning

confidence: 99%

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Solid-State NMR Studies Reveal Native-like β-Sheet Structures in Transthyretin Amyloid

et al. 2016

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“…Our previous solid-state studies revealed that the native-like CBEF and DAGH β-structure is retained in WT TTR amyloid state, while AB loop regions that are hydrogen-bonded with strand A undergo a structural transition. 33 The partly exposed strand A might be involved in intermolecular interactions essential to TTR amyloid formation. In our present solid-state NMR studies reported here, the amyloid states of the two TTR variants are also shown to adopt the similar native-like CBEF and AGH β-sheet structure.…”

Section: Discussionmentioning

confidence: 99%

“…The TTR amyloid was examined by transmission electron microscopy (TEM) and thioflavin T (ThT) binding assay in our previous studies. 33 …”

Section: Methodsmentioning

confidence: 99%

Pathogenic Mutations Induce Partial Structural Changes in the Native β-Sheet Structure of Transthyretin and Accelerate Aggregation

Lim

Dasari

et al. 2017

Biochemistry

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Amyloid formation of natively folded proteins involves global and/or local unfolding of the native state to form aggregation-prone intermediates. Here we report solid-state NMR structural studies of amyloid derived from wild-type (WT) and more aggressive mutant forms of transthyretin (TTR) to investigate the structural changes associated with effective TTR aggregation. We employed selective 13C-labeling schemes to investigate structural features of β-structured core regions in amyloid states of WT and two mutant forms (V30M and L55P) of TTR. Analyses of the 13C-13C correlation solid-state NMR spectra revealed that WT TTR aggregates contain an amyloid core consisting of native-like CBEF and DAGH β-sheet structures and the mutant TTR amyloids adopt a similar amyloid core structure with native-like CBEF and AGH β-structures. However, the V30M mutant amyloid was shown to have a different DA β-structure. In addition, strand D is more disordered even in the native state of L55P TTR, indicating that the pathogenic mutations affect the DA β-structure, leading to more effective amyloid formation. The NMR results are consistent with our mass spectrometry-based thermodynamic analyses that showed the amyloidogenic precursor states of WT and mutant TTRs adopt folded structures, but the mutant precursor states are less stable than that of WT TTR. Analyses of the oxidation rate of methionine sidechain also revealed that the sidechain of residue Met-30 pointing between strands D and A is not protected from the oxidation in V30M mutant, while protected in the native state, supporting that the DA β-structure might be disrupted in V30M mutant amyloid.

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Biophysical characterization and modulation of Transthyretin Ala97Ser

Liu

Yen

Ricardo

et al. 2019

Ann Clin Transl Neurol

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ObjectiveAla97Ser (A97S) is the major transthyretin (TTR) mutation in Taiwanese patients of familial amyloid polyneuropathy (FAP), characterized by a late‐onset but rapidly deteriorated neuropathy. Tafamidis can restore the stability of some mutant TTR tetramers and slow down the progression of TTR‐FAP. However, there is little understanding of the biophysical features of A97S‐TTR mutant and the pharmacological modulation effect of tafamidis on it. This study aims to delineate the biophysical characteristics of A97S‐TTR and the pharmacological modulation effect of tafamidis on this mutant.MethodThe stability of TTR tetramers was assessed by urea denaturation and differential scanning calorimetry. Isothermal titration calorimetry (ITC) was used to measure the binding constant of tafamidis to TTR. Nuclear magnetic resonance spectroscopy (NMR) titration experiment was used to map out the tafamidis binding site.ResultsChemical and thermal denaturation confirmed the destabilization effect of A97S. Consistent with other the amyloidogenic mutant, A97S‐TTR has slightly lower conformational stability. NMR revealed the binding site of A97S‐TTR with tafamidis is at the thyroxine binding pocket. The ITC experiments documented the high affinity of the binding which can effectively stabilize the A97S‐TTR tetramer.InterpretationThis study confirmed the structural modulation effect of tafamidis on A97S‐TTR and implied the potential therapeutic benefit of tafamidis for A97S TTR‐FAP. This approach can be applied to investigate the modulation effect of tafamidis on other rare TTR variants and help to make individualized choices of available treatments for FAP patients.

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Structural Changes Associated with Transthyretin Misfolding and Amyloid Formation Revealed by Solution and Solid-State NMR

Cited by 41 publications

References 26 publications

Solid-State NMR Studies Reveal Native-like β-Sheet Structures in Transthyretin Amyloid

Solid-State NMR Studies Reveal Native-like β-Sheet Structures in Transthyretin Amyloid

Pathogenic Mutations Induce Partial Structural Changes in the Native β-Sheet Structure of Transthyretin and Accelerate Aggregation

Biophysical characterization and modulation of Transthyretin Ala97Ser

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