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Impact of Deuteration on the Assembly Kinetics of Transthyretin Monitored by Native Mass Spectrometry and Implications for Amyloidoses.

ABSTRACT: It is well established that the formation of transthyretin (TTR) amyloid fibrils is linked to the destabilization and dissociation of its tetrameric structure into insoluble aggregates. Isotope labeling is used for the study of TTR by NMR, neutron diffraction, and mass spectrometry (MS). Here MS, thioflavin T fluorescence, and crystallographic data demonstrate that while the X-ray structures of unlabeled and deuterium-labeled TTR are essentially identical, subunit exchange kinetics and amyloid formation are accelerated for the deuterated protein. However, a slower subunit exchange is noted in deuterated solvent, reflecting the poorer solubility of non-polar protein side chains in such an environment. These observations are important for the interpretation of kinetic studies involving deuteration. The destabilizing effects of TTR deuteration are rather similar in character to those observed for aggressive mutations of TTR such as L55P (associated with familial amyloid polyneuropathy).

SUBMITTER: Yee AW

PROVIDER: S-EPMC5094506 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Impact of Deuteration on the Assembly Kinetics of Transthyretin Monitored by Native Mass Spectrometry and Implications for Amyloidoses.

Yee Ai Woon AW Moulin Martine M Breteau Nina N Haertlein Michael M Mitchell Edward P EP Cooper Jonathan B JB Boeri Erba Elisabetta E Forsyth V Trevor VT

Angewandte Chemie (International ed. in English) 20160617 32

It is well established that the formation of transthyretin (TTR) amyloid fibrils is linked to the destabilization and dissociation of its tetrameric structure into insoluble aggregates. Isotope labeling is used for the study of TTR by NMR, neutron diffraction, and mass spectrometry (MS). Here MS, thioflavin T fluorescence, and crystallographic data demonstrate that while the X-ray structures of unlabeled and deuterium-labeled TTR are essentially identical, subunit exchange kinetics and amyloid f ...[more]

PMID: 27311939

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Project description:Viral capsid assembly, in which viral proteins self-assemble into complexes of well defined architecture, is a fascinating biological process. Although viral structure and assembly processes have been the subject of many excellent structural biology studies in the past, questions still remain regarding the intricate mechanisms that underlie viral structure, stability, and assembly. Here we used native mass spectrometry-based techniques to study the structure, stability, and assembly of Norwalk virus-like particles. Although detailed structural information on the fully assembled capsid exists, less information is available on potential capsid (dis)assembly intermediates, largely because of the inherent heterogeneity and complexity of the disassembly pathways. We used native mass spectrometry and atomic force microscopy to investigate the (dis)assembly of the Norwalk virus-like particles as a function of solution pH, ionic strength, and VP1 protein concentration. Native MS analysis at physiological pH revealed the presence of the complete capsid (T = 3) consisting of 180 copies of VP1. The mass of these capsid particles extends over 10 million Da, ranking them among the largest protein complexes ever analyzed by native MS. Although very stable under acidic conditions, the capsid was found to be sensitive to alkaline treatment. At elevated pH, intermediate structures consisting of 2, 4, 6, 18, 40, 60, and 80 copies of VP1 were observed with the VP1(60) (3.36-MDa) and VP1(80) (4.48-MDa) species being most abundant. Atomic force microscopy imaging and ion mobility mass spectrometry confirmed the formation of these latter midsize spherical particles at elevated pH. All these VP1 oligomers could be reversely assembled into the original capsid (VP1(180)). From the MS data collected over a range of experimental conditions, we suggest a disassembly model in which the T = 3 VP1(180) particles dissociate into smaller oligomers, predominantly dimers, upon alkaline treatment prior to reassembly into VP1(60) and VP1(80) species.

Dataset Information

Impact of Deuteration on the Assembly Kinetics of Transthyretin Monitored by Native Mass Spectrometry and Implications for Amyloidoses.

Publications

Impact of Deuteration on the Assembly Kinetics of Transthyretin Monitored by Native Mass Spectrometry and Implications for Amyloidoses.

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