Unravelling the shape and structural assembly of the photosynthetic GAPDH–CP12–PRK complex fromArabidopsis thalianaby small-angle X-ray scattering analysis

Oxygenic photosynthetic organisms produce sugars through the Calvin–Benson cycle, a metabolism that is tightly linked to the light reactions of photosynthesis and is regulated by different mechanisms, including the formation of protein complexes. Two enzymes of the cycle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), form a supramolecular complex with the regulatory protein CP12 with the formula (GAPDH–CP122–PRK)2, in which both enzyme activities are transiently inhibited during the night. Small-angle X-ray scattering analysis performed on both the GAPDH–CP12–PRK complex and its components, GAPDH–CP12 and PRK, fromArabidopsis thalianashowed that (i) PRK has an elongated, bent and screwed shape, (ii) the oxidized N-terminal region of CP12 that is not embedded in the GAPDH–CP12 complex prefers a compact conformation and (iii) the interaction of PRK with the N-terminal region of CP12 favours the approach of two GAPDH tetramers. The interaction between the GAPDH tetramers may contribute to the overall stabilization of the GAPDH–CP12–PRK complex, the structure of which is presented here for the first time.

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Small-Angle X-ray Scattering Study of Protein Complexes with Tea Polyphenols

Journal of Agricultural and Food Chemistry ◽

10.1021/acs.jafc.6b04630 ◽

2017 ◽

Vol 65 (3) ◽

pp. 656-665 ◽

Cited By ~ 8

Author(s):

Ce Shi ◽

Haifeng Tang ◽

Jie Xiao ◽

Fengchao Cui ◽

Kecheng Yang ◽

...

Keyword(s):

Small Angle ◽

Protein Complexes ◽

Tea Polyphenols ◽

X Ray ◽

X Ray Scattering ◽

Scattering Study ◽

Ray Scattering

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Differential ultracentrifugation coupled to small-angle X-ray scattering on macromolecular complexes

Journal of Applied Crystallography ◽

10.1107/s1600576715005051 ◽

2015 ◽

Vol 48 (3) ◽

pp. 769-775 ◽

Cited By ~ 5

Author(s):

Robert M. G. Hynson ◽

Anthony P. Duff ◽

Nigel Kirby ◽

Stephan Mudie ◽

Lawrence K. Lee

Keyword(s):

Small Angle ◽

Quaternary Structure ◽

Protein Complexes ◽

New Method ◽

Size Exclusion ◽

Saxs Data ◽

X Ray ◽

X Ray Scattering ◽

Macromolecular Protein ◽

Ray Scattering

Small-angle X-ray scattering (SAXS) can provide accurate structural information and low-resolution shapes of macromolecules in solution. The technique is particularly amenable to large protein assemblies, which produce a strong scattering signal. Hence, SAXS can be a powerful tool to elucidate quaternary structure, especially when used in combination with high-resolution structural techniques such as X-ray crystallography and NMR. Sample requirements for SAXS experiments are stringent and only monodispersed samples can be satisfactorily analysed. Often, it is not possible to obtain a stable monodispersed sample of the protein of interest, in particular for multi-subunit protein complexes. In these circumstances, when the complex is less than approximately 1 MDa, size exclusion chromatography (SEC) coupled with SAXS (SEC-SAXS) can facilitate the separation of monodispersed protein from a polydispersed sample for a sufficient amount of time to collect useful SAXS data. However, many very large multi-subunit macromolecular assemblies have not been successfully purified with SEC, and hence despite being well suited to SAXS there is often no way to produce sample of sufficient quality. Rather than SEC, differential ultracentrifugation (DU) is the method of choice for the final step in the purification of large macromolecular protein complexes. Here, a new method is described for collecting SAXS data on samples directly from the fractionated elution of ultracentrifuge tubes after DU. It is demonstrated using apoferritin as a model protein that, like SEC-SAXS, DU-coupled SAXS can facilitate simultaneous purification and data collection. It is envisaged that this new method will enable high-quality SAXS data to be collected on a host of large macromolecular protein complex assemblies for the first time.

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