Tetrameric Structures of Inorganic CBS-Pyrophosphatases from Various Bacterial Species Revealed by Small-Angle X-ray Scattering in Solution

Quaternary structure of CBS-pyrophosphatases (CBS-PPases), which belong to the PPases of family II, plays an important role in their function ensuring cooperative behavior of the enzymes. Despite an intensive research, high resolution structures of the full-length CBS-PPases are not yet available making it difficult to determine the signal transmission path from the regulatory to the active center. In the present work, small-angle X-ray scattering (SAXS) combined with size-exclusion chromatography was applied to determine the solution structures of the full-length wild-type CBS-PPases from three different bacterial species. Previously, in the absence of an experimentally determined full-length CBS-PPase structure, a homodimeric model of the enzyme based on known crystal structures of the CBS domain and family II PPase without this domain has been proposed. Our SAXS analyses demonstrate, for the first time, the existence of stable tetramers in solution for all studied CBS-PPases from different sources. Our findings show that further studies are required to establish the functional properties of these enzymes. This is important not only to enhance our understanding of the relation between CBS-PPases structure and function under normal conditions but also because some human pathogens harbor this class of enzymes.

Download Full-text

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.

Download Full-text

Arguments for an additional long-lived intermediate in the photocycle of the full-length aureochrome 1c receptor: A time-resolved small-angle X-ray scattering study

Structural Dynamics ◽

10.1063/1.5095063 ◽

2019 ◽

Vol 6 (3) ◽

pp. 034701 ◽

Cited By ~ 1

Author(s):

Saskia Bannister ◽

Elena Böhm ◽

Thomas Zinn ◽

Thomas Hellweg ◽

Tilman Kottke

Keyword(s):

Small Angle ◽

Full Length ◽

Time Resolved ◽

X Ray ◽

X Ray Scattering ◽

Scattering Study ◽

Ray Scattering

Download Full-text

Studies by Small-Angle X-Ray Scattering of the Quaternary Structure of Dissociation Products of the beta-Haemocyanin of Helix pomatia

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1977.tb11313.x ◽

1977 ◽

Vol 73 (1) ◽

pp. 247-253 ◽

Cited By ~ 10

Author(s):

J. BERGER ◽

I. PILZ ◽

Raphael WITTERS ◽

Rene LONTIE

Keyword(s):

Small Angle ◽

Quaternary Structure ◽

Helix Pomatia ◽

X Ray ◽

X Ray Scattering ◽

Ray Scattering

Download Full-text

Nanoscale Structure Determination of Murray Valley Encephalitis and Powassan Virus Non-coding RNAs

10.1101/2020.01.10.901082 ◽

2020 ◽

Author(s):

Tyler Mrozowich ◽

Amy Henrickson ◽

Borries Demeler ◽

Trushar R Patel

Keyword(s):

Computational Modeling ◽

Small Angle ◽

Genetic Material ◽

Size Exclusion ◽

Low Resolution ◽

Modeling Tools ◽

X Ray ◽

X Ray Scattering ◽

Powassan Virus ◽

Ray Scattering

AbstractViral infections are responsible for numerous deaths worldwide. Flaviviruses, which contain RNA as their genetic material, are one of the most pathogenic families of viruses. There is an increasing amount of evidence suggesting that their 5’ and 3’ non-coding terminal regions are critical for their survival. In this study, the 5’ and 3’ terminal regions of Murray Valley Encephalitis and Powassan virus were examined using biophysical and computational modeling methods. First, the purity of in-vitro transcribed RNAs were investigated using size exclusion chromatography and analytical ultracentrifuge methods. Next, we employed small-angle X-ray scattering techniques to study solution conformation and low-resolution structures of these RNAs, which suggested that the 3’ terminal regions are highly extended, compared to the 5’ terminal regions for both viruses. Using computational modeling tools, we reconstructed 3-dimensional structures of each RNA fragment and compared them with derived small-angle X-ray scattering low-resolution structures. This approach allowed us to further reinforce that the 5’ terminal regions adopt more dynamic structures compared to the mainly double-stranded structures of the 3’ terminal regions.

Download Full-text

Investigation of Ribulose-1,5-bisphosphate Carboxylase-Oxygenase from Tobacco by Small Angle X-Ray Scattering: A Structural Model for the Enzyme in Solution

Zeitschrift für Naturforschung C ◽

10.1515/znc-1988-5-609 ◽

1988 ◽

Vol 43 (5-6) ◽

pp. 373-376 ◽

Cited By ~ 4

Author(s):

P. M. Abuja ◽

I. Pilz

Keyword(s):

Small Angle ◽

Quaternary Structure ◽

Higher Plants ◽

Maximum Diameter ◽

Radius Of Gyration ◽

Bisphosphate Carboxylase ◽

X Ray ◽

X Ray Scattering ◽

Inactive Form ◽

Ray Scattering

The quaternary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco (Nicotiana tabacum) was investigated in solution by means of small angle X-ray scattering. The most important molecular parameters as the radius of gyration (Rg) and the maximum diameter (Dmax) were determined. Both the active and the inactive form of the enzyme were measured at 5 °C and at 20 °C. A more distinct difference in size could be detected between the inactive forms at these two temperatures (Rg = 4.80 nm (5 °C) and 4.68 nm (20 °C)) than between the active forms (Rg = 4.73 nm and 4.69 nm). The maximum diameters were determined to be 13.1 nm for the inactive form at 5 °C and 12.8 nm for the other forms. A model is proposed consisting of eight large and eight small subunits arranged in the way that seems to be typical for this enzyme in higher plants.

Download Full-text