Abstract
The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS) has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome) protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N- and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.
Highlights
Small Angle X-ray Scattering (SAXS) is a powerful technique that is used to obtain structural information of both ordered and disordered biological molecules at low resolution
In contrast to the fungi Bcp1 involved in ribosome biogenesis, the human and mouse homologues seem to promote cell cycle arrest by enhancing the inhibition of CDK2 activity by CDKN1A/p21 [32] and may be required for DNA damage repair by homologous recombination together with BRCA2 [33]
SAXS is a solution technique that provides structural information on molecules such as proteins, and the resolution is low, it is very useful for evaluating conformational changes in response to external conditions and conformational ensembles in flexible proteins
Summary
Small Angle X-ray Scattering (SAXS) is a powerful technique that is used to obtain structural information of both ordered and disordered biological molecules at low resolution. It provides information about the size and shape of proteins and complexes, as well as about structural changes that occur at different experimental conditions [1]. SAXS requires small (milligram) amounts of purified and monodisperse samples. Even data analysis, when the quality of the sample is good enough, can be fast, thanks to powerful specialized software [3]. SAXS is a useful technique for the characterization of multidomain proteins [4], which consist of two or more domains connected
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