Abstract
High-resolution structures of photosynthetic pigment–protein complexes are often determined using crystallography or cryo-electron microscopy (cryo-EM), which are restricted to the use of protein crystals or to low temperatures, respectively. However, functional studies and biotechnological applications of photosystems necessitate the use of proteins isolated in aqueous solution, so that the relevance of high-resolution structures has to be independently verified. In this regard, small-angle neutron and X-ray scattering (SANS and SAXS, respectively) can serve as the missing link because of their capability to provide structural information for proteins in aqueous solution at physiological temperatures. In the present review, we discuss the principles and prototypical applications of SANS and SAXS using the photosynthetic pigment–protein complexes phycocyanin (PC) and Photosystem I (PSI) as model systems for a water-soluble and for a membrane protein, respectively. For example, the solution structure of PSI was studied using SAXS and SANS with contrast matching. A Guinier analysis reveals that PSI in solution is virtually free of aggregation and characterized by a radius of gyration of about 75 Å. The latter value is about 10% larger than expected from the crystal structure. This is corroborated by an ab initio structure reconstitution, which also shows a slight expansion of Photosystem I in buffer solution at room temperature. In part, this may be due to conformational states accessible by thermally activated protein dynamics in solution at physiological temperatures. The size of the detergent belt is derived by comparison with SANS measurements without detergent match, revealing a monolayer of detergent molecules under proper solubilization conditions.
Highlights
A proper understanding of protein function requires detailed knowledge about the three-dimensional static protein structure
Before we proceed with the solution structure of the Photosystem I (PSI)–detergent complex determined from the SANS data, we recall that our earlier SAXS data [62] indicated the presence of free detergent micelles in the preparation, which were visible as a distinct peak at about 0.17 Å−1 corresponding to a small spherical particle with an average size of only about 35–40 Å
InIn this short review, we have presented selected applications forfor asas powerful experimental tool to study protein structures in aqueous solution at physiological powerful experimental tool to study protein structures in aqueous solution at physiologtemperatures using the photosynthetic pigment–protein complexes phycocyanin (PC) and ical temperatures using the photosynthetic pigment–protein complexes phycocyanin (PC)
Summary
A proper understanding of protein function requires detailed knowledge about the three-dimensional static protein structure. The general relevance of the high-resolution structures for solubilized PSI has to be independently verified, because the protein structure may generally deviate from its crystal structure in buffer solution at physiological temperatures [16,17,18,19] In this regard, small-angle neutron and X-ray scattering (SANS and SAXS, respectively) become the missing link by providing structural information on proteins in aqueous solution at physiological temperatures [20,21,22], including membrane proteins solubilized using detergent molecules [21]. One can use Pymol program [47] to visualize and compare the results of the Dammif analysis with the known pdb structure of a given protein complex While both SANS and SAXS techniques provide information about the size and shape of biomolecules, their nature of interaction with a sample is different. The purification of PSI from T. elongatus was performed as described earlier [15,52,53] so that the results are directly comparable to [15]
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