Abstract
As an essential component of protein cofactors, iron is important for all photosynthetic organisms. In Chlamydomonas reinhardtii, iron levels are strictly controlled by proteins such as iron-assimilating protein 1 (FEA1). This periplasmic protein is expressed under conditions of iron deficiency, but its mechanisms of function remain unknown. Because FEA1 has no amino-acid similarity to protein structures in the Protein Data Bank, its crystal structure cannot be solved by molecular replacement. Here, recombinant FEA1 protein lacking the N-terminal signal sequence was successfully purified and crystals of apo FEA1 were obtained by hanging-drop vapor diffusion. Neither selenomethionine substitution nor heavy-atom derivatization was successful; therefore, the phase problem of FEA1 crystals was solved by the native sulfur SAD method using long-wavelength X-rays (2.7 Å). Laser-cutting technology was used to increase the signal-to-noise ratio and derive an initial structure. This study will lead to further structural studies of FEA1 to understand its function and its links to the iron-assimilation pathway.
Highlights
The green alga Chlamydomonas reinhardtii is important in many fields of research, including metal metabolism
Metals play essential roles in cells as part of protein cofactors; their concentrations are tightly controlled as an excess amount can be toxic, while a deficiency leads to inactive metalloenzymes (Hanikenne, 2003; Merchant et al, 2006)
Rubinelli and coworkers initially characterized the regulation of the expression of HCR1-like protein (H43), which is induced by an iron-deficient medium (Rubinelli et al, 2002)
Summary
The green alga Chlamydomonas reinhardtii is important in many fields of research, including metal metabolism. FEA1 is a homologue of HCR1 from the marine alga Chlorococcum littorale Both proteins are highly induced by high CO2 levels and iron deficiency (Rubinelli et al, 2002; Kobayashi et al, 1997; Baba et al, 2011; Sasaki et al, 1998). Studies have shown that the abundance of FEA1 mRNA and protein is greatly increased under conditions of iron deficiency in C. reinhardtii (Allen et al, 2007; Urzica et al, 2012). Protein structures are mostly solved by molecular replacement; proteins with no homology to structures in the Protein Data Bank (PDB) require experimental phasing One such phasing method is single-wavelength anomalous dispersion (SAD) using selenomethionine (SeMet) or heavy-atom derivatized crystals. We report the crystallization and successful phasing of FEA1 using SSAD at a long wavelength of 2.7 Acoupled with laser-cutting technology
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