Biophysical Characterization and Conformational Analysis of the GTPase Domain of Pre-Protein Import Receptors atToc33G and psToc34G

Abstract

atToc33 and psToc34 are GTPases-, that act as receptors for pre-protein import in the chloroplast outer envelope. Various lines of evidence suggest that the GTPase domain of atToc33 (atToc33G) dimerizes, including native PAGE studies (Weibel et al., 2003), gel-filtration experiments (Yeh et al., 2007) and filter binding assays (Koenig et al., 2008). The crystal structure of atToc33 shows that it is a monomer (Koenig et al., 2008) while psToc34 crystallizes as a dimer (Sun et al., 2002). The R130A atToc33 mutant apparently leads to monomer formation in vitro (Reddick et al., 2007; Weibel et al., 2003). Both-, atToc33-wt and atToc33-R130A bind GTP and GDP with high affinity and hydrolyse GTP with similar efficiencies (Weibel et al., 2003). To further investigate the conformations of these GTPases, and to characterize their optical spectroscopic properties, we first, expressed atToc33G and its R130A mutant in high yield. Initial characterization of these proteins by CD spectroscopy revealed significant α-helical content in their secondary structure. We also examined the effect of an increase in protein concentration of atToc33G and its R130A mutant using CD and fluorescence spectroscopy. CD spectral line shape does not change with increasing protein concentrations indicating that no or only small conformational changes occurs as the concentration increases. Fluorescence spectra for both proteins are superimposable with an intensity maximum at ∼320nm. The addition of GDP to both the proteins leads to reduction of intensity. No wavelength shift in emission spectrum was observed. Our spectroscopic results and anisotropy experiments show that both atToc33G and atToc33G R130A are monomeric, irrespective of the protein concentration and also in presence of GDP or GTP. Experiments to compare atToc33G and atToc33G R130A with their corresponding homologues from Pisumsativum are also presented.