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COR15A and COR15B form a tandem repeat of highly homologous genes in Arabidopsis thaliana. Both genes are highly cold induced and the encoded proteins belong to the Pfam LEA_4 group (group 3) of the late embryogenesis abundant (LEA) proteins. Both proteins were predicted to be intrinsically disordered in solution. Only COR15A has previously been characterized and it was shown to be localized in the soluble stroma fraction of chloroplasts. Ectopic expression of COR15A in Arabidopsis resulted in increased freezing tolerance of both chloroplasts after freezing and thawing of intact leaves and of isolated protoplasts frozen and thawed in vitro. In the present study we have generated recombinant mature COR15A and COR15B for a comparative study of their structure and possible function as membrane protectants. CD spectroscopy showed that both proteins are predominantly unstructured in solution and mainly a-helical after drying. Both proteins showed similar effects on the thermotropic phase behavior of dry liposomes. A decrease in the gel to liquid-crystalline phase transition temperature depended on both the unsaturation of the fatty acyl chains and lipid headgroup structure. FTIR spectroscopy indicated no strong interactions between the proteins and the lipid phosphate and carbonyl groups, but significant interactions with the galactose headgroup of the chloroplast lipid monogalactosyldiacylglycerol. These findings were rationalized by modeling the secondary structure of COR15A and COR15B. Helical wheel projection indicated the presence of amphipathic a-helices in both proteins. The helices lacked a clear separation of positive and negative charges on the hydrophilic face, but contained several hydroxylated amino acids.
Summary Using five different steps, ;-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeniety with approximately 90-fold purificationwith a specific activity of 281 units mg;1 protein. A single bandwas observed in native PAGE. Activity staining of the native gel with 5-bromo4-chloro 3-indoxyl ;-D-galactopyranoside (X-Gal) at pH 4.0 also produceda single band. Analytical gel filtration in Superdex G-75 revealed the molecularmass of the native protein to be approximately 75 kD. 10 percnt; SDS-PAGE under reducingconditions showed two subunits of molecular masses, 45 and 30 kD, respectively.Hence, ;-galactosidase from kidney beans is a heterodimer. A typical proteinprofile with ;max at 280 nm was observed and A280/A260ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86 percnt; sequencehomology with an Arabidopsis thaliana and 85 percnt; with Lycopersiconesculentum putative ;-galactosidase sequences. The Electrospray MassSpectrometric analysis of this band also revealed a peptide fragment that had90 percnt; sequence homology with an Arabidopsis thaliana putative ;- galactosidasesequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometricanalysis both by MALDI- TOF and ES MS revealed certain sequences that matchedwith phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0and it hydrolysed o- and p-nitrophenyl ;-D galactopyranosidewith a Km value of 0.63 mmol/L and 0.74 mmol/L, respectively.The energy of activation calculated from the Arrhenius equation was 14.8 kcal/molenzyme site. The enzyme was found to be comparatively thermostable showing maximumactivity at 67 °C. Thermal denaturation of the enzyme at 65 °C obeyssingle exponential decay with first order-rate constant 0.105 min;1.Galactose, a hydrolytic product of this enzyme was a competitive inhibitor witha Ki of 2.7 mmol/L.
Bacteriophage Sf6 tailspike protein is functionally equivalent to the well characterized tailspike ofSalmonella phage P22, mediating attachment of the viral particle to host cell-surface polysaccharide. However, there is significant sequence similarity between the two 70-kDa polypeptides only in the N-terminal putative capsid-binding domains. The major, central part of P22 tailspike protein, which forms a parallel ;-helix and is responsible for saccharide binding and hydrolysis, lacks detectable sequence homology to the Sf6 protein. After recombinant expression in Escherichia coli as a soluble protein, the Sf6 protein was purified to homogeneity. As shown by circular dichroism and Fourier transform infrared spectroscopy, the secondary structure contents of Sf6 and P22 tailspike proteins are very similar. Both tailspikes are thermostable homotrimers and resist denaturation by SDS at room temperature. The specific endorhamnosidase activities of Sf6 tailspike protein toward fluorescence-labeled dodeca-, deca-, and octasaccharide fragments of Shigella O-antigen suggest a similar active site topology of both proteins. Upon deletion of the N-terminal putative capsid-binding domain, the protein still forms a thermostable, SDS-resistant trimer that has been crystallized. The observations strongly suggest that the tailspike of phage Sf6 is a trimeric parallel ;-helix protein with high structural similarity to its functional homolog from phage P22.