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| The Secondery Structure Study of Iron-binding Protein MtsA from Streptococcus pyogenes |
| WANG Hong-cui, ZHANG Jing, XU Qian, XU Li-na, WANG Nan-jie, SUN Xue-song |
| Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, China |
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Abstract Streptococcus pyogenes is a Gram-positive human pathogen, and iron is essential for its survival and infection. MtsA is a lipoprotein of Streptococcus pyogenes, which is responsible for iron binding. MtsA was amplified by PCR from Streptococcus pyogenes MGAS5005 and constructed the recombinant plasmid pGEX-MtsA. The recombinant plasmid was transformed into Escherichia coli BL21 to express the fusion protein after induction with IPTG. The protein was purified using affinity chromatography. The conservative of the MtsA iron binding center was analyzed using multiple alignment. The mutant proteins were constructed by site-directed mutagenesis. Circular dichroism was used to collect the changes of mutants’ secondery structure when compared to wild-type protein. The result of multiple alignment showed the four binding amino acids were conserved and were in the hollow of MtsA space structure. The CD spectra of WT MtsA and mutants were collected respectively. The α-helix content increased when Fe2+ was added to the apo-protein solution, which indicated that the metal binding induced some conformational change. For the apo mutant proteins H68A, E206A and D281A, the contents of α-helix more or less decreased, reflecting that mutations caused alterations of the secondary structures at some extends but not substantially disturbed the protein conformation. The secondary-structural change in mutant H140A was unexpectedly barely detectable when compared to WT MtsA. These results provided a valuable information for the understanding of iron transport in bacteria, which may be helpful for the development of novel strategies in the control of bacterial infection.
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Received: 20 September 2012
Published: 25 December 2012
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