Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen - Analytical Methods & Tools for Lyophilization

Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen
Received 17 February 2006; accepted 11 April 2006. Available online 5 June 2006.
Hongmin Lia, b, , , Yiwei Zhaoa, 1, Yi Guoa, Sandra J. VanVrankena, Zhong Lia, Leslie Eiselea and Walid Mouradc
Molecular Immunology
Volume 44, Issue 5 , February 2007
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Copyright ? 2006 Elsevier Ltd All rights reserved.
aWadsworth Center, New York State Department of Health, Empire State Plaza, PO Box 509, Albany, NY 12201-0509, United States
bDepartment of Biomedical Sciences, School of Public Health, University at Albany, State University of New York, Empire State Plaza, PO Box 509, Albany, NY 12201-0509, United States
cUniversit? de Montreal, CHUM, Campus St-Luc, PEA, 264, Boul. Ren? L?vesque Est, Bureau 313, Montr?al, Qu?. H2X 1P1, Canada
Abstract
Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen (SAg) that can activate large fractions of T cells bearing particular TCR V? elements. Here we report the mutagenesis, biochemical and biophysical studies on the dimerization of MAM in solution. Our studies showed that although MAM mainly exists as a monomer in solution, a small percentage of MAM molecules form homodimer at high protein concentration, regardless of the presence of Zn2+. A distinct peak corresponding to a MAM homodimer was detected in the presence of EDTA, using both chemical cross-linking and analytical ultracentrifugation methods. Further mutagenesis studies revealed that single mutation of residues at the interface of the crystallographic dimer of MAM does not significantly affect the dimerization of MAM in solution. Circular dichroism (CD) analysis indicated that addition of Zn2+ does not induce conformational changes of MAM from its apo-state. Thermal denaturation experiments indicated that addition of Zn2+ to MAM solution resulted in a decrease of melting point (Tm), whereas addition of EDTA did not affect the Tm of MAM. These results imply that there is no defined Zn2+-binding site on MAM.
Keywords: MAM; Superantigen; Dimerization; Analytical ultracentrifugation; Sedimentation; Cross-linking; CD
Abbreviations: MAM, Mycoplasma arthritidis-derived mitogen; SAg, superantigen; TCR, T-cell receptor; EDTA, ethylendiaminetetraacetic acid; CD, circular dichroism; Tm, melting point; MHC, major histocompatibility complex; HA, haemagglutinin peptide 306?318; APC, antigen-presenting cell; DSG, disuccinimidyl glutarate; DMSO, dimethyl sulfoxide; SV, sedimentation velocity; AUC, analytical ultracentrifuge; HBS, Hepes-buffered saline; S(20,w), sedimentation coefficient; KD, dissociation constant
Corresponding author. Tel.: +1 518 486 9154; fax: +1 518 474 7992.
1 Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Ave., Boston, MA 02115, United States.
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