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J. Biol. Chem., Vol. 280, Issue 24, 23032-23040, June 17, 2005

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Monoclonal Antibody Epitope Mapping Describes Tailspike -Helix Folding and Aggregation Intermediates^*

Madhulika Jain, Michael S. Evans¶, Jonathan King, and Patricia L. Clark¶||

From the ^¶Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

There is growing interest in understanding how the cellular environment affects protein folding mechanisms, but most spectroscopic methods for monitoring folding in vitro are unsuitable for experiments in vivo or in other complex mixtures. Monoclonal antibody binding represents a sensitive structural probe that can be detected against the background of other cellular components. A panel of antibodies has been raised against Salmonella typhimurium phage P22 tailspike. In this report, nine -tailspike antibody binding epitopes were characterized by measuring the binding of these monoclonal antibodies to tailspike variants bearing surface point mutations. These results reveal that the antibody epitopes are distributed throughout the tailspike structure, with several clustered in the central parallel -helix domain. The ability of each antibody to distinguish between tailspike conformational states was assessed by measuring antibody binding to tailspike in vitro refolding intermediates. Interestingly, the binding of all but one of the nine antibodies is sensitive to the tailspike conformational state. Whereas several antibodies bind preferentially to the tailspike native structure, the structural features that comprise the binding epitopes form with different rates. In addition, two antibodies preferentially recognize early refolding intermediates. Combined with the epitope mapping, these results indicate portions of the -helix form early during refolding, perhaps serving as a scaffold for the formation of additional structure. Finally, three of the antibodies show enhanced binding to non-native, potentially aggregation-prone tailspike conformations. The refolding results indicate these non-native conformations form early during the refolding reaction, long before the appearance of native tailspike.

Received for publication, February 22, 2005 , and in revised form, April 11, 2005.

^* This work was supported in part by National Institutes of Health Grant GM17980 (to J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplementary Fig. S1.

Present address: Dept. of Biochemistry and Biophysics, Box 2200, University of California at San Francisco, San Francisco, CA 94143-2200.

^|| Supported by National Institutes of Health Postdoctoral Fellowship GM19715 at Massachusetts Institute of Technology and by an award from the Clare Boothe Luce Program of the Luce Foundation at the University of Notre Dame. To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556. Tel.: 574-631-8353; Fax: 574-631-6652; E-mail: pclark1{at}nd.edu.

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