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Published online before print May 7, 2004, 10.1110/ps.03579304
Protein Science (2004), 13:1538-1546. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Pressure dissociation studies provide insight into oligomerization competence of temperature-sensitive folding mutants of P22 tailspike

Brian G. Lefebvre, Noelle K. Comolli, Matthew J. Gage and Anne Skaja Robinson

Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA

(RECEIVED December 17, 2003; FINAL REVISION February 24, 2004; ACCEPTED February 24, 2004)



Abstract

Several temperature-sensitive folding (tsf) mutants of the tailspike protein from bacteriophage P22 have been found to fold with lower efficiency than the wild-type sequence, even at lowered temperatures. Previous refolding studies initiated from the unfolded monomer have indicated that the tsf mutations decrease the rate of structured monomer formation. We demonstrate that pressure treatment of the tailspike aggregates provides a useful tool to explore the effects of tsf mutants on the assembly pathway of the P22 tailspike trimer. The effects of pressure on two different tsf mutants, G244R and E196K, were explored. Pressure treatment of both G244R and E196K aggregates produced a folded trimer. E196K forms almost no native trimer in in vitro refolding experiments, yet it forms a trimer following pressure in a manner similar to the native tailspike protein. In contrast, trimer formation from pressure-treated G244R aggregates was not rapid, despite the presence of a G244R dimer after pressure treatment. The center-of-mass shifts of the fluorescence spectra under pressure are nearly identical for both tsf aggregates, indicating that pressure generates similar intermediates. Taken together, these results suggest that E196K has a primary defect in formation of the {beta}-helix during monomer collapse, while G244R is primarily an assembly defect.

Keywords: P22 tailspike; temperature-sensitive mutations; protein folding kinetics; aggregation; hydrostatic pressure

Reprint requests to: Anne Skaja Robinson, Colburn Laboratory, Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA; e-mail: robinson{at}che.udel.edu; fax: (302) 831-1048.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03579304.


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