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Protein Science (2007), 16:1543-1556. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Domain folding and flexibility of Escherichia coli FtsZ determined by tryptophan site-directed mutagenesis

Rodrigo Díaz-Espinoza1,3, Andrea P. Garcés1,3, José J. Arbildua1,3, Felipe Montecinos1, Juan E. Brunet2, Rosalba Lagos1, and Octavio Monasterio1

1 Laboratorio de Biología Estructural y Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
2 Instituto de Química, Facultad de Ciencias Básicas y Matemáticas, Pontificia Universidad Católica de Valparaíso, Casilla 4059, Valparaíso, Chile

(RECEIVED February 5, 2007; FINAL REVISION April 26, 2007; ACCEPTED May 21, 2007)

FtsZ has two domains, the amino GTPase domain with a Rossmann fold, and the carboxyl domain that resembles the chorismate mutase fold. Bioinformatics analyses suggest that the interdomain interaction is stronger than the interaction of the protofilament longitudinal interfaces. Crystal B factor analysis of FtsZ and detected conformational changes suggest a connection between these domains. The unfolding/folding characteristics of each domain of FtsZ were tested by introducing tryptophans into the flexible region of the amino (F135W) and the carboxyl (F275W and I294W) domains. As a control, the mutation F40W was introduced in a more rigid part of the amino domain. These mutants showed a native-like structure with denaturation and renaturation curves similar to wild type. However, the I294W mutant showed a strong loss of functionality, both in vivo and in vitro when compared to the other mutants. The functionality was recovered with the double mutant I294W/F275A, which showed full in vivo complementation with a slight increment of in vitro GTPase activity with respect to the single mutant. The formation of a stabilizing aromatic interaction involving a stacking between the tryptophan introduced at position 294 and phenylalanine 275 could account for these results. Folding/unfolding of these mutants induced by guanidinium chloride was compatible with a mechanism in which both domains within the protein show the same stability during FtsZ denaturation and renaturation, probably because of strong interface interactions.

Keywords: FtsZ; folding; protein flexibility; GTPase


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