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Journal of Bacteriology, July 2003, p. 4226-4232, Vol. 185, No. 14
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.14.4226-4232.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

A Bacterial TrwC Relaxase Domain Contains a Thermally Stable -Helical Core

Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, E-48080 Bilbao,¹ Departamento de Biología Molecular (Unidad Asociada al C.I.B., CSIC), Universidad de Cantabria, E-39011, Santander, Spain²

Received 10 February 2003/ Accepted 16 April 2003

The TrwC protein is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. The TrwC-N275 fragment comprises the 275-amino-acid N-terminal domain of the protein that contains the DNA cleavage and strand transfer activities (the relaxase domain). It can be easily purified by keeping a cell lysate at 90°C for 10 min. Infrared spectroscopy shows that this domain has a predominantly /ß structure with some amount of unordered structure. Fast heating and cooling does not change the secondary structure, whereas slow heating produces two bands in the infrared spectrum characteristic of protein aggregation. The denaturation temperature is increased in the protein after the fast-heating thermal shock. Two-dimensional infrared correlation spectroscopy shows that thermal unfolding is a very cooperative two-state process without any appreciable steps prior to aggregation. After aggregation, the -helix percentage is not altered and -helix signal does not show in the correlation maps, meaning that the helices are not affected by heating. The results indicate that the domain has an -helix core resistant to temperature and responsible for folding after fast heating and an outer layer of ß-sheet and unordered structure that aggregates under slow heating. The combination of a compact core and a flexible outer layer could be related to the structural requirements of DNA-protein binding.

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