A bacterial TrwC relaxase domain contains a thermally stable alpha-helical core.
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 degrees C for 10 min. Infrared spectroscopy shows that this domain has a predominantly alpha/beta 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 alpha-helix percentage is not altered and alpha-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 alpha-helix core resistant to temperature and responsible for folding after fast heating and an outer layer of beta-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.
Arrondo
José-Luis R
JL
Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, E-48080 Bilbao, Spain. gbproarj@lg.ehu.es
Echabe
Izaskun
I
Iloro
Ibón
I
Hernando
Miguel-Angel
MA
de la Cruz
Fernando
F
Goñi
Félix M
FM
D001426:Bacterial Proteins; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D045522:Recombinases; D013055:Spectrophotometry, Infrared / Q000379:methods; D013329:Structure-Activity Relationship; D013696:Temperature
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D045522:Recombinases; D004254:DNA Nucleotidyltransferases; D004706:Endodeoxyribonucleases; C120220:MobM protein, bacteria