Secondary and tertiary structure changes of reconstituted LmrA induced by nucleotide binding or hydrolysis. A fourier transform attenuated total reflection infrared spectroscopy and tryptophan fluorescence quenching analysis.
LmrA, a membrane protein of Lactococcus lactis, extrudes amphiphilic compounds from the inner leaflet of the cytoplasmic membrane, using energy derived from ATP hydrolysis. A combination of total reflection Fourier transform infrared spectroscopy, (2)H/H exchange, and fluorescence quenching experiments was used to investigate the effect of nucleotide binding and/or hydrolysis on the structure of LmrA reconstituted into proteoliposomes. These measurements allowed us to describe secondary structure changes of LmrA during the catalytic cycle. The structure of LmrA is enriched in beta-sheet after ATP binding, and the protein recovers its initial secondary structure after ATP hydrolysis, when P(i) has been released. (2)H/H exchange and fluorescence quenching studies indicate that the protein undergoes two distinct tertiary structure changes during the hydrolysis process. Indeed, the protein alone is poorly accessible to the aqueous medium but adopts a more accessible conformation when ATP hydrolysis takes place. After ATP hydrolysis, but when P(i) is still associated with the protein, the accessibility is intermediate between these two states.
Vigano
C
C
Laboratoire de Chimie Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, B1050 Brussels, Belgium. catherine.vigano@ulb.ac.be
Margolles
A
A
van Veen
H W
HW
Konings
W N
WN
Ruysschaert
J M
JM
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D001426:Bacterial Proteins; D005453:Fluorescence; D006868:Hydrolysis; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared; D014364:Tryptophan
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D014364:Tryptophan; C101824:LmrA protein, Lactococcus lactis