Study of amide-proton exchange of Escherichia coli melibiose permease by attenuated total reflection-Fourier transform infrared spectroscopy: evidence of structure modulation by substrate binding.
The accessibility of Escherichia coli melibiose permease to aqueous solvent was studied following hydrogen-deuterium exchange kinetics monitored by attenuated total reflection-Fourier transform infrared spectroscopy under four distinct conditions where MelB forms different complexes with its substrates (H(+), Na(+), melibiose). Analysis of the amide II band upon (2)H(2)O exposure discloses a significant sugar protection of the protein against aqueous solvent, resulting in an 8% less exchange of the corresponding H(+)*melibiose*MelB complex compared with the protein in the absence of sugar. Investigation of the amide I exchange reveals clear substrate effects on beta-sheet accessibility, with the complex H(+)*melibiose*MelB being the most protected state against exchange, followed by Na(+)*melibiose*MelB. Although of smaller magnitude, similar changes in alpha-helices plus non-ordered structures are detected. Finally, no differences are observed when analyzing reverse turn structures. The results suggest that sugar binding induces a remarkable compactness of the carrier's structure, affecting mainly beta-sheet domains of the transporter, which, according to secondary structure predictions, may include cytoplasmic loops 4-5 and 10-11. A possible catalytic role of these two loops in the functioning of MelB is hypothesized.
Dave
Natàlia
N
Unitat de Biofisica, Departament de Bioquimica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain.
Lórenz-Fonfría
Víctor A
VA
Villaverde
Joaquim
J
Lemonnier
Raymonde
R
Leblanc
Gérard
G
Padrós
Esteve
E
D000577:Amides / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D027981:Symporters / Q000737:chemistry / Q000378:metabolism*
D000577:Amides; D008081:Liposomes; D011510:Proteolipids; D011994:Recombinant Proteins; D027981:Symporters; C020485:proteoliposomes; C021548:melibiose permease