The role of calcium in the conformational dynamics and thermal stability of the D-galactose/D-glucose-binding protein from Escherichia coli.
We have characterized stability and conformational dynamics of the calcium depleted D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli. The structural stability of the protein was investigated by steady state and time resolved fluorescence, and far-UV circular dichroism in the temperature range from 20 degrees C to 70 degrees C. We have found that the absence of the Ca(2+) ion results in a significant destabilization of the C-terminal domain of the protein. In particular, the melting temperature decreases by about 10 degrees C with the simultaneous loss of the melting cooperativity. Time resolved fluorescence quenching revealed significant loosening of the protein when highly shielded Trp residue(s) became accessible to acrylamide at higher temperatures. We have documented a significant stabilizing effect of glucose that mostly reverts the effect of calcium, that is, the thermal stability of the protein increases by about 10 degrees C and the melting cooperativity is restored. Moreover, the protein structure remains compact with low amplitude of the segmental mobility up to high temperatures. We have used molecular dynamics to identify the structural feature responsible for changes in the temperature stability. Disintegration of the Ca(2+)-binding loop seems to be responsible for the loss of the stability in the absence of calcium. The new insights on the structural properties and temperature stability of the calcium depleted GGBP contribute to better understanding of the protein function and constitute important information for the development of new biotechnological applications of this class of proteins.
Herman
Petr
P
Faculty of Mathematics and Physics, Institute of Physics, Charles University, Prague, Czech Republic. herman@karlov.mff.cuni.cz
Vecer
Jaroslav
J
Barvik
Ivan
I
Scognamiglio
Viviana
V
Staiano
Maria
M
de Champdoré
Marcella
M
Varriale
Antonio
A
Rossi
Mose'
M
D'Auria
Sabato
S
D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology*; D002413:Cations, Divalent; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D005454:Fluorescence Polarization; D005690:Galactose / Q000737:chemistry / Q000494:pharmacology*; D005947:Glucose / Q000737:chemistry / Q000494:pharmacology*; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; C509064:mglB protein, E coli; D005947:Glucose; D002118:Calcium; D005690:Galactose