The role of calcium in the conformational dynamics and thermal stability of the D‐galactose/D‐glucose‐binding protein from Escherichia coli

Abstract

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.

D‐Trehalose/D‐maltose‐binding protein from the hyperthermophilic archaeon Thermococcus litoralis: The binding of trehalose and maltose results in different protein conformational states

“…The smaller k q increase than the predicted one is a consequence of Trp shielding and a sign of dense residue packing. Similar behavior has previously been found for the glucose‐galactose binding protein in the presence of glucose 39. Quenching data indicate that at 90°C the TMBP structure partially opens, which results in a dramatic increase of the bimolecular quenching constant.…”

Thermal Motions of the E. coli Glucose-Galactose Binding Protein Studied Using Well-Sampled Semi-Atomistic Simulations

“…Additionally, it was also recently observed that the presence of Ca +2 in this protein enhances its thermal stability – neglecting this in our model could have an effect as well. [24] It is also possible that by adding additional chemistry to our coarse-grained model, such as Ramachandran potentials, hydrogen bonding, or residue-specific interactions, we might be able to observe a more realistic sampling of the fluctuations. We are currently working on such improvements.…”

Pressure effect on the stability and the conformational dynamics of the D‐Galactose/D‐Glucose‐binding protein from Escherichia coli

“…The net of residues involved in the Ca 2ϩ coordination is not able to prevent calcium depletion. A previous work 29 showed that Ca 2ϩ depletion markedly reduces the thermal stability of the whole GGBP protein and especially the stability of the C-terminal domain. Although the metal ion is inserted in a binding site at the surface of the protein, it exerts a stabilizing effect that encompasses the whole structure of the protein.…”

“…Binding of glucose results in a minor 5% fluorescence quenching (data not shown), which is consistent with data published on the calcium depleted GGBP. 29 This quenching is probably the result of a stacking interaction between the pyranose ring of the bound sugar and the aromatic residues Phe 16 and Trp 183 of the protein. 13…”