Intra- versus intermolecular interactions in monellin: contribution of surface charges to protein assembly.
The relative significance of weak non-covalent interactions in biological context has been much debated. Here, we have addressed the contribution of Coulombic interactions to protein stability and assembly experimentally. The sweet protein monellin, a non-covalently linked heterodimeric protein, was chosen for this study because of its ability to spontaneously reconstitute from separated fragments. The reconstitution of monellin mutants containing large surface charge perturbations was compared to the thermostability of structurally equivalent single-chain monellin containing the same sets of mutations under varying salt concentrations. The affinity between monellin fragments is found to correlate with the thermostability of single chain monellin, indicating the involvement of the same underlying Coulombic interactions. This confirms that there are no principal differences in the interactions involved in folding and binding. Based on comparison with a previous mutational study involving hydrophobic core residues, the relative contribution of Coulombic interactions to stability and affinity is modest. However, the Coulombic perturbations only affect the association rates of reconstitution in contrast to perturbations involving hydrophobic residues, which affect primarily the dissociation rates. These results indicate that Coulombic interactions are likely to be of main importance for the association of protein assembly, relevant for functions of proteins.
Department of Biophysical Chemistry, Center for Chemistry and Chemical Engineering, Lund University, Lund SE-22100, Sweden. wei-feng.xue@bpc.lu.se
Wei-Feng
WF
Xue
Olga
O
Szczepankiewicz
Mikael C
MC
Bauer
Eva
E
Thulin
Sara
S
Linse
D004355:Drug Stability; D029583:Menispermaceae / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics
D010940:Plant Proteins; D011994:Recombinant Proteins; C002541:monellin protein, Dioscoreophyllum cumminsii