Open and Closed Form of Maltose Binding Protein in Its Native and Molten Globule State As Studied by Electron Paramagnetic Resonance Spectroscopy

Abstract

An intensively investigated intermediate state of protein folding is the molten globule (MG) state, which contains secondary but hardly any tertiary structure. In previous work, we have determined the distances between interacting spins within maltose binding protein (MBP) in its native state using continuous wave and double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy. Seven double mutants had been employed to investigate the structure within the two domains of MBP. DEER data nicely corroborated the previously available X-ray data. Even in its MG state, MBP is known to still bind its ligand maltose. We therefore hypothesized that there must be a defined structure around the binding pocket of MBP already in the absence of tertiary structure. Here we have investigated the functional and structural difference between native and MG state in the open and closed form with a new set of MBP mutants. In these, the spin-label positions were placed near the active site. Binding of its ligands leads to a conformational change from open to closed state, where the two domains are more closely together. The complete set of MBP mutants was analyzed at pH 3.2 (MG) and pH 7.4 (native state) using double-quantum coherence EPR. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structures of MBP in open and closed form and were found to be in excellent agreement. Measurements show a defined structure around the binding pocket of MBP in MG, which explains maltose binding. A new and important finding is that in both states ligand-free MBP can be found in open and closed form, while ligand-bound MBP appears only in closed form because of maltose binding.

Figure 1:

Overview of the complete set of MBP mutants in open state MBP (PDB: 1OMP). Blue lines indicate C(alpha)-amino-acid-distances of mutants MBP 01 – 06. Green lines indicate C(alpha)-amino-acid-distances of mutants MBP 07 – 11.

Figure 2:

Distance distribution of MTS distances for MBP 07 (P298C S238C) pH 7 without maltose. The rotamers of closed and open state MBP are marked.

Figure 3:

Distance distribution of MTS distances in mutants MBP 01 – 11 in native (pH 7) and MG (pH 3.2) state with and without maltose. More rotamers can be seen in measurements without maltose (blue & green) indicating open state distances.

Figure 3:

Distance distribution of MTS distances in mutants MBP 01 – 11 in native (pH 7) and MG (pH 3.2) state with and without maltose. More rotamers can be seen in measurements without maltose (blue & green) indicating open state distances.