Tritium NMR spectroscopy of ligand binding to maltose-binding protein

Abstract

Tritium-labeled alpha- and beta-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR spectroscopy of the labeled sugars showed large upfield chemical shift changes upon binding and strong anomeric specificity. At 10 degrees C, MBP bound alpha-maltose with 2.7 +/- 0.5-fold higher affinity than beta-maltose, and, for longer maltodextrins, the ratio of affinities (KD beta/KD alpha) was even larger (between 10 and 30). The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound alpha-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound beta-maltotriose resonances in rapid exchange. We interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the beta-maltodextrin is bound by its reducing end, and, in the other complex, the beta-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins.

Role of Water in Ligand Binding to Maltose-Binding Protein: Insight from a New Docking Protocol Based on the 3D-RISM-KH Molecular Theory of Solvation

“…In particular, we describe an alternative binding mode of maltotriose at the open state of MBP, the existence of which was previously suggested based on NMR spectroscopic experiments. 7 We show that such a binding event results in triggering the domains motion of MBP and evolution of the structure toward the holo-closed state, which is in excellent agreement with the experimental X-ray structure. 11 Validating the ability of this new approach to provide systematic improvement of docking results for a general pool of biomolecular systems where structural solvation plays an .…”

Two Modes of Ligand Binding in Maltose-binding Protein ofEscherichia coli

“…In contrast, when it bound to MBP purely through the B mode, it failed to be transported through a wild type transporter complex, even when the binding to MBP occurred with very high affinity. We have also argued (1) that the R and B modes most likely correspond to the "end-on" and "middle" modes of binding, previously defined on the basis of NMR chemical shift of the 3 H atom on the anomeric carbon of the ligand molecules (3).…”

“…This correlation suggests that the R and B modes may produce closing and nonclosing of the two lobes of MBP, respectively. However, because fluorescence, UV absorption, and chemical shift of the anomeric 3 H are all influenced mainly by the local environment surrounding the ligand, these results did not provide concrete data on the global conformation of MBP.…”

NMR-Active Nuclei for Biological and Biomedical Applications

“…At a temperature of 10 ºC, MBP bound α-maltose with 2.7 ± 0.5-fold higher affinity than -maltose and longer maltodextrins had a ratio of affinities (Kd /Kdα) that was significantly greater (10-to 30-fold). Further interpretation of the spectra also revealed how MBP is able to bind both linear and circular maltodextrins [59]. 3 H NMR has been used to study two nucleic acid molecules, an 8 kDa DNA oligomer and a 20 kDa 'hammer-head' RNA.…”

“…This Figure Co NMR has been used for in vivo thermometry with liposomes containing cobalt complexes, which is useful for making localised temperature measurements in tissue during hyperthermia treatment of cancer. The method uses the complex tris(ethylenediamine) cobalt(III) trichloride as a temperature sensor by determining the temperature dependence of its 59 Co NMR chemical shift (Figure 19). Encapsulation within liposomes allows targeting of the agent to the reticuloendothelial system and temperature changes in the order of 0.1 ºC have been measured in vivo in rats [268].…”