Escherichia coli maltose binding protein (MBP) is commonly used to promote the solubility of its fusion partners. To investigate the mechanism of solubility enhancement by MBP, we compared the properties of MBP fusion proteins refolded in vitro with those of the corresponding fusion proteins purified under native conditions. We fused five aggregation-prone passenger proteins to 3 different N-terminal tags: His6-MBP, His6-GST and His6. After purifying the 15 fusion proteins under denaturing conditions and refolding them by rapid dilution, we recovered far more of the soluble MBP fusion proteins than their GST- or His-tagged counterparts. Hence, we can reproduce the solubilizing activity of MBP in a simple in vitro system, indicating that no additional factors are required to mediate this effect. We assayed both the soluble fusion proteins and their TEV protease digestion products (i.e., with the N-terminal tag removed) for biological activity. Little or no activity was detected for some fusion proteins whereas others were quite active. When the MBP fusions proteins were purified from E. coli under native conditions they were all substantially active. These results indicate that the ability of MBP to promote the solubility of its fusion partners in vitro sometimes, but not always, results in their proper folding. We show that the folding of some passenger proteins is mediated by endogenous chaperones in vivo. Hence, MBP serves as a passive participant in the folding process; passenger proteins either fold spontaneously or with the assistance of chaperones.

Nuclear magnetic resonance (NMR) spectroscopy of protons in protonated solids is challenging. Fast magic angle spinning (MAS) and homonuclear decoupling schemes, in conjunction, with high magnetic fields have improved the proton resolution. However, experiments to quantitatively measure 1 H− 1 H distances still remain elusive due to the dense proton−proton dipolar coupling network. A novel MAS solidstate NMR pulse sequence is proposed to selectively recouple and measure interproton distances in protonated samples. The phase-modulated sequence combined with a judicious choice of transmitter frequency is used to measure quantitative 1 H− 1 H distances on the order of 3 Å in Lhistidine•HCl•H 2 O, despite the presence of other strongly coupled protons. This method provides a major boost to NMR crystallography approaches for structural determination of pharmaceutical molecules by directly measuring 1 H− 1 H distances. The band-selective nature of the sequence also enables observation of selective 1 H− 1 H correlations (e.g., H N −H N /H N −H α /Η Ν −Η Methyl ) in peptides and proteins, which should serve as useful restraints in structure determination.

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