Enteropeptidase is a serine protease used in different biotechnological applications. For many applications the smaller light chain can be used to avoid the expression of the rather large holoenzyme. Recombinant human enteropeptidase light chain (hEPL) shows high activity but low solubility and refolding yields, currently limiting its use in biotechnological applications. Here we describe several protein modifications that lead to improved solubility and refolding yield of human hEPL whilst retaining the enzyme activity. Specifically, protein surface supercharging (N6D, G21D, G22D, N141D, K209E) of the protein increased the solubility more than 100-fold. Replacement of a free cysteine residue with serine (C112S) improved the refolding yield by 50%. The heat stability of this C112S variant was also significantly improved by supercharging. This study shows that even mild protein surface supercharging can have pronounced effects on protein solubility and stability.

Proteoliposomes represent nanoscale assemblies of indispensable value for studying membrane proteins in general and membrane transporters in particular. Since no universal protocol exists, conditions for proteoliposome formation must be determined on a case-by-case basis. This process will be significantly expedited if the size and composition of the assemblies can be analyzed in a single step using only microliters of sample. Here we show that dual-color fluorescence cross-correlation spectroscopy (FCCS) is of great value for optimizing the reconstitution process, because it distinguishes micelles, liposomes and aggregates in heterogeneous mixtures and permits direct monitoring of the co-localization of proteins and lipids in the diffusing assemblies. As proof-of-principle, liposomes containing the functional multidrug resistance transporter NorA from Staphylococcus aureus were prepared, demonstrating that FCCS is an excellent tool to guide the development of reconstitution protocols.

The highly specific serine protease human enteropeptidase light chain cleaves the Asp4Lys recognition sequence and represents an interesting enzyme for biotechnological applications. The human enzyme shows 10 times faster kinetics compared to other animal sources but low solubility under low salt conditions, which hampers protein production and crystallization. Therefore, a supercharged variant (N6D/G21D/G22D/N142D/K210E/C112S) with increased solubility was used for crystallization. The structure (resolution, 1.9 Å) displays a typical α/β trypsin-like serine protease-fold. The mutations introduced for protein supercharging generate larger clusters of negative potential on both sites of the active cleft but do not affect the structural integrity of the protein.

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Synthetic model peptides such as GWALP23 (acetyl-GGALW 5 LALALALA LALALW 19 LAGA-amide)provide a useful host framework for investigations of the influence of polar amino acids, for example histidine residues, within the hydrophobic core of a transmembrane helix. Importantly, membranespanning GWALP23 is quite sensitive to single-residue replacements, in part because the transmembrane helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2 H quadrupolar splitting (Biophys. J. 101, 2939). We inserted His residues into position 12 and/or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2 H-Ala labels within the helical core sequence. Solid-state 2 H NMR spectra of GWALP23-H12 reveal a marked difference in peptide behavior between acidic and neutral pH conditions. At neutral pH, GWALP23-H12 exhibits a well-defined tilted transmembrane orientation in both DOPC and DLPC bilayer membranes. To prevent the acid catalyzed degradation of lipids, we employed ether-linked DOPC bilayers to observe the effect of low pH on the L12H mutant. Under acidic conditions GWALP23-H12 is highly dynamic and exhibits multiple states. Indeed, the multi-state behavior of GWALP23-H12, when His is charged between pH 1.5 and pH 3, resembles closely that of GWALP23-R12 at neutral pH (J. Am. Chem. Soc. 132, 5803). The dramatic change in the behavior of GWALP23-H12 indicates a pK a value less than 3 for His near the center of a lipid bilayer. Investigations are in progress to chemically exchange the C2 imidazole hydrogen of His for deuterium in the peptide, toward a goal of enabling direct observation of the His ring by solid-state 2 H NMR over a range of pH and buffer conditions.