Insight into the Structure and Activity of Surface-Engineered Lipase Biofluids.

Despite a successful application of solvent-free liquid protein (biofluids) concept to a number of commercial enzymes, the technical advantages of enzyme biofluids as hyperthermal stable biocatalysts cannot be fully utilized as up to 90-99% of native activities are lost when enzymes were made into biofluids. With a two-step strategy (site-directed mutagenesis and synthesis of variant biofluids) on Bacillus subtilis lipase A (BsLA), we elucidated a strong dependency of structure and activity on the number and distribution of polymer surfactant binding sites on BsLA surface. Here, it is demonstrated that improved BsLA variants can be engineered via site-mutagenesis by a rational design, either with enhanced activity in aqueous solution in native form, or with improved physical property and increased activity in solvent-free system in the form of a protein liquid. This work answered some fundamental questions about the surface characteristics for construction of biofluids, useful for identifying new strategies for developing advantageous biocatalysts.

Role of Charge and Hydrophobicity in Liprotide Formation: A Molecular Dynamics Study with Experimental Constraints.

Bovine α-lactalbumin (aLA) and oleate (OA) form a complex that has been intensively studied for its tumoricidal activity. Small-angle X-ray scattering (SAXS) has revealed that this complex consists of a lipid core surrounded by partially unfolded protein. We call this type of complex a liprotide. Little is known of the molecular interactions between OA and aLA, and no technique has so far provided any high-resolution structure of a liprotide. Here we have used coarse-grained (CG) molecular dynamics (MD) simulations, isothermal titration calorimetry (ITC) and SAXS to investigate the interactions between aLA and OA during the process of liprotide formation. With ITC we found that the strongest enthalpic interactions occurred at a molar ratio of 12.0±1.4:1 OA/aLA. Liprotides formed between OA and aLA at several OA/aLA ratios in silico were stable both in CG and in all-atom simulations. From the simulated structures we calculated SAXS spectra that show good agreement with experimentally measured patterns of matching liprotides. The simulations showed that aLA assumes a molten globular (MG) state, exposing several hydrophobic patches involved in interactions with OA. Initial binding of aLA to OA occurs in an area of aLA in which a high amount of positive charge is located, and only later do hydrophobic interactions become important. The results reveal how unfolding of aLA to expose hydrophobic residues is important for complex formation between aLA and OA. Our findings suggest a general mechanism for liprotide formation and might explain the ability of a large number of proteins to form liprotides with OA.

Glycolipid Biosurfactants Activate, Dimerize, and Stabilize Thermomyces lanuginosus Lipase in a pH-Dependent Fashion.

We present a study of the interactions between the lipase from Thermomyces lanuginosus (TlL) and the two microbially produced biosurfactants (BSs), rhamnolipid (RL) and sophorolipid (SL). Both RL and SL are glycolipids; however, RL is anionic, while SL is a mixture of anionic and non-ionic species. We investigate the interactions of RL and SL with TlL at pH 6 and 8 and observe different effects at the two pH values. At pH 8, neither RL nor SL had any major effect on TlL stability or activity. At pH 6, in contrast, both surfactants increase TlL's thermal stability and fluorescence and activity measurements indicate interfacial activation of TlL, resulting in 3- and 6-fold improved activity in SL and RL, respectively. Nevertheless, isothermal titration calorimetry reveals binding of only a few BS molecules per lipase. Size-exclusion chromatography and small-angle X-ray scattering suggest formation of TlL dimers with binding of small amounts of either RL or SL at the dimeric interface, forming an elongated complex. We conclude that RL and SL are compatible with TlL and constitute promising green alternatives to traditional surfactants.

Impact of Chemical Composition on the Nanostructure and Biological Activity of α-Galactosidase-Loaded Nanovesicles for Fabry Disease Treatment.

Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems-quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development.

Plant Polyphenols Inhibit Functional Amyloid and Biofilm Formation in Pseudomonas Strains by Directing Monomers to Off-Pathway Oligomers.

Self-assembly of proteins to ß-sheet rich amyloid fibrils is commonly observed in various neurodegenerative diseases. However, amyloid also occurs in the extracellular matrix of bacterial biofilm, which protects bacteria from environmental stress and antibiotics. Many Pseudomonas strains produce functional amyloid where the main component is the highly fibrillation-prone protein FapC. FapC fibrillation may be inhibited by small molecules such as plant polyphenols, which are already known to inhibit formation of pathogenic amyloid, but the mechanism and biological impact of inhibition is unclear. Here, we elucidate how polyphenols modify the self-assembly of functional amyloid, with particular focus on epigallocatechin gallate (EGCG), penta-O-galloyl-ß-d-glucose (PGG), baicalein, oleuropein, and procyanidin B2. We find EGCG and PGG to be the best inhibitors. These compounds inhibit amyloid formation by redirecting the aggregation of FapC monomers into oligomeric species, which according to small-angle X-ray scattering (SAXS) measurements organize into core-shell complexes of short axis diameters 25-26 nm consisting of ~7 monomers. Using peptide arrays, we identify EGCG-binding sites in FapC's linker regions, C and N-terminal parts, and high amyloidogenic sequences located in the R2 and R3 repeats. We correlate our biophysical observations to biological impact by demonstrating that the extent of amyloid inhibition by the different inhibitors correlated with their ability to reduce biofilm, highlighting the potential of anti-amyloid polyphenols as therapeutic agents against biofilm infections.

Liprotides assist in folding of outer membrane proteins.

Proteins and lipids can form complexes called liprotides, in which the partially denatured protein forms a shell encasing a lipid core. This effectively stabilizes a lipid micelle in an aqueous solvent and suggests that liprotides may provide a suitable vessel for membrane proteins. Accordingly we have investigated if liprotides consisting of α-lactalbumin and oleate could aid folding of four different outer membrane proteins (OMPs) tOmpA, PagP, BamA, and OmpF. tOmpA was able to fold in the presence of the liprotide, and folding did not occur if only oleate or α-lactalbumin were added. Although the liprotides did not fold the other three OMPs on its own, it was able to assist their folding in the presence of vesicles. Incubation with liprotides before folding into vesicles increased the folding yield of the outer membrane proteins to a level higher than using micelles of the non-ionic surfactant DDM. Even though the liprotide was stable at both high urea concentrations and high pH, it failed to efficiently fold OmpA at high pH. Instead, optimal folding was seen at pH 8-9, suggesting that important changes in the liprotide occurred when increasing the pH. We conclude that an otherwise folding-inactive fatty acid can be activated when presented by a liprotide and thereby work as an in vitro chaperone for outer membrane proteins.