The Use of Surfactants to Solubilise a Glucagon Analogue.

PURPOSE: The peptide hormone glucagon, used to treat hypoglycaemic incidents, is prone to aggregation. Generating alternatives with better stability is of pharmaceutical interest in the treatment of diabetes. Here we investigate the impact of six different surfactants on the solubility and stability of ZP-GA-1, a stable version of glucagon. METHODS: We use chemical surfactants (sodium dodecyl sulphate, dodecyl maltoside and polysorbate 20) and the biosurfactants rhamnolipid, sophorolipid and surfactin. We investigate their interaction with ZP-GA-1 by pyrene fluorescence, circular dichroism and isothermal titration calorimetry. RESULTS: All six surfactants induce α-helical structure in ZP-GA-1, SDS having the biggest impact and polysorbate 20 the smallest. SDS keeps ZP-GA-1 solubilised over >48 days as opposed to 29 days in DDM, 3 days in polysorbate 20 and 0 days in buffer. Similarly, much less SDS than DDM, polysorbate 20 or biosurfactant is needed to redissolve aggregated ZP-GA-1. ITC confirms this trend, with SDS exhibiting very strong, and polysorbate 20 very weak interactions. CONCLUSION: Simple surfactant structures promote stronger peptide interactions. ITC shows promise as a general strategy to predict surfactants' solubilising powers. Stronger enthalpic interactions improved the absolute solubility of ZP-GA-1 and their strength correlated to the absolute solubility of the peptides though not to the kinetics of precipitation.

Myoglobin and α-Lactalbumin Form Smaller Complexes with the Biosurfactant Rhamnolipid Than with SDS.

Biosurfactants (BSs) attract increasing attention as sustainable alternatives to petroleum-derived surfactants. This necessitates structural insight into how BSs interact with proteins encountered by current chemical surfactants. Thus, small-angle x-ray scattering (SAXS) has been used for studying the structures of complexes made of the proteins α-Lactalbumin (αLA) and myoglobin (Mb) with the biosurfactant rhamnolipid (RL). For comparison, complexes between αLA and the chemical surfactant sodium dodecyl sulfate (SDS) were also investigated. The SAXS data for pure RL micelles can be described by prolate core-shell structures with a core radius of 7.7 Å and a shell thickness of 12 Å, giving an aggregation number of 11. The small core radius is attributed to RL's complex hydrophobic tail. Data for the αLA-RL complex agree with a 12-molecule micelle with a single protein molecule in the shell. For Mb-RL, the analysis gives complexes of two connected micelles, each containing 10 RL and one protein in the shells. αLA-RL and Mb-RL form surfactant-saturated complexes above 5.6 and 4.7 mM RL, respectively, leaving the remaining RL in free micelles. The SAXS data for SDS agree with oblate-shaped micelles with a core of 20 Å, core eccentricity 0.7, and shell thickness of 5.45 Å, with an aggregation number of 74. The αLA-SDS complexes contain a prolate micelle with a core radius of 11-14 Å and a shell of 8-12 Å with up to 3 αLA per particle and up to 43 SDS per αLA, both considerably larger than for RL. Unlike the RL-protein complexes, the number of surfactant molecules in αLA-SDS complexes increases with surfactant concentration, and saturate at higher surfactant concentrations than αLA-RL complexes. The results highlight how RL and SDS follow similar overall rules of self-assembly and interactions with proteins, but that differences in the strength of protein-surfactant interactions affect the formed structures.

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.

Promoting protein self-association in non-glycosylated Thermomyces lanuginosus lipase based on crystal lattice contacts.

We have used the crystal structure of Thermomyces lanuginosus lipase (TlL) to identify and strengthen potential protein-protein interaction sites in solution. As wildtype we used a deglycosylated mutant of TlL (N33Q). We designed a number of TlL mutants to promote interactions via interfaces detected in the crystal-lattice structure, through strengthening of hydrophobic, polar or electrostatic contacts or truncation of sterically blocking residues. We identify a mutant predicted to lead to increased interfacial hydrophobic contacts (N92F) that shows markedly increased self-association properties on native gradient gels. While wildtype TlL mainly forms monomer and <5% dimers, N92F forms stable trimers and dimers according to Size-Exclusion Chromatography and Small-Angle X-ray Scattering. These oligomers account for ~25% of the population and their enzymatic activity is comparable to that of the monomer. Self-association stabilizes TlL against thermal denaturation. Furthermore, the trimer is stable to dilution and requires high concentrations (>2M) of urea to dissociate. We conclude that crystal lattice contacts are a good starting point for design strategies to promote protein self-association.