Assessing membrane protein stability is among the major challenges in protein science due to their inherent complexity, which complicates the application of conventional biophysical tools. In this work, sodium dodecyl sulfate-induced denaturation of AfCopA, a Cu(I)-transport ATPase from Archaeoglobus fulgidus, was explored using a combined model-free spectral phasor analysis and a model-dependent thermodynamic analysis. Decrease in tryptophan and 1-anilino-naphthalene-8-sulfonate fluorescence intensity, displacements in the spectral phasor space, and the loss of ATPase activity were reversibly induced by this detergent. Refolding from the SDS-induced denatured state yields an active enzyme that is functionally and spectroscopically indistinguishable from the native state of the protein. Phasor analysis of Trp spectra allowed us to identify two intermediate states in the SDS-induced denaturation of AfCopA, a result further supported by principal component analysis. In contrast, traditional thermodynamic analysis detected only one intermediate state, and including the second one led to overparameterization. Additionally, ANS fluorescence spectral analysis detected one more intermediate and a gradual change at the level of the hydrophobic transmembrane surface of the protein. Based on this evidence, a model for acquiring the native structure of AfCopA in a membrane-like environment is proposed.

Realising a fully circular bioeconomy requires the valorisation of lignocellulosic biomass. Cellulose is the most attractive component of lignocellulose but depolymerisation is inefficient, expensive and resource intensive requiring substantial volumes of potable water. Seawater is an attractive prospective replacement, however seawater tolerant enzymes are required for the development of seawater-based biorefineries. Here, we report a halophilic cellobiohydrolase SMECel6A, identified and isolated from a salt marsh meta-exo-proteome dataset with high sequence divergence to previously characterised cellobiohydrolases. SMECel6A contains a glycoside hydrolase family 6 (GH6) domain and a carbohydrate binding module family 2 (CBM2) domain. Characterisation of recombinant SMECel6A revealed SMECel6A to be active upon crystalline and amorphous cellulose. Mono- and oligosaccharide product profiles revealed cellobiose as the major hydrolysis product confirming SMECel6A as a cellobiohydrolase. We show SMECel6A to be halophilic with optimal activity achieved in 0.5X seawater displaying 80.6 ± 6.93% activity in 1 × seawater. Structural predictions revealed similarity to a characterised halophilic cellobiohydrolase despite sharing only 57% sequence identity. Sequential thermocycling revealed SMECel6A had the ability to partially reversibly denature exclusively in seawater retaining significant activity. Our study confirms that salt marsh ecosystems harbour enzymes with attractive traits with biotechnological potential for implementation in ionic solution based bioprocessing systems.

Anti-IZUMO1PFF VHH (variable domain of camelid heavy chain antibody) clones, N6 and N15, from immunized alpaca (Lama pacos) phage library were efficiently expressed and their VHH products were secreted into the culture medium of Brevibacillus choshinensis HPD31-SP3, e.g., at a level of 26-95mg in 100ml conventional flask culture. With a 3-L scale fed-batch culture for 65h, the N15 VHH protein with C-terminal His-tag was produced at ∼3g/l culture medium. The N6 and N15 proteins were easily purified to apparent homogeneity by cation exchange and Ni-affinity chromatographies. Both proteins showed specific antigen-binding activity by ELISA and high antigen binding affinity, KD=6.0-8.6nM, by surface plasmon resonance analysis. Size exclusion chromatography-multi-angle laser light scattering analysis revealed that N6 and N15 proteins purified were exclusively monomeric form in phosphate buffered saline. CD spectrum showed beta-sheet rich structure, consistent with a typical antibody structure and also suggested aromatic-aromatic interactions, as indicated by a positive peak at 232nm. Thermal melting analysis of the N15 protein with C-terminal His-tag demonstrated a clear thermal transition with a Tm at 67°C. The heat-denatured sample recovered antigen binding activity upon cooling, indicating a reversible denaturation.