Biosurfactants, are
surface active molecules that can be produced by renewable, industrially
scalable biologic processes. DAMP4, a designer biosurfactant, enables the
modification of interfaces via genetic or chemical fusion to functional
moieties. However, bioconjugation of addressable amines introduces
heterogeneity that limits the precision of functionalization as well as the resolution
of interfacial characterization. Here we designed DAMP4 variants with cysteine
point mutations to allow for site-specific bioconjugation. The DAMP4 variants
were shown to retain the structural stability and interfacial activity characteristic
of the parent molecule, while permitting efficient and specific conjugation of
polyethylene glycol (PEG). PEGylation results in a considerable reduction on
the interfacial activity of both single and double mutants. Comparison of
conjugates with one or two conjugation sites shows that both the number of
conjugates as well as the mass of conjugated material impacts the interfacial activity
of DAMP4. As a result, the ability of DAMP4 variants with multiple PEG
conjugates to impart colloidal stability on peptide-stabilized emulsions is
reduced. We suggest that this is due to constraints on the structure of
amphiphilic helices at the interface. Specific and efficient bioconjugation permits
the exploration and investigation of the interfacial properties of designer
protein biosurfactants with molecular precision. Our findings should therefore
inform the design and modification of biosurfactants for their increasing use
in industrial processes, and nutritional and pharmaceutical formulations.