Elucidating structure-functionality relationships of phycocyanin through size-exclusion chromatography coupled with in-line small-angle X-ray scattering

Abstract

Phycocyanin (PC), a key protein in the antenna-shaped light-harvesting system of algae, has been proven to be a competitive blue colorant and emulsifier suitable for the food industry. Controlled disassembly and denaturation allow converting PC from intact polymers to unfolded subunits to enhance its emulsifying capacity, which often comes at the cost of its blue color. We report here, that by increasing the concentration of urea, a common denaturant, stepwise disassembly, denaturation, and reorganization of PC from large and uneven particles into small and uniform particles (<10 nm) were achieved as visualized using transmission electron microscopy. Soluble and similarly sized aggregates, however, were found at high urea concentrations by polyacrylamide gel electrophoresis and size-exclusion chromatography. Size-exclusion chromatography was used in-line with a small-angle X-ray scattering system to characterize shape and tertiary structural changes in PC. As PC converged from a large and uneven structure to a smaller and homogeneous structure, the conformation changed from an intact globular to a denatured elongated state. Three modeling algorithms, DAMMIN, GASBOR, and DENSS, were used to compare and align with the already-solved crystal structures, achieving good fitting scores (χ2 < 1.2). The smaller size of PC in its partially denatured structure balanced its hydrophilic-lipophilic character and increased the unit surface area, which elevated interfacial capacity. This resulted in lower air-water and oil-water interfacial energy needed (∼39 and ∼2.4 mN m−1) and improved dynamic interfacial adsorbing parameters, which promoted emulsifying activity and emulsion stability, as well as foaming capacity and foam stability.