Selective Detection of Protein Secondary Structural Changes in Solution Protein−Polysaccharide Complexes Using Vibrational Circular Dichroism (VCD)

Abstract

A major challenge to understanding the fundamental structural basis of interactions between macromolecules in solution is how to measure their separate contributions. Particularly challenging is the interaction between proteins and polysaccharides. The polysaccharide component is often both very large (10 kDa−MDa) and immobile, or it undergoes anisotropic motion in solution, causing line broadening in NMR. Furthermore, they often exhibit signals in the very spectral regions normally employed for protein secondary structural analysis (FTIR and CD), and these signals cannot simply be subtracted because they occupy variable positions. The selective detection of protein secondary structural changes in aqueous complexes of proteins and polysaccharides, particularly the biologically important glycosaminoglycan class, is demonstrated here, exploiting a property of vibrational circular dichroism (VCD) that allows signals from proteins to be selectively detected. We show that polysaccharides, in contrast to proteins, which show well-documented and characteristic VCD signals for distinct secondary structural types, exhibit no VCD signals in the amide I‘ region despite containing N-acetyl groups. This is because the chromophores in the polysaccharides (in the CO bonds of N-acetyl and carboxylic acids groups) lack the regular geometric relationship to each other that characterizes stretches of defined protein secondary structure. We have exploited this hitherto unreported feature of VCD to enhance the contrast between proteins and bound polysaccharides in protein−polysaccharide complexes in solution. This enables the direct observation of protein secondary structural changes in protein−polysaccharide complexes in solution and will advance understanding of the structural basis of these interactions.