Conformational Flexibility of Glycosylated Unstructured Peptides

Abstract

Glycosylation of proteins plays an important role in molecular recognition among proteins with implications for a variety of cellular processes. The large amount of glycan variants enables interactions of exquisite specificity. Glycoproteins often are partially or fully disordered and intrinsically unstructured regions in proteins are known for mediating many protein-protein or protein-nucleotide interactions during regulation of transcription, translation, and cellular signal transduction. The question arises how conjugated glycans influence protein and peptide conformational dynamics and by that modify their biological activity. We compare the conformational dynamics of unstructured polypeptides consisting of eight glycine-serine repeat units with and without glycosylated serine units. We synthesized glycine-serine repeats with O-conjugated beta-galactose at every serine residue by solid-phase synthesis with glycosylated dipeptides as building blocks. Introducing an organic oxazine dye and tryptophan at either end of these peptides allows measurements of end-to-end contact kinetics. Upon van-der-Waals contact between dye and tryptophan fluorescence is quenched by photo induced electron transfer (PET). Fluorescence intensity fluctuations are analyzed using fluorescence correlation spectroscopy (FCS) and contact formation rate constants are determined. We studied influences from solvent viscosity and temperature on end-to-end contact formation rates and found a decrease of rate constants upon glycosylation. Arrhenius analysis of end-to-end contact rates yields enhanced activation energy for the glycosylated sample. The viscosity dependence of the relaxation rates shows that contact formation still is viscosity controlled. This study confirms previous reports that glycosylation has a significant influence on peptide dynamics mostly through steric hindrance.