Conformational Flexibility of Glycosylated Peptides

Abstract

Glycosylation adds carbohydrate moieties to proteins and functional peptides. Glycans, highly branched carbohydrate heteropolymers that can be attached to amino acids either through N-or O-glycosylation, serve as coding entity, providing specificity for carbohydrate-protein interactions, or modifying dynamic and structural behaviour of proteins. Glycoconjugates present glycans of extraordinary large structural variety, enabling information coding by steering molecular recognition.[1] Glycosylated proteins and lipids usually present their glycan moieties solvent-exposed at the surface where they can serve as highly specific binding partners. Specific protein-glycan interactions generally are enthalpically driven through hydrogen bonding and van der Waals interactions. Strong interactions only come about through cooperative formation of many hydrogen bonds or hydrophobic interactions. Glycoproteins and -lipids contribute to a range of signaling interactions mostly in eukaryotic cells, help regulate cell-cell communication and immune response, [2] or control protein folding and turnover.[3]Often a protein's functional state or specific complex is stabilized by glycosylation analogous to the effects of molecular crowding. [3b,c] Glycoproteins also serve as antifreeze reagent when interacting with ice crystals. [4] Since glycoproteins often are partially or fully disordered and glycans themselves are highly flexible structures, [5] the question arises how conjugated glycans influence protein and peptide conformational dynamics. Extensive O-glycosylation of mucin-like glycopeptides seems to rigidify the peptide backbone and increase the overall radius of gyration.[6] The unfolded state in a dynamic folding/unfolding protein equilibrium is influenced by glycosylation, too.[3b] However, various and partly contradictory effects of N/O-glycosylation on peptide conformations were observed experimentally and in computer simulations. [5,7] Overall, there is no comprehensive understanding on the molecular scale how glycoconjugate conformational dynamics are shaped by glycans and we seek to address this issue by studying well-defined synthetic model peptides.Herein we give an account of the influence that b-galactose O-conjugated to serine residues impose on peptide conformational dynamics. We compare intramolecular dynamics of unstructured peptides made of either glycine-serine repeats [GlySer] or made of glycosylated serine-glycine repeats [Ser(b-Gal)-Gly)] and characterize influences from attached glycans on peptide dynamics. Conformational dynamics are monitored and analyzed by fluorescence correlation spectroscopy (FCS). [8] As reporter system for dynamic processes we introduce a fluorophore-quencher pair on opposite ends of the unstructured peptides that report on end-to-end contacts by on/off changes in the fluorescence emission. We use the oxazine fluorophore MR121 (Figure 1), attached via site-specific labeling chemistry to the N-terminus, and the fluorescence-quenching amino acid tryptophan (Trp), incorporated as...

The roughness of the protein energy landscape results in anomalous diffusion of the polypeptide backbone

“…48 On the other hand, glycosylated peptides have slower loop formation dynamics. 62 As noted above, the speed of peptide segment diffusion is governed by two main effects, namely solvent friction, i.e. the viscosity of the solvent through which the peptide chain moves, and internal friction, i.e.…”

“…retardation of peptide segment motion arising from peptide internal interactions, which is directly related to the roughness of the local energy landscape. The influence of solvent friction manifests itself in the viscosity dependence of the loop formation rate of unstructured peptides, 39,40,42,47,49,53,55,62 in particular the different rates found in H 2 O and D 2 O which have significantly different viscosity. 46,58 On the other hand, a significant contribution from internal friction, i.e.…”

“…Finally, deviations from exponential dynamics on time scales of 100 ns or slower have in fact been reported in many of the loop formation experiments. 13,[15][16][17]20,22,37,43,44,49,50,52,55,62,[75][76][77][78][79][80]82,85,148 These can often be ascribed to experimental artefacts inherent in the method, such as contributions from photoproducts other than the reporter state, or to sample heterogeneity, impurities or intermolecular effects. However, these additional dynamic components further limit the conclusions that can be drawn on complete conformational equilibration of polypeptides on the nano-to microsecond time scale.…”

Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

“…A classic example for such a molecular ruler is Förster-type resonance energy transfer (FRET) 5 , which allows achieving structural information with a spatial resolution in the nanometre range and (sub)millisecond temporal resolution 6 7 8 9 10 . However, other photophysical effects such as photo-induced electron transfer (PET) 11 12 13 14 or protein-induced fluorescence enhancement (PIFE) 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 can be used for similar purposes. Since the fluorescent signal can be read out with high time-resolution, even fast conformational changes 33 34 35 36 37 38 39 , as well as interactions between biomolecules, can be mapped in physiologically relevant environments in vitro 40 41 and in vivo 42 43 with a sensitivity allowing to address individual molecules.…”

Fluorescence resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

“…However, other photophysical effects such as photo-induced electron transfer (PET) [11][12][13][14] or protein-induced fluorescence enhancement (PIFE) [15][16][17][18][19] can be used for a similar purpose. Since the fluorescent signal can be read out with high time-resolution, even fast conformational changes [20][21][22][23][24][25][26] , as well as interactions between biomolecules can be mapped in physiologically relevant environments in vitro 27,28 and in vivo with a sensitivity of individual molecules.…”