Influence of PEGylation on Domain Dynamics of Phosphoglycerate Kinase: PEG Acts Like Entropic Spring for the Protein

Protein–polymer conjugation is a widely used technique to develop protein therapeutics with improved pharmacokinetic properties as prolonged half-life, higher stability, water solubility, lower immunogenicity, and antigenicity. Combining biochemical methods, small angle scattering (SAXS/SANS), and neutron spin–echo spectroscopy, here we examine the impact of PEGylation (i.e., the covalent conjugation with poly(ethylene glycol) or PEG) on structure and internal domain dynamics of phosphoglycerate kinase (PGK) to elucidate the reason for reduced activity that is connected to PEGylation. PGK is a protein with a hinge motion between the two main domains that is directly related to function. We find that secondary structure and ligand access to the binding sites are not affected. The ligand induced cleft closing is unchanged. We observe an additional internal motion between covalent bonded PEG and the protein compatible with Brownian motion of PGK in a harmonic potential. Entropic interaction with the full PEG chain leads to a force constant of about 8 pN/nm independent of PEG chain length. This additional force preserves protein structure and has negligible effects on the functional domain dynamics of the protein. PEGylation seems to reduce activity just by acting as a local crowder for the ligands. The newly identified interaction mechanism might open possibilities to improve rational design of protein–polymer conjugates

Discussion of paper by M. Staropoli, A. Raba, C. H. Hovelmann, M.-S. Appavou, J. Allgaier, M. Krutyeva, W. Pyckhout-Hintzen, A. Wischnewski, and D. Richter, entitled ‘Melt dynamics of supramolecular comb polymers: Viscoelastic and dielectric response’

The combination of dielectric and mechanical spectroscopies is very powerful. However, it is not quite clear in your paper how the time scales of your supramolecular combs compare from these two spectroscopies. Maybe you can make a plot of the temperature dependence of the time scales for arm retraction and for terminal relaxation that are obtained by these two methods? That would really help the reader understand not only how they compare but what temperature ranges can be studied for these two vital time scales...

Melt dynamics of supramolecular comb polymers: Viscoelastic and dielectric response

The structure and the dynamics of supramolecular comblike polymers in the melt state is studied by a combination of linear rheology, dielectric spectroscopy, and small angle neutron scattering. The system consists of blends of 1,2-polybutyleneoxide (PBO) entangled backbones, randomly functionalized with thymine (thy) and barely entangled PBO graft chains—modified with 2,4-diamino-1,3,5-triazine (DAT) end groups. These bioinspired groups associate into a transiently branched comb architecture through heterocomplementary interaction involving the two different hydrogen bonding groups thy and DAT. In the present manuscript, we focus on the comparison of the macroscopic dynamics of the associating blends and permanent comb analogs. The viscoelastic and dielectric response of covalent and reversible combs are found to be comparable. The viscoelastic response of mixtures of thy-functionalized entangled backbones and DAT-end-modified barely entangled chains show a relaxation mechanism, which is mostly attributed to the association/breakage dynamics of the transient bonds with characteristic time ∼1 s  at T=−25 °C. In the parallel dielectric investigation, the reversible branched structure is still evident from the comparison with the corresponding permanent combs and allows the distinction between fixed arms relaxation and the lifetime. A α∗  process of the thy-thy association is likewise detected. The time scale of the supramolecular association makes the thy-DAT pair an ideal candidate for the development of responsive materials that combine permanent and transient linkages for novel applications and self-healing propertie

Influence of PEGylation on Domain Dynamics of Phosphoglycerate Kinase: PEG Acts Like Entropic Spring for the Protein

Protein–polymer conjugation is a widely used technique to develop protein therapeutics with improved pharmacokinetic properties as prolonged half-life, higher stability, water solubility, lower immunogenicity, and antigenicity. Combining biochemical methods, small angle scattering (SAXS/SANS), and neutron spin–echo spectroscopy, here we examine the impact of PEGylation (i.e., the covalent conjugation with poly­(ethylene glycol) or PEG) on structure and internal domain dynamics of phosphoglycerate kinase (PGK) to elucidate the reason for reduced activity that is connected to PEGylation. PGK is a protein with a hinge motion between the two main domains that is directly related to function. We find that secondary structure and ligand access to the binding sites are not affected. The ligand induced cleft closing is unchanged. We observe an additional internal motion between covalent bonded PEG and the protein compatible with Brownian motion of PGK in a harmonic potential. Entropic interaction with the full PEG chain leads to a force constant of about 8 pN/nm independent of PEG chain length. This additional force preserves protein structure and has negligible effects on the functional domain dynamics of the protein. PEGylation seems to reduce activity just by acting as a local crowder for the ligands. The newly identified interaction mechanism might open possibilities to improve rational design of protein–polymer conjugates