The power and elegance of protein-polymer conjugates has solved many vexing problems for society. Rational design of these complex covalent hybrids depends on a deep understanding of how polymer physicochemical properties impact the conjugate structure-function-dynamic relationships. We have generated a large family of chymotrypsin-polymer conjugates which differ in polymer length and charge, using grafting-from atom-transfer radical polymerization, to elucidate how the polymers influenced enzyme structure and function at pHs that would unfold and inactivate the enzyme. We also used molecular dynamics simulations to deepen our understanding of protein-polymer intramolecular interactions. Remarkably, the data revealed that, contrary to current thoughts on how polymers stabilize proteins, appropriately designed polymers actually stabilize partially unfolded intermediates and assist in refolding to an active conformation. Long, hydrophilic polymers minimized interfacial interactions in partially unfolded conjugates leading to increased stabilization. The design of covalently attached intramolecular biomimetic chaperones that drive protein refolding could have far reaching consequences.
Therapeutic proteins have increasingly
been used in modern medical
applications, but their effectiveness is limited by factors such as
stability and blood circulation time. Recently, there has been significant
research into covalently linking polyethylene glycol polymer chains
(PEG) to proteins, known as PEGylation, to mitigate these issues.
In this work, an atomistic molecular dynamics study of N-terminal
conjugated PEG-BSA (bovine serum albumin) was conducted with varying
PEG molecular weights (2, 5, 10, and 20 kDa) to probe PEG-BSA interactions
and evaluate the effect of polymer length on dynamics. It was found
that the affinity of PEG toward the protein surface increased as a
function of PEG molecular weight and that a certain weight (around
10 kDa) was required to promote protein–polymer interactions.
Additionally, preferential interactions were monitored through formed
contacts and hotspots were identified. PEG chains coordinating in
looplike conformations were found near lysine residues. Also, it was
found that hydrophobic interactions played an important role in promoting
PEG-BSA interactions as the PEG molecular weight increased. The results
provide insight into underlying mechanisms behind transitions in PEG
conformations and will aid in future design of effective PEGylated
drug molecules.
Almost all commercial proteins are purified using ammonium sulfate precipitation. Protein-polymer conjugates are synthesized from pure starting materials, and the struggle to separate conjugates from polymer, native protein, and from isomers has vexed scientists for decades. We have discovered that covalent polymer attachment has a transformational effect on protein solubility in salt solutions. Here, protein-polymer conjugates with a variety of polymers, grafting densities, and polymer lengths are generated using atom transfer radical polymerization. Charged polymers increase conjugate solubility in ammonium sulfate and completely prevent precipitation even at 100% saturation. Atomistic molecular dynamic simulations show the impact is driven by an anti-polyelectrolyte effect from zwitterionic polymers. Uncharged polymers exhibit polymer length-dependent decreased solubility. The differences in salting-out are then used to simply purify mixtures of conjugates and native proteins into single species. Increasing protein solubility in salt solutions through polymer conjugation could lead to many new applications of protein-polymer conjugates.
Atomistic molecular dynamics simulations improve our understanding of protein–polymer conjugates, and can predict how charged polymers affect the native dynamics of the protein.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.