(548a) Molecular Sieving on the Surface of Nano-Armored Protein | AIChE

(548a) Molecular Sieving on the Surface of Nano-Armored Protein

Authors 

Kaupbayeva, B. - Presenter, Carnegie Mellon University
Murata, H., Carnegie Mellon University
Lucas, A., Carnegie Mellon University
Matyjaszewski, K., Carnegie Mellon University
Minden, J., Carnegie Mellon University
Russell, A., Carnegie Mellon University
Protein-polymer conjugates have proven their worth across a range of applications, with the most notorious example being PEGylated proteins for therapeutics. There are currently 14 PEGylated proteins that are FDA approved contributing to a market that exceeds $15 billion annually. Polymers are conjugated to proteins to alter their physicochemical properties, leading to conjugates with enhanced bioactivity, increased stability and solubility, as well as imparting added functionality through the use of stimuli responsive polymers. Although many protein-polymer conjugates have been synthesized and used for therapeutics, the understanding of permeability of molecules through the polymer chains towards the surface of the protein is still not well understood. As a result, the synthesis of these molecules remains a stochastic guesswork. The molecular sieving of polymers is an important criterion affecting the efficacy of protein drugs. For example, the enzyme-polymer conjugates used in therapy need to repel the protein-antibody interactions and protease-mediated hydrolysis, while allowing the passage of their substrates to the active site.

In this work, we have focused on determining the permeability of molecules towards the protein surface as a step forward in understanding the role of polymer chain length and grafting density on a surface of a protein in changing the binding rates of ligands. We have used atom transfer radical polymerization (ATRP) to generate well-defined, avidin-polymer conjugates with varying polymer lengths. The number of chains grown from a protein using “grafting from” ATRP with amino-reactive, single-headed initiators cannot exceed the number of accessible amine groups on the surface of the protein. To overcome this limitation and better understand the impact of polymer grafting density on the rate of sieving by attached polymers, we designed a novel, NHS-functionalized, double-headed ATRP initiator that supported the growth of two polymers from one initiation point. We systematically characterized the bindings rates of molecules with different sizes and shapes towards the protein surface by exploiting the high binding affinity of biotin toward avidin protein. For molecular permeation studies, globular proteins: aprotinin, histone and horse radish peroxidase (hydrodynamic sizes of 2.4, 4.6 and 5.1 nm respectively) were selected based on their sizes and biotinylated with Biotin-PEG-NHS. Another substrate that was used to test molecular sieving was linear biotin-PEG with different PEG size 550-30000 Da.

Until now, there have not been ways to detect the permeation rates of biomacromolecules through polymer layers to the protein surface. Herein we describe an approach that quantifies the rate of binding of molecules through a layer of polymer chains to the binding site of avidin protein using a stopped-flow system observing the change in tryptophan fluorescence.

We conclude that the rate of ligand binding is strongly dependent on the polymer grafting density and the size of the substrate, but, interestingly, far less dependent on the length of the polymer and substrate shape. This study unveils a deeper understanding of the relationship between polymer characteristics and binding kinetics, discovering important steps in rational design of protein-polymer-conjugates.