Mapping protein–polymer conformations in bioconjugates with atomic precision

Abstract

Protein–polymer conjugates are explored using magnetic resonance methods to investigate the conformations of the polymer relative to the protein's surface.

Two‐Distinct Polymer Ubiquitin Conjugates by Photochemical Grafting‐From

“…In the first step, the NHS-functionalized ATRP initiator was reacted with the N-terminal amine and lysine -amines of Ub 77C . Based on prior work in our group using wild-type Ub, [34] we expect K 11 , K 48 , and K 63 to be fast-reacting sites, while the N-terminus is predicted to be intermediate, however in this case the N-terminus is located at the end of a TEV recognition sequence and His 6 tag, where it is far more accessible than in wild-type Ub. The MALDI-TOF-MS spectrum (Figure 2a) of BiB-Ub shows that addition of 2-3 BiB is most common, though small amounts (<10%) of native remains, and species with 4-5 BiB have also been observed.…”

“…It is notable for its resilience against chemical and thermal denaturation, robust resistance to protease, and longevity in solution. [34,[38][39][40][41][42][43][44] Wild type ubiquitin has no native cysteine residues, so in order to enable our scheme we used a variant of ubiquitin with a cysteine residue added at the C-terminus as the 77th residue (Ub 77C ). This variant remains easy to overexpress in high yield, similar to wild type ubiquitin, and since the C-terminus of ubiquitin is fully solvent exposed and highly flexible, [45] we expect this to allow for more favorable reaction at the cysteine.…”

“…The potential disadvantages are thus: the properties of a statistical copolymer will differ from that of two homopolymers whose roles are distinct and may be detrimental to stabilization character or phase behavior; an attached block copolymer places only one block of the copolymer in close proximity to the protein, while the other block will be further away, and proximity may be relevant to obtaining the desired conjugation benefit. [ 34 ] It is also of inherent interest to synthesize novel conjugate topologies such as tri‐blocks where protein is the middle block or well‐defined hetero‐arm star polymers with a protein core. In light of these considerations, we set out to design a scheme to modify a protein with two different polymers using orthogonal chemistries.…”

Saturation Transfer Difference NMR Spectroscopy for the Elucidation of Supramolecular Albumin–Polymer Interactions

“…Protein−polymer interactions of block copolymers and ubiquitin have been previously investigated via paramagnetic relaxation enhancement (PRE) NMR spectroscopy. 12 The technique is advantageous for polymers as it can provide distance information on up to 30 Å, allowing conclusions to be drawn on protein−polymer conjugation and the stability of the resulting conjugate. 12 A supplement to the information acquired from PRE-NMR would be to understand how each functional group of the polymer contributes to the protein− polymer interaction.…”

“…12 The technique is advantageous for polymers as it can provide distance information on up to 30 Å, allowing conclusions to be drawn on protein−polymer conjugation and the stability of the resulting conjugate. 12 A supplement to the information acquired from PRE-NMR would be to understand how each functional group of the polymer contributes to the protein− polymer interaction. This can be achieved through saturation transfer difference NMR (STD-NMR), a powerful, robust, and attractive technique which is applied in high throughput screening for active ligands in mixtures of small molecules.…”