Valence-controlled protein conjugation on nanoparticles via re-arrangeable multivalent interactions of tandem repeat protein chains

Abstract

Precise control of the number of conjugated proteins on a nanoparticle surface has long been a highly challenging task. Here, we developed a one-pot, purification-free strategy for valency-controlled conjugation of tandem repeat protein chains on gold nanoparticles. Protein chains were designed to contain multiple, regularly spaced binding modules, which can multivalently interact with coating molecules on nanoparticle surfaces. We discovered that a slow increase of this interaction strength facilitates full participation of repeated binding modules on a protein chain for surface binding (as well as dynamic rearrangement) on a single nanoparticle, which resulted in stable protein chain wrapping around nanoparticles. By varying the protein chain length, a defined number of protein chains were conjugated on gold nanoparticles with difference sizes. Various high-order nanoparticle structures were accurately assembled with these valence-controlled protein-particle conjugates. The present strategy offers a highly dynamic but controlled protein coating approach on solid surfaces of diverse nanostructures. In addition, this work also provides a valuable clue to understand dynamic binding processes of multivalent repeat proteins.

Scheme 1. Schematic representation of valence-controlled protein chain wrapping around gold nanoparticles. Under slowly decreasing imidazole concentration, a protein chain wraps around a gold nanoparticle via dynamic and re-arrangeable binding of repeated 6His tags on a particle surface. A functional protein (e.g. nanobody), repeated 6His tags (red line) on protein chains and Ni-NTA coating (translucent gray cover) on gold nanoparticles are indicated.

Fig. 1. Construction of repeat modular protein chains with multiple 6His for multivalent binding to Ni-NTA–AuNPs. (a) 6His-fused protein (6His–GFP) binding to 5 nm Ni-NTA–AuNPs. 5 nm Ni-NTA–AuNPs were mixed with 6His–GFP or 6His-cleaved GFP, and the resulting particles were analyzed on an agarose gel. Ctrl: 5 nm Ni-NTA–AuNP. (b) Construction of 6His–SC inserted ST-repeat protein chains. ST-repeat backbone scaffolds (STn) were reacted with excess 6His–SC, and unbound 6His–SC was removed by 180 mM imidazole washing. A SDS-PAGE data for 6His–SC–ST10–ALFA construction is shown in the right. (c) Agarose gel shift analysis of 5 nm Ni-NTA–AuNP binding to 6His–SC monomer (left) or to 6His 10mer (right) under different imidazole concentration from 0 to 300 mM. Ctrl: 5 nm Ni-NTA–AuNP.

Fig. 2. Valence-controlled wrapping of protein chains on gold nanoparticles. (a) TEM images of 5 nm Ni-NTA–AuNP binding to 6His 15mer without (left) or with (right) 50 mM imidazole. Potential protein chain wrapping routes are schematically described at the top. Scale bars: 100 nm. (b) TEM images of 5 nm Ni-NTA–AuNP binding to 6His 15mer with slowly decreasing imidazole concentration (from 1 M to 25 mM) at protein : particle ratios 1 : 1 (left) or at 0.5 : 1 (right). Scale bars: 100 nm. (c) Agarose gel shift analysis of 5 nm Ni-NTA–AuNP binding to 6His 15mer (6His–SC–ST15–NbALFA) at varying protein : particle ratios. (d) Representative TEM images of 5 nm Ni-NTA–AuNPs without protein binding (left), with 6His 15mer (6His–SC–ST15–ALFA) binding (middle), and large 6His 15mer (6His–SC–mCherry–ST15–ALFA) binding (right). Scale bars: 10 nm. (e) Agarose gel shift analysis of 5 nm Ni-NTA–AuNP binding to 6His 15mer (6His–SC–ST15–ALFA; left), 6His 10mer (middle), or 6His 7mer (right) at varying protein : particle ratios. (f) Representative TEM images of 5 nm AuNP assemblies between monovalent NbALFA–AuNP and monovalent ALFA–AuNP (left), divalent ALFA–AuNP (middle), or trivalent-ALFA–AuNP (right). Scale bars: 20 nm. AuNP (yellow circle), Ni-NTA coating (light gray), protein chains (dark gray), and fused NbALFA (red) or ALFA (blue) are schematically indicated.

Fig. 3. Valance-controlled protein chain conjugation to particles with different sizes. (a) Agarose gel shift analysis of 10 nm Ni-NTA–AuNP (left) or 15 nm Ni-NTA–AuNP (right) binding to 6His 15mer (6His–SC–ST15–ALFA) at varying protein : particle ratios. Band shifts by additional protein chain binding are indicated with arrows. (b) Representative TEM images of AuNP assemblies between monovalent 5 nm NbALFA–AuNP and divalent 10 nm ALFA–AuNP (top) or tetravalent 15 nm ALFA–AuNP (middle). Assemblies between divalent 10 nm NbALFA–AuNP and divalent 10 nm ALFA–AuNP are also shown in the bottom. Scale bars: 20 nm.

Fig. 4. Multimeric protein targeting with valence-controlled AuNPs. (a) Representative TEM images of monovalent 5 nm biotin–AuNP (top) or divalent 10 nm biotin–AuNP (bottom) binding to dimeric rhizavidin. (b) Representative TEM images of monovalent 5 nm NbALFA–AuNP binding to trimeric ALFA–5K7V (top) or tetrameric ALFA–Gab (bottom). (c) Representative TEM images of monovalent 5 nm Z_R–AuNP binding to trimeric Z_E–5K7V. Scale bars: 20 nm.