Protein Scaffold Engineering Towards Tunable Surface Attachment

Abstract

The desire to control, predict, and manipulate protein adsorption to specific surfaces has been the main driving force for intensive research in the past few years directed at gaining a better understanding and control over such proteinsurface interactions. [1,2] Controlling the affinity of proteins to surfaces is of great importance for applications such as memory arrays, biosensors, and novel composite materials. [3] The main strategies towards immobilizing proteins are either by surface modifications or through engineering specific surface-binding groups at different locations on the protein structure. The main drawback of the former is the alteration of the bulk chemical surface characteristics and the need of an extensive surface processing, typically involved with multiple steps. The latter strategy typically requires a single step, but is often untunable and frequently results in low surface affinity. Overcoming the need for surface modifications combined with control of the protein surface affinity would enable the exploitation of protein immobilization for new materials and will increase future fabrication throughput.SP1, a ring-like protein that is highly stable to boiling and protease resistant, [4][5][6][7] was recently proposed as a new selfassembled molecular scaffold for nanobiotechnology and biomaterials applications. [8][9][10][11] Herein we present a novel strategy to control the interfacial adsorption of SP1 to an unmodified surface with high selectivity and controlled affinity. By genetically fusing specific affinity peptides to retractable N termini, we were able to control the protein surface affinity for the first time by simply changing the solvent conditions. Understanding the dependence of the protein surface affinity on its structure is a key element in engineering a novel surface-binding scaffold. To fully understand the availability of the protein structure to the surface, the protein affinity to gold surfaces was investigated in a straightforward fashion by integration of thiol groups. Cysteine (Cys)-free wild-type SP1 (wt SP1) shows no significant affinity to gold surfaces. Therefore, surface accessibility of the SP1 to gold was endowed by introducing Cys residues (using site-directed mutagenesis) as anchoring points at two different sites in the protein structure. In the first variant, Methionine 43, which is located in the protein inner pore, was replaced with a cysteine (mutant name: M43CSP1). In the second variant, Leucine 81, which is located on the protein rim, was replaced with a cysteine (mutant name: L81CSP1). Figure 1 a-c shows the protein structure and the positions of the Cys amino acids in the two mutants. Because SP1 is a dodecamer, each complex presents 12 Cys amino acids, with six on each face of the protein ring. Whilst the Cys thiol groups in M43CSP1 are confined to the inner pore, the L81CSP1 thiol groups are exposed on the outer rim of the protein ring. The two mutants were expressed in E. coli and have high stability characteristics (Supporting Information, Figu...

Highly ordered protein cage assemblies: A toolkit for new materials

“…SP1 ring-like protein is highly stable at high temperatures (Wang et al, 2006). Furthermore, site-directed mutagenesis of the amino acid sequence allows for selective binding to metal or insulating surfaces (i.e., cysteine to bind to Au and silicon-binding peptide to bind SiO 2 , respectively), rendering single-protein monolayers (Heyman et al, 2009).…”

Clues for Biomimetics From Natural Composite Materials

“…The identification of silaffin proteins, which mineralize silica in aqueous solutions, enabled the development of biomimietic silica-based technologies utilizing both natural silica-binding peptides such as the R5 silaffin-1 precursor polypeptide from Cerithiopsis fusiformis [24], as well as new silaffin-like peptides identified from combinatorial phage display libraries that precipitated or bound silica in vitro . These findings enabled the production of new silicon-containing biomimetic materials [24–29]. For example, Wong Po Foo et al .…”

“…SP1 protein displays 12 N-terminal arms, six on each side of the protein ring, and therefore any N-terminal fusion to SP1 forms a multivaliant nanoparticle. SP1 has a unique structure and is stable, which allowed the development of self-assembling molecular scaffolds for nanobiotechnology and biomimetic materials [29,31–33]. TBP-1 was fused to the N-termini of the monomers forming multisilica-binding nanoparticles enabling solvent controlled selective binding to silicon surfaces [29].…”

“…SP1 has a unique structure and is stable, which allowed the development of self-assembling molecular scaffolds for nanobiotechnology and biomimetic materials [29,31–33]. TBP-1 was fused to the N-termini of the monomers forming multisilica-binding nanoparticles enabling solvent controlled selective binding to silicon surfaces [29]. Furthermore, it was shown that TBP-1–SP1 can form nano-memory units (these memory units relying on the finite capacity of a silicon nanoparticle embedded in an insulating SP1 protein pore, and the ability of atomic force microscopy to locally address a dot) [34].…”

Effects of the 3D sizing of polyacrylonitrile fabric with carbon nanotube–SP1 protein complex on the interfacial properties of polyacrylonitrile/phenolic composites

“…7 The oligomeric form of SP1 is an exceptionally stable structure, resistant to proteases, high temperatures, organic solvents, and high levels of ionic detergents. 8 Genetically modified SP1 variants that bind a variety of compounds and materials, such as gold, silicon oxide, or titanium oxide, have been constructed [9][10][11][12][13] by fusion of material-specific peptides to its N-terminus, yielding multivalent, high-binding avidity. Moreover, an effective crossbridge reagent was obtained by constructing a silicone-binding SP1 variant, which exposes six silicon oxide-binding sites on each side of the ringlike structure, allowing for silicon oxide nanoparticles binding to the silicone oxide surface.…”