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Influence of silk-silica fusion protein design on silica condensation in vitro and cellular calcification.

ABSTRACT: Biomaterial design via genetic engineering can be utilized for the rational functionalization of proteins to promote biomaterial integration and tissue regeneration. Spider silk has been extensively studied for its biocompatibility, biodegradability and extraordinary material properties. As a protein-based biomaterial, recombinant DNA derived derivatives of spider silks have been modified with biomineralization domains which lead to silica deposition and potentially accelerated bone regeneration. However, the influence of the location of the R5 (SSKKSGSYSGSKGSKRRIL) silicifying domain fused with the spider silk protein sequence on the biosilicification process remains to be determined. Here we designed two silk-R5 fusion proteins that differed in the location of the R5 peptide, C- vs. N-terminus, where the spider silk domain consisted of a 15mer repeat of a 33 amino acid consensus sequence of the major ampullate dragline Spidroin 1 from Nephila clavipes (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT). The chemical, physical and silica deposition properties of these recombinant proteins were assessed and compared to a silk 15mer control without the R5 present. The location of the R5 peptide did not have a significant effect on wettability and surface energies, while the C-terminal location of the R5 promoted more controlled silica precipitation, suggesting differences in protein folding and possibly different access to charged amino acids that drive the silicification process. Further, cell compatibility in vitro, as well as the ability to promote human bone marrow derived mesenchymal stem cell (hMSC) differentiation were demonstrated for both variants of the fusion proteins.

SUBMITTER: Plowright R

PROVIDER: S-EPMC4792305 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Influence of silk-silica fusion protein design on silica condensation <i>in vitro</i> and cellular calcification.

Plowright Robyn R Dinjaski Nina N Zhou Shun S Belton David J DJ Kaplan David L DL Perry Carole C CC

RSC advances 20160101 26

Biomaterial design via genetic engineering can be utilized for the rational functionalization of proteins to promote biomaterial integration and tissue regeneration. Spider silk has been extensively studied for its biocompatibility, biodegradability and extraordinary material properties. As a protein-based biomaterial, recombinant DNA derived derivatives of spider silks have been modified with biomineralization domains which lead to silica deposition and potentially accelerated bone regeneration ...[more]

PMID: 26989487

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Project description:Requiring mild synthesis conditions and possessing a high level of organization and functionality, biosilicas constitute a source of wonder and inspiration for both materials scientists and biologists. In order to understand how such biomaterials are formed and to apply this knowledge to the generation of novel bioinspired materials, a detailed study of the materials, as formed under biologically relevant conditions, is required. In this contribution, data from a detailed study of silica speciation and condensation using a model bioinspired silica precursor (silicon catechol complex, SCC) is presented. The silicon complex quickly and controllably dissociates under neutral pH conditions to well-defined, metastable solutions of orthosilicic acid. The formation of silicomolybdous (blue) complexes was used to monitor and study different stages of silicic acid condensation. In parallel, the rates of silicomolybdic (yellow) complex formation, with mathematical modeling of the species present, was used to follow the solution speciation of polysilicic acids. The results obtained from the two assays correlate well. Monomeric silicic acid, trimeric silicic acids, and different classes of oligomeric polysilicic acids and silica nuclei can be identified and their periods of stability during the early stages of silica condensation measured. For experiments performed at a range of temperatures (273-323 K), an activation energy of 77 kJ.mol(-1) was obtained for the formation of trimers. The activation energies for the forward and reverse condensation reactions for addition of monomers to polysilicic acids (273-293 +/- 1 K) were 55.0 and 58.6 kJ.mol(-1), respectively. For temperatures above 293 K, these energies were reduced to 6.1 and 7.3 kJ.mol(-1), indicating a probable change in the prevailing condensation mechanism. The impact of pH on the rates of condensation were measured. There was a direct correlation between the apparent third-order rate constant for trimer formation and pH (4.7-6.9 +/- 0.1) while values for the reversible first-order rates reached a plateau at circumneutral pH. These different behaviors are discussed with reference to the generally accepted mechanism for silica condensation in which anionic silicate solution species are central to the condensation process. The results presented in this paper support the use of precursors such as silicon catecholate complexes in the study of biosilicification in vitro. Further detailed experimentation is needed to increase our understanding of specific biomolecule silica interactions that ultimately generate the complex, finely detailed siliceous structures we observe in the world around us.

Dataset Information

Influence of silk-silica fusion protein design on silica condensation in vitro and cellular calcification.

Publications

Influence of silk-silica fusion protein design on silica condensation <i>in vitro</i> and cellular calcification.

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