Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Abstract

Integral membrane proteins (IMPs) comprise highly important classes of proteins such as transporters, sensors, and channels, but their investigation and biotechnological application are complicated by the difficulty to stabilize them in solution. We set out to develop a biomimetic procedure to encapsulate functional integral membrane proteins in silica to facilitate their handling under otherwise detrimental conditions and thereby extend their applicability. To this end, we designed and expressed new fusion constructs of the membrane scaffold protein MSP with silica-precipitating peptides based on the R5 sequence from the diatom Cylindrotheca fusiformis . Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed that membrane lipid nanodiscs surrounded by our MSP variants fused to an R5 peptide, so-called nanodiscs, were formed. Exposing them to silicic acid led to silica-encapsulated nanodiscs, a new material for stabilizing membrane structures and a first step toward incorporating membrane proteins in such structures. In an alternative approach, four fusion constructs based on the amphiphilic β-sheet peptide BP-1 and the R5 peptide were generated and successfully employed toward silica encapsulation of functional diacylglycerol kinase (DGK). Silica-encapsulated DGK was significantly more stable against protease exposure and incubation with simulated gastric fluid (SGF) and intestinal fluid (SIF).

Enzyme-Assisted Metabolically Coated Bimetallic Thalassiosira pseudonana Nanosilica as a Surface-Enhanced Raman Scattering Substrate for Specific Screening of Prostate Cancer Individuals

“…The presence of protein tags compared to those without tags does not influence the native conformation of the SoX protein or its activities (Figure S4c). This aligns with previous studies on fusing silaffin tags to multiple proteins 26,38 without affecting the native structure. Immobilization of both tagged SoX from crude protein extract measured by Bradford assay showed 90% selective uptake, indicating that both tags have high affinity for T. pseudonana frustules.…”

Sustainability of in vitro light-dependent NADPH generation by the thylakoid membrane of Synechocystis sp. PCC6803

“…To protect the lipid bilayer and protein-pigment complexes of TM from external stress, they were kept in a defined space via coating with a rigid silica shield. Silaffin-derived peptide R5 (SSKKSGSYSGSKGSKRRIL) has been shown to mediate silicification, which can encapsulate proteins and bacteria to enhance stability and survivability [ 26 , 46 ]. Inspired by this process, pTMe was biosilicified using R5 with a TMOS (Additional file 1 : Fig.…”

“…Encapsulated proteins confined by silica shells are known to possess considerable resistance to acidic environment, digestive enzymes, and denaturing agents [ 25 – 27 ]. However, if the thickness of silica shell exceeds a certain limit, the movement of reactants across silica is restricted and would slow down the reaction [ 26 ].…”

A sticky tale : Biomaterial coatings from modular proteins

“…Since silicification was proved to increase the thermal and chemical stability of fluorescent proteins, enzymes, viruses, and eukaryotic cells, this approach was tested with therapeutical compounds. [211][212][213][214] For example, the use of a bifunctional polypeptide allowed to stabilize a model hydrophobic drug into polypeptide assemblies and induce the formation of a silica shell around the polypeptide/drug core, with positive results both in vitro and in vivo. 215,216 More recently, other molecular scaffolds were combined with silicification.…”