Design of a Redox-Sensitive Supramolecular Protein Assembly System Operating in Live Cells

A fusion construct between Citrine (a YFP variant) and human ferritin (H-chain) was recently shown to form supramolecular assemblies of micrometer size when expressed in mammalian cells. The assembly process is driven by weak hydrophobic interactions leading to dimerization of YFP. Protein assembly could be suppressed at the gene level by mutation in the primary sequence of the construct. In this work, we describe the engineering of a self-assembly interface sensitive to redox state in the cell. Key hydrophobic residues of YFP were mutated systematically to cysteines. Supramolecular assembly of the Citrine–ferritin construct was in some cases preserved by formation of disulfide bonds in place of hydrophobic interactions. In others cases, assembly was abolished, resulting in a diffuse distribution of the expressed protein. A specific variant that remained diffuse under normally reducing intracellular conditions was found to self-assemble rapidly upon exposure to a thiol-specific oxidizing reagent

Supramolecular Assembly and Coalescence of Ferritin Cages Driven by Designed Protein–Protein Interactions

A genetically encoded system for expression of supramolecular protein assemblies (SMPAs) based on a fusion construct between ferritin and citrine (YFP) was transferred from a mammalian to a bacterial host. The assembly process is revealed to be independent of the expression host, while dimensions and level of order of the assembled structures were influenced by the host organism. An additional level of interactions, namely, coalescence between the preformed SMPAs, was observed during the purification process. SAXS investigation revealed that upon coalescence, the local order of the individual SMPAs was preserved. Finally, the chaotropic agent urea effectively disrupted both the macroscopic coalescence and the interactions at the nanoscale until the level of the single ferritin cage

Probing Protein Conformation in Cells by EPR Distance Measurements using Gd³⁺ Spin Labeling

Protein structure investigations are usually carried out <i>in vitro</i> under conditions far from their native environment in the cell. Differences between <i>in-cell</i> and <i>in vitro</i> structures of proteins can be generated by crowding effects, local pH changes, specific and nonspecific protein and ligand binding events, and chemical modifications. Double electron–electron resonance (DEER), in conjunction with site-directed spin-labeling, has emerged in the past decade as a powerful technique for exploring protein conformations in frozen solutions. The major challenges facing the application of this methodology to <i>in-cell</i> measurements are the instabilities of the standard nitroxide spin labels in the cell environment and the limited sensitivity at conventional X-band frequencies. We present a new approach for <i>in-cell</i> DEER distance measurement in human cells, based on the use of: (i) reduction resistant Gd³⁺ chelates as spin labels, (ii) high frequency (94.9 GHz) for sensitivity enhancement, and (iii) hypo-osmotic shock for efficient delivery of the labeled protein into the cell. The proof of concept is demonstrated on doubly labeled ubiquitin in HeLa cells

Optimization of Localized Surface Plasmon Resonance Transducers for Studying Carbohydrate–Protein Interactions

Noble metal nanostructures supporting localized surface plasmons (SPs) have been widely applied to chemical and biological sensing. Changes in the refractive index near the nanostructures affect the SP extinction band, making localized surface plasmon resonance (LSPR) spectroscopy a convenient tool for studying biological interactions. Carbohydrate–protein interactions are of major importance in living organisms; their study is crucial for understanding of basic biological processes and for the construction of biosensors for diagnostics and drug development. Here LSPR transducers based on gold island films prepared by evaporation on glass and annealing were optimized for monitoring the specific interaction between Concanavalin A (Con A) and D-(+)-mannose. The sugar was modified with a PEG-thiol linker and immobilized on the Au islands. Sensing assays were performed under stationary and flow conditions, the latter providing kinetic parameters for protein binding and dissociation. Ellipsometry and Fourier transform-infrared (FT-IR) data, as well as scanning electron microscopy (SEM) imaging of fixated and stained samples, furnished independent evidence for the protein-sugar recognition. Enhanced response and visual detection of protein binding was demonstrated using Au nanoparticles stabilized with the linker-modified mannose molecules. Mannose-coated transducers display an excellent selectivity toward Con A in the presence of a large excess of bovine serum albumin (BSA)