Atomic-Level Quality Assessment of Enzymes Encapsulated in Bioinspired Silica.

Among protein immobilization strategies, encapsulation in bioinspired silica is increasingly popular. Encapsulation offers high yields and the solid support is created through a protein-catalyzed polycondensation reaction that occurs under mild conditions. An integrated strategy is reported for the characterization of both the protein and bioinspired silica scaffold generated by the encapsulation of enzymes with an external silica-forming promoter or with the promoter expressed as a fusion to the enzyme. This strategy is applied to the catalytic domain of matrix metalloproteinase 12. Analysis reveals that the structure of the protein encapsulated by either method is not significantly altered with respect to the native form. The structural features of silica obtained by either strategy are also similar, but differ from those obtained by other approaches. In case of the covalently linked R5-enzyme construct, immobilization yields are higher. Encapsulation through a fusion protein, therefore, appears to be the method of choice.

Solid-State NMR of PEGylated Proteins.

PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic-level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid-state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use.

Biosilica-Entrapped Enzymes Studied by Using Dynamic Nuclear-Polarization-Enhanced High-Field NMR Spectroscopy.

Enzymes are used as environmentally friendly catalysts in many industrial applications, and are frequently immobilized in a matrix to improve their chemical stability for long-term storage and reusability. Recently, it was shown that an atomic-level description of proteins immobilized in a biosilica matrix can be attained by examining their magic-angle spinning (MAS) NMR spectra. However, even though MAS NMR is an excellent tool for determining structure, it is severely hampered by sensitivity. In this work we provide the proof of principle that NMR characterization of biosilica-entrapped enzymes could be assisted by high-field dynamic nuclear polarization (DNP).

Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria.

The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, implement combinatorial regulation, and reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.

(1)H-detected solid-state NMR of proteins entrapped in bioinspired silica: a new tool for biomaterials characterization.

Proton-detection in solid-state NMR, enabled by high magnetic fields (>18 T) and fast magic angle spinning (>50 kHz), allows for the acquisition of traditional (1)H-(15)N experiments on systems that are too big to be observed in solution. Among those, proteins entrapped in a bioinspired silica matrix are an attractive target that is receiving a large share of attention. We demonstrate that (1)H-detected SSNMR provides a novel approach to the rapid assessment of structural integrity in proteins entrapped in bioinspired silica.

Bilayer Membrane Modulation of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Structure and Proteolytic Activity.

Cell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple-helices occurs within blades I and II of this domain. Examination of simultaneous membrane interaction and triple-helix binding revealed a possible regulation of proteolysis due to steric effects of the membrane. At bicelle concentrations of 1%, enzymatic activity towards triple-helices was increased 1.5-fold. A single mutation in the putative membrane interaction region of MT1-MMP (Ser466Pro) resulted in lower enzyme activation by bicelles. An initial structural framework has thus been developed to define the role(s) of cell membranes in modulating proteolysis.

NMR of sedimented, fibrillized, silica-entrapped and microcrystalline (metallo)proteins.

Resolution and sensitivity in solid state NMR (SSNMR) can rival the results achieved by solution NMR, and even outperform them in the case of large systems. However, several factors affect the spectral quality in SSNMR samples, and not all systems turn out to be equally amenable for this methodology. In this review we attempt at analyzing the causes of this variable behavior and at providing hints to increase the chances of experimental success.

SSNMR of biosilica-entrapped enzymes permits an easy assessment of preservation of native conformation in atomic detail.

Bioinspired silica coprecipitates of enzymes find a wide range of applications for analysis and catalysis in industrial and academic research. Here we used SSNMR as a proxy for the 3D structure of enzymes trapped in bioinspired silica. We show that it is easy to assess whether the enzymes retain their native conformation in atomic detail. We thus propose SSNMR as a rapid and reliable tool to analyze this kind of samples at high resolution.