A Major Shell Protein of 1,2-Propanediol Utilization Microcompartment Conserves the Activity of Its Signature Enzyme at Higher Temperatures.

A classic example of an all-protein natural nano-bioreactor, the bacterial microcompartment is a prokaryotic organelle that confines enzymes in a small volume enveloped by an outer protein shell. These protein compartments metabolize specific organic molecules, allowing bacteria to survive in restricted nutrient environments. In this work, 1,2-propanediol utilization microcompartment (PduMCP) was used as a model to study the effect of molecular confinement on the stability and catalytic activity of native enzymes in the microcompartment. A combination of enzyme assays, spectroscopic techniques, binding assays, and computational analysis were used to evaluate the impact of the major shell protein PduBB' on the stability and activity of PduMCP's signature enzyme, dioldehydratase PduCDE. While free PduCDE shows ∼45 % reduction in its optimum activity (activity at 37 °C) when exposed to a temperature of 45 °C, it retains similar activity up to 50 °C when encapsulated within PduMCP. PduBB', a major component of the outer shell of PduMCP, preserves the catalytic efficiency of PduCDE under thermal stress and prevents temperature-induced unfolding and aggregation of PduCDE in vitro. We observed that while both PduB and PduB' interact with the enzyme with micromolar affinity, only the PduBB' combination influences its activity and stability, highlighting the importance of the unique PduBB' combination in the functioning of PduMCP.

Varying protein architectures in 3-dimensions for scaffolding and modulating properties of catalytic gold nanoparticles.

Fabrication and development of nanoscale materials with tunable structural and functional properties require a dynamic arrangement of nanoparticles on architectural templates. The function of nanoparticles not only depends on the property of the nanoparticles but also on their spatial orientations. Proteins with self-assembling properties which can be genetically engineered to varying architectural designs for scaffolds can be used to develop different orientations of nanoparticles in three dimensions. Here, we report the use of naturally self-assembling bacterial micro-compartment shell protein (PduA) assemblies in 2D and its single-point mutant variant (PduA[K26A]) in 3D architectures for the reduction and fabrication of gold nanoparticles. Interestingly, the different spatial organization of gold nanoparticles resulted in a smaller size in the 3D architect scaffold. Here, we observed a two-fold increase in catalytic activity and six-fold higher affinity toward TMB (3,3',5,5'-tetramethylbenzidine) substrate as a measure of higher peroxidase activity (nanozymatic) in the case of PduA[K26A] 3D scaffold. This approach demonstrates that the hierarchical organization of scaffold enables the fine-tuning of nanoparticle properties, thus paving the way toward the design of new nanoscale materials.

Light-controlled shape-changing azomacrocycles exhibiting reversible modulation of pyrene fluorescence emission.

We report the design, synthesis, and study of light-induced shape-changing azomacrocycles. These systems have been incorporated with azobenzene photoswitches using alkoxy tethers and triazole units to afford flexibility and binding. We envision that such azomacrocycles are capable of reversibly binding with the guest molecule. Remarkably, we have demonstrated fully light-controlled fluorescence quenching and enhancement in the monomeric emission of pyrene (guest). Such modulations have been achieved by the photoisomerization of the azomacrocycle and, in turn, host-guest interactions. Also, the azomacrocycles tend to aggregate and can also be controlled by light or heat. We uncovered such phenomena using spectroscopic, microscopic, and isothermal titration calorimetry (ITC) studies and computations.

Weakening of interaction networks with aging in tip-link protein induces hearing loss.

Age-related hearing loss (ARHL) is a common condition in humans marking the gradual decrease in hearing with age. Perturbations in the tip-link protein cadherin-23 that absorbs the mechanical tension from sound and maintains the integrity of hearing is associated with ARHL. Here, in search of molecular origins for ARHL, we dissect the conformational behavior of cadherin-23 along with the mutant S47P that progresses the hearing loss drastically. Using an array of experimental and computational approaches, we highlight a lower thermodynamic stability, significant weakening in the hydrogen-bond network and inter-residue correlations among ß-strands, due to the S47P mutation. The loss in correlated motions translates to not only a remarkable two orders of magnitude slower folding in the mutant but also to a proportionately complex unfolding mechanism. We thus propose that loss in correlated motions within cadherin-23 with aging may trigger ARHL, a molecular feature that likely holds true for other disease-mutations in ß-strand-rich proteins.

The Wrappers of the 1,2-Propanediol Utilization Bacterial Microcompartments.

The propanediol utilization bacterial microcompartments are specialized protein-based organelles in Salmonella that facilitate the catabolism of 1,2-propanediol when available as the sole carbon source. This smart prokaryotic cell organelle compartmentalizes essential enzymes and substrates in a volume of a few attoliters compared to the femtoliter volume of a bacterial cell thereby enhancing the enzyme kinetics and properly orchestrating the downstream pathways. A shell or coat, which is composed of a few thousand protein subunits, wraps a chain of consecutively acting enzymes and serves as ducts for the diffusion of substrates, cofactors, and products into and out of the core of the microcompartment. In this article we bring together the properties of the wrappers of the propanediol utilization bacterial microcompartments to update our understanding on the mechanism of the formation of these unique wraps, their assembly, and interaction with the encapsulated enzymes.

Protein morphology drives the structure and catalytic activity of bio-inorganic hybrids.

Bio-hybrid materials have received a lot of attention in view of their bio-mimicking nature. One such biomimetic material with catalytic activity are the protein derived floral nanohybrid. Copper phosphate coordinated flakes can be curated to distinct floral morphology using proteins. Structurally two different proteins with similar size and with no known enzymatic activity are used to evaluate the role of protein structure and morphology, on the structure-activity relationship of the developed hybrid nanoflowers. Globular protein BSA and bacterial microcompartment domain protein PduBB' are selected. PduBB' because of self-assembling nature forms extended sheets, whereas BSA lacks specific assembly. The developed hybrid NFs differ in their morphology and also in their mimicry as a biological catalyst. The present investigation highlights the importance of the quaternary structure of proteins in tailoring the structure and function of the h-NFs. The results in this manuscript will motivate and guide designing, engineering and selection of glue material for fabricating biomacromolecule derived biohybrid material to mimic natural enzymes of potential industrial application.

Probe into a multi-protein prokaryotic organelle using thermal scanning assay reveals distinct properties of the core and the shell.

BACKGROUND: Bacterial microcompartments represent the only reported category of prokaryotic organelles that are capable of functioning as independent bioreactors. In this organelle, a biochemical pathway with all the enzyme machinery is encapsulated within an all protein shell. The shell proteins and the enzymes have distinct structural features. It is hypothesized that flat shell proteins align sideways to form extended sheets and, the globular enzymes fill up the central core of the organelle. METHODS: Using differential scanning fluorimetry, we explored the structure and functional alteration of Pdu BMC, involving tertiary or quaternary structures. RESULTS: Our findings exhibit that these intact BMCs as a whole behave similar to a globular protein with a rich hydrophobic core, which is exposed upon thermal insult. The encapsulated enzymes itself have a strong hydrophobic core, which is in line with the hydrophobic-collapse model of protein folding. The shell proteins, on the other hand, do not have a strong hydrophobic core and show a significant portion of exposed hydrophobic patches. CONCLUSION: We show for the first time the thermal unfolding profile of the BMC domain proteins and the unique exposure of hydrophobic patches in them might be required for anchoring the enzymes leading to better packaging of the micro-compartments. GENERAL SIGNIFICANCE: These observations indicate that the genesis of these unique bacterial organelles is driven by the hydrophobic interactions between the shell and the enzymes. Insights from this work will aid in the genetic and biochemical engineering of thermostable efficient enzymatic biomaterials.

Functional protein shells fabricated from the self-assembling protein sheets of prokaryotic organelles.

Fabricating protein compartments from protein units is challenging and limited by the use of external stimuli and crosslinkers. Here we explore the fabrication of all-protein compartments using self-assembled proteins of prokaryotic organelles. These proteins have intrinsic interacting domains which are ionic in nature, and spontaneously self-assemble into sheets when over-expressed. Using a one-step approach, we maneuvered the formation of the protein shells from the sheets without any external stimuli or crosslinker. The spontaneous self-assembly of the native protein sheets into protein shells not only preserves the native functional properties of the protein but also enhances their thermal stability compared to the sheets. We further demonstrate that these compartments can encapsulate macromolecular enzymes and, more interestingly, permit the free exchange of small molecules and substrates through their intrinsic conduit channels. The porous nature of the shell housing active enzymes and allowing movement of small molecules makes them suitable as active bioreactors. Furthermore, to extend the tunability of these protein-compartments with respect to stability, enzyme-encapsulation, and permeability, we fabricated three different compartments using three different sheet proteins, PduA/B/B' and compared their properties. Interestingly we find that all three protein shells show similar behaviour with respect to an encapsulated diol-dehydratase enzyme and vitamin B12, which are native to the Pdu BMC system. Furthermore, for the non-native enzyme CytC, the small molecule R6G dye, doxorubicin, NR and curcumin they behave diversely. Insights from this analysis will allow us to design and develop sheet protein based synthetic active bioreactors requiring meticulous, compartmentalization in process optimization.

Cellulose-metallothionein matrix for metal binding.

In this report, we have modified bacterial cellulose to a metal binding matrix by covalently conjugating physiological metal chelators known as metallothioneins. The hydroxyl groups of the native bacterial cellulose from Gluconobacter xylinus are epoxidized, followed by the covalent conjugation with the amine groups of the proteins. For the first time, a covalent conjugation of protein with bacterial cellulose is achieved using the epoxy-amine conjugation chemistry. Using this protocol, 50% mass by mass of the metallothionein could be attached to bacterial cellulose. The morphological features and porosity of the modified cellulose are different compared to pristine bacterial cellulose. Also, the conjugated material has better thermal stability. A five-fold enhancement in the metal binding capacity of the metallothionein conjugated bacterial cellulose is achieved as compared to pristine bacterial cellulose. Cellular metabolic assay and membrane integrity assay on MCF and HeLa cell lines showed no significant toxicity of the conjugate material. This bacterial cellulose-metallothionein conjugate can be explored for health care applications where management of metal toxicity is crucial. Further, the epoxy-amine chemistry for covalent conjugation of protein can be applied for several other types of proteins to develop specific functional biocompatible and biodegradable cellulose matrices.