Emergence of slip-ideal-slip behavior in tip-links serve as force filters of sound in hearing.

Tip-links in the inner ear convey force from sound and trigger mechanotransduction. Here, we present evidence that tip-links (collectively as heterotetrameric complexes of cadherins) function as force filters during mechanotransduction. Our force-clamp experiments reveal that the tip-link complexes show slip-ideal-slip bond dynamics. At low forces, the lifetime of the tip-link complex drops monotonically, indicating slip-bond dynamics. The ideal bond, rare in nature, is seen in an intermediate force regime where the survival of the complex remains constant over a wide range. At large forces, tip-links follow a slip bond and dissociate entirely to cut-off force transmission. In contrast, the individual tip-links (heterodimers) display slip-catch-slip bonds to the applied forces. While with a phenotypic mutant, we showed the importance of the slip-catch-slip bonds in uninterrupted hearing, our coarse-grained Langevin dynamics simulations demonstrated that the slip-ideal-slip bonds emerge as a collective feature from the slip-catch-slip bonds of individual tip-links.

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.

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.

Broken force dispersal network in tip-links by the mutations at the Ca2+-binding residues induces hearing-loss.

Tip-link as force-sensor in hearing conveys the mechanical force originating from sound to ion-channels while maintaining the integrity of the entire sensory assembly in the inner ear. This delicate balance between structure and function of tip-links is regulated by Ca2+-ions present in endolymph. Mutations at the Ca2+-binding sites of tip-links often lead to congenital deafness, sometimes syndromic defects impairing vision along with hearing. Although such mutations are already identified, it is still not clear how the mutants alter the structure-function properties of the force-sensors associated with diseases. With an aim to decipher the differences in force-conveying properties of the force-sensors in molecular details, we identified the conformational variability of mutant and wild-type tip-links at the single-molecule level using FRET at the endolymphatic Ca2+ concentrations and subsequently measured the force-responsive behavior using single-molecule force spectroscopy with an Atomic Force Microscope (AFM). AFM allowed us to mimic the high and wide range of force ramps (103-106 pN s-1) as experienced in the inner ear. We performed in silico network analysis to learn that alterations in the conformations of the mutants interrupt the natural force-propagation paths through the sensors and make the mutant tip-links vulnerable to input forces from sound stimuli. We also demonstrated that a Ca2+ rich environment can restore the force-response of the mutant tip-links which may eventually facilitate the designing of better therapeutic strategies to the hearing loss.

Force-activated catalytic pathway accelerates bacterial adhesion against flow.

Mechanical cues often influence the factors affecting the transition states of catalytic reactions and alter the activation pathway. However, tracking the real-time dynamics of such activation pathways is limited. Using single-molecule trapping of reaction intermediates, we developed a method that enabled us to perform one reaction at one site and simultaneously study the real-time dynamics of the catalytic pathway. Using this, we showed single-molecule calligraphy at nanometer resolution and deciphered the mechanism of the sortase A enzymatic reaction that, counter-intuitively, accelerates bacterial adhesion under shear tension. Our method captured a force-induced dissociation of the enzyme-substrate bond that accelerates the forward reaction 100×, proposing a new mechano-activated catalytic pathway. In corroboration, our molecular dynamics simulations in the presence of force identified a force-induced conformational switch in the enzyme that accelerates proton transfer between CYS184 (acceptor) and HIS120 (donor) catalytic dyads by reducing the inter-residue distances. Overall, the present study opens up the possibility of studying the influence of factors affecting transition states in real time and paves the way for the rational design of enzymes with enhanced efficiency.

ESCORTing proteins directly from whole cell-lysate for single-molecule studies.

We have developed a method for Enzymatic Sortase-assisted Covalent Orientation-specific Restraint Tethering (ESCORT) recombinant proteins onto surfaces directly from cell-lysate. With an improved surface passivation method, we obviate the cumbersome purification steps even for single molecule studies that demand high purity in the sample. We demonstrated high-specificity of the method, high-passivity of the surface and uncompromised functional integrity of anchored proteins using single molecule fluorescence and force-mapping. We anticipate that this method will substantially reduce the investment by way of time, money and energy in the area of single molecule studies.