Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers.

Influenza hemagglutinin (HA) is the canonical type I viral envelope glycoprotein and provides a template for the membrane-fusion mechanisms of numerous viruses. The current model of HA-mediated membrane fusion describes a static "spring-loaded" fusion domain (HA2) at neutral pH. Acidic pH triggers a singular irreversible conformational rearrangement in HA2 that fuses viral and cellular membranes. Here, using single-molecule Förster resonance energy transfer (smFRET)-imaging, we directly visualized pH-triggered conformational changes of HA trimers on the viral surface. Our analyses reveal reversible exchange between the pre-fusion and two intermediate conformations of HA2. Acidification of pH and receptor binding shifts the dynamic equilibrium of HA2 in favor of forward progression along the membrane-fusion reaction coordinate. Interaction with the target membrane promotes irreversible transition of HA2 to the post-fusion state. The reversibility of HA2 conformation may protect against transition to the post-fusion state prior to arrival at the target membrane.

Site-selective peptide bond hydrolysis and ligation in water by short peptide-based assemblies.

The evolution of complex chemical inventory from Darwin's nutrient-rich warm pond necessitated rudimentary yet efficient catalytic folds. Short peptides and their self-organized microstructures, ranging from spherical colloids to amyloidogenic aggregates might have played a crucial role in the emergence of contemporary catalytic entities. However, the question of how short peptide fragments had functions akin to contemporary complex enzymes to catalyze cleavage and formation of highly stable peptide bonds that constitute the backbone of all proteins remains an unresolved yet fundamentally important question in terms of the origins of enzymes. We report short-peptide-based spherical assemblies that demonstrated residue-specific cleavage and formation of peptide bonds of diverse peptide-based substrates under aqueous environment. Despite the short sequence length, the assemblies utilized the synergistic collaboration of four residues which included the catalytic triad of extant serine proteases with a nonproteinogenic amino acid (quinone moiety), to facilitate proteolysis, ligation, and a three-step (hydrolysis-ligation-hydrolysis) cascade. Such short-peptide-based catalytic assemblies argue for their candidacy as the earliest protein folds and open up avenues for biotechnological applications.

Estimates of differential toxin expression governing heterogeneous intracellular lifespans of Streptococcus pneumoniae.

Following invasion of the host cell, pore-forming toxins secreted by pathogens compromise vacuole integrity and expose the microbe to diverse intracellular defence mechanisms. However, the quantitative correlation between toxin expression levels and consequent pore dynamics, fostering the intracellular life of pathogens, remains largely unexplored. In this study, using Streptococcus pneumoniae and its secreted pore-forming toxin pneumolysin (Ply) as a model system, we explored various facets of host-pathogen interactions in the host cytosol. Using time-lapse fluorescence imaging, we monitored pore formation dynamics and lifespans of different pneumococcal subpopulations inside host cells. Based on experimental histograms of various event timescales such as pore formation time, vacuolar death or cytosolic escape time and total degradation time, we developed a mathematical model based on first-passage processes that could correlate the event timescales to intravacuolar toxin accumulation. This allowed us to estimate Ply production rate, burst size and threshold Ply quantities that trigger these outcomes. Collectively, we present a general method that illustrates a correlation between toxin expression levels and pore dynamics, dictating intracellular lifespans of pathogens.

Temporal (Dis)Assembly of Peptide Nanostructures Dictated by Native Multistep Catalytic Transformations.

Emergence of complex catalytic machinery via simple building blocks under non-equilibrium conditions can contribute toward the system level understanding of the extant biocatalytic reaction network that fuels metabolism. Herein, we report temporal (dis)assembly of peptide nanostructures in presence of a cofactor dictated by native multistep cascade transformations. The short peptide can form a dynamic covalent bond with the thermodynamically activated substrate and recruit cofactor hemin to access non-equilibrium catalytic nanostructures (positive feedback). The neighboring imidazole and hemin moieties in the assembled state rapidly converted the substrate to product(s) via a two-step cascade reaction (hydrolase-peroxidase like) that subsequently triggered the disassembly of the catalytic nanostructures (negative feedback). The feedback coupled reaction cycle involving intrinsic catalytic prowess of short peptides to realize the advanced trait of two-stage cascade degradation of a thermodynamically activated substrate foreshadows the complex non-equilibrium protometabolic networks that might have preceded the chemical emergence of life.

Allosteric Control of the Catalytic Properties of Dipeptide-Based Supramolecular Assemblies.

Allostery, as seen in extant biology, governs the activity regulation of enzymes through the redistribution of conformational equilibria upon binding an effector. Herein, a minimal design is demonstrated where a dipeptide can exploit dynamic imine linkage to condense with simple aldehydes to access spherical aggregates as catalytically active states, which facilitates an orthogonal reaction due to the closer proximity of catalytic residues (imidazoles). The allosteric site (amine) of the minimal catalyst can concomitantly bind to an inhibitor via a dynamic exchange, which leads to the alternation of the energy landscape of the self-assembled state, resulting in downregulation of catalytic activity. Further, temporal control over allosteric regulation is realized via a feedback-controlled autonomous reaction network that utilizes the hydrolytic activity of the (in)active state as a function of time.

Exact Distribution of the Quantal Content in Synaptic Transmission.

During electrochemical signal transmission through synapses, triggered by an action potential (AP), a stochastic number of synaptic vesicles (SVs), called the "quantal content," release neurotransmitters in the synaptic cleft. It is widely accepted that the quantal content probability distribution is a binomial based on the number of ready-release SVs in the presynaptic terminal. But the latter number itself fluctuates due to its stochastic replenishment, hence the actual distribution of quantal content is unknown. We show that exact distribution of quantal content can be derived for general stochastic AP inputs in the steady state. For fixed interval AP train, we prove that the distribution is a binomial, and corroborate our predictions by comparison with electrophysiological recordings from MNTB-LSO synapses of juvenile mice. For a Poisson train, we show that the distribution is nonbinomial. Moreover, we find exact moments of the quantal content in the Poisson and other general cases, which may be used to obtain the model parameters from experiments.

Nature of barriers determines first passage times in heterogeneous media.

Intuition suggests that passage times across a region increase with the number of barriers along the path. Can this fail depending on the nature of the barrier? To probe this fundamental question, we exactly solve for the first passage time in general d-dimensions for diffusive transport through a spatially patterned array of obstacles - either entropic or energetic, depending on the nature of the obstacles. For energetic barriers, we show that first passage times vary non-monotonically with the number of barriers, while for entropic barriers it increases monotonically. This non-monotonicity for energetic barriers is further reflected in the behaviour of effective diffusivity as well. We then design a simple experiment where a robotic bug navigates in a heterogeneous environment through a spatially patterned array of obstacles to validate our predictions. Finally, using numerical simulations, we show that this non-monotonic behaviour for energetic barriers is general and extends to even super-diffusive transport.

Molecular design of the γδT cell receptor ectodomain encodes biologically fit ligand recognition in the absence of mechanosensing.

High-acuity αßT cell receptor (TCR) recognition of peptides bound to major histocompatibility complex molecules (pMHCs) requires mechanosensing, a process whereby piconewton (pN) bioforces exert physical load on αßTCR-pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αßTCRs and pre-TCRs within the αßT lineage, its role in γδT cells is unknown. Here, we show that the human DP10.7 γδTCR specific for the sulfoglycolipid sulfatide bound to CD1d only sustains a significant load and undergoes force-induced structural transitions when the binding interface-distal γδ constant domain (C) module is replaced with that of αß. The chimeric γδ-αßTCR also signals more robustly than does the wild-type (WT) γδTCR, as revealed by RNA-sequencing (RNA-seq) analysis of TCR-transduced Rag2-/- thymocytes, consistent with structural, single-molecule, and molecular dynamics studies reflective of γδTCRs as mediating recognition via a more canonical immunoglobulin-like receptor interaction. Absence of robust, force-related catch bonds, as well as γδTCR structural transitions, implies that γδT cells do not use mechanosensing for ligand recognition. This distinction is consonant with the fact that their innate-type ligands, including markers of cellular stress, are expressed at a high copy number relative to the sparse pMHC ligands of αßT cells arrayed on activating target cells. We posit that mechanosensing emerged over ∼200 million years of vertebrate evolution to fulfill indispensable adaptive immune recognition requirements for pMHC in the αßT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection.

Temporal Self-Regulation of Mechanical Properties via Catalytic Amyloid Polymers of a Short Peptide.

We report a short peptide that accessed dynamic catalytic polymers to demonstrate four-stage (sol-gel-weak gel-strong gel) temporal self-regulation of its mechanical properties. The peptide exploited its intrinsic catalytic capabilities of manipulating C-C bonds (retro-aldolase-like) that resulted in a nonlinear variation in the catalytic rate. The seven-residue sequence exploited two lysines for binding and cleaving the thermodynamically activated substrate that subsequently led to the self-regulation of the mechanical strengths of the polymerized states as a function of time and reaction progress. Interestingly, the polymerization events were modulated by the different catalytic potentials of the two terminal lysines to cleave the substrate, covalently trap the electrophilic products, and subsequently control the mechanical properties of the system.

Emergence of Catalytic Triad by Short Peptide Based Nanofibrillar Assemblies.

Through millions of years of the evolutionary journey, contemporary enzymes observed in extant metabolic pathways have evolved to become specialized, in contrast to their ancestors, which displayed promiscuous activities with wider substrate specificities. However, there remain critical gaps in our understanding of how these early enzymes could show such catalytic versatility despite lacking the complex three-dimensional folds of the existing modern-day enzymes. Herein, we report the emergence of a promiscuous catalytic triad by short amyloid peptide based nanofibers that access paracrystalline folds of ß-sheets to expose three residues (lysine, imidazole, and tyrosine) toward solvent. The ordered folded nanostructures could simultaneously catalyze two metabolically relevant chemical transformations via C-O and C-C bond manipulations, displaying both hydrolase and retro-aldolase-like activities. Further, the latent catalytic capabilities of the short peptide based promiscuous folds also helped in processing a cascade transformation, suggesting the important role they might have played in protometabolism and early evolutionary processes.

Emergence of Photomodulated Protometabolism by Short Peptide-Based Assemblies.

In the early Earth, rudimentary enzymes must have utilized the available light energy source to modulate protometabolic processes. Herein, we report the light-responsive C-C bond manipulation via short peptide-based assemblies bound to the photosensitive molecular cofactor (azo-based photoswitch) where the energy of the light source regulated the binding sites which subsequently modulated the retro-aldolase activity. In the presence of a continual source of high-energy photons, temporal realization of a catalytically more proficient state could be achieved under nonequilibrium conditions. Further, the hydrophobic surface of peptide assemblies facilitated the binding of an orthogonal molecular catalyst that showed augmented activity (promiscuous hydrolytic activity) upon binding. This latent activity was utilized for the in situ generation of light-sensitive cofactor that subsequently modulated the retro-aldolase activity, thus creating a reaction network.

Covalent Organic Framework Cladding on Peptide-Amphiphile-Based Biomimetic Catalysts.

Peptide-based biomimetic catalysts are promising materials for efficient catalytic activity in various biochemical transformations. However, their lack of operational stability and fragile nature in non-aqueous media limit their practical applications. In this study, we have developed a cladding technique to stabilize biomimetic catalysts within porous covalent organic framework (COF) scaffolds. This methodology allows for the homogeneous distribution of peptide nanotubes inside the COF (TpAzo and TpDPP) backbone, creating strong noncovalent interactions that prevent leaching. We synthesized two different peptide-amphiphiles, C10FFVK and C10FFVR, with lysine (K) and arginine (R) at the C-termini, respectively, which formed nanotubular morphologies. The C10FFVK peptide-amphiphile nanotubes exhibit enzyme-like behavior and efficiently catalyze C-C bond cleavage in a buffer medium (pH 7.5). We produced nanotubular structures of TpAzo-C10FFVK and TpDPP-C10FFVK through COF cladding by using interfacial crystallization (IC). The peptide nanotubes encased in the COF catalyze C-C bond cleavage in a buffer medium as well as in different organic solvents (such as acetonitrile, acetone, and dichloromethane). The TpAzo-C10FFVK catalyst, being heterogeneous, is easily recoverable, enabling the reaction to be performed for multiple cycles. Additionally, the synthesis of TpAzo-C10FFVK thin films facilitates catalysis in flow. As control, we synthesized another peptide-amphiphile, C10FFVR, which also forms tubular assemblies. By depositing TpAzo COF crystallites on C10FFVR nanotubes through IC, we produced TpAzo-C10FFVR nanotubular structures that expectedly did not show catalysis, suggesting the critical role of the lysines in the TpAzo-C10FFVK.

Conformational changes in the Ebola virus membrane fusion machine induced by pH, Ca2+, and receptor binding.

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, removing the glycan cap and exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. NPC1 binding to cleaved GP1 is required for entry. How this interaction translates to GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a bulk fluorescence dequenching assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding synergistically induce conformational changes in GP2 and permit virus-liposome lipid mixing. Acidic pH and Ca2+ shifted the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. Glycan cap cleavage on GP1 enabled GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the postfusion 6-helix bundle; NPC1 binding further promoted transition to the irreversible conformation. Thus, the glycan cap of GP1 may allosterically protect against inactivation of EBOV by premature triggering of GP2.

Systems chemistry of peptide-assemblies for biochemical transformations.

During the billions of years of the evolutionary journey, primitive polymers, involved in proto metabolic pathways with low catalytic activity, played critical roles in the emergence of modern enzymes with remarkable substrate specificity. The precise positioning of amino acid residues and the complex orchestrated interplay in the binding pockets of evolved enzymes promote covalent and non-covalent interactions to foster a diverse set of complex catalytic transformations. Recent efforts to emulate the structural and functional information of extant enzymes by minimal peptide based assemblies have attempted to provide a holistic approach that could help in discerning the prebiotic origins of catalytically active binding pockets of advanced proteins. In addition to the impressive sets of advanced biochemical transformations, catalytic promiscuity and cascade catalysis by such small molecule based dynamic systems can foreshadow the ancestral catalytic processes required for the onset of protometabolism. Looking beyond minimal systems that work close to equilibrium, catalytic systems and compartments under non-equilibrium conditions utilizing simple prebiotically relevant precursors have attempted to shed light on how bioenergetics played an essential role in chemical emergence of complex behaviour. Herein, we map out these recent works and progress where diverse sets of complex enzymatic transformations were demonstrated by utilizing minimal peptide based self-assembled systems. Further, we have attempted to cover the examples of peptide assemblies that could feature promiscuous activity and promote complex multistep cascade reaction networks. The review also covers a few recent examples of minimal transient catalytic assemblies under non-equilibrium conditions. This review attempts to provide a broad perspective for potentially programming functionality via rational selection of amino acid sequences leading towards minimal catalytic systems that resemble the traits of contemporary enzymes.

Exploitation of Catalytic Dyads by Short Peptide-Based Nanotubes for Enantioselective Covalent Catalysis.

Extant enzymes with precisely arranged multiple residues in their three-dimensional binding pockets are capable of exhibiting remarkable stereoselectivity towards a racemic mixture of substrates. However, how early protein folds that possibly featured short peptide fragments facilitated enantioselective catalytic transformations important for the emergence of homochirality still remains an intriguing open question. Herein, enantioselective hydrolysis was shown by short peptide-based nanotubes that could exploit multiple solvent-exposed residues to create chiral binding grooves to covalently interact and subsequently hydrolyse one enantiomer preferentially from a racemic pool. Single or double-site chiral mutations led to opposite but diminished and even complete loss of enantioselectivities, suggesting the critical roles of the binding enthalpies from the precise localization of the active site residues, despite the short sequence lengths. This work underpins the enantioselective catalytic prowess of short peptide-based folds and argues their possible role in the emergence of homochiral chemical inventory.

Nonequilibrium Catalytic Supramolecular Assemblies of Melamine- and Imidazole-Based Dynamic Building Blocks.

The development of synthetic nonequilibrium systems has gathered increasing attention due to their potential to illustrate the dynamic, complex, and emergent traits of biological systems. Simple building blocks capable of interacting via dynamic covalent chemistry and physical assembly in a reaction network under nonequilibrium conditions can contribute to our understanding of complex systems of life and its origin. Herein, we have demonstrated the nonequilibrium generation of catalytic supramolecular assemblies from simple heterocycle melamine driven by a thermodynamically activated ester. Utilizing a reversible covalent linkage, an imidazole moiety was recruited by the assemblies to access a catalytic transient state that dissipated energy via accelerated hydrolysis of the activated ester. The nonequilibrium assemblies were further capable of temporally binding to a hydrophobic guest to modulate its photophysical properties. Notably, the presence of an exogenous aromatic base augmented the lifetime of the catalytic microphases, reflecting their higher kinetic stability.

Nonequilibrium Amyloid Polymers Exploit Dynamic Covalent Linkage to Temporally Control Charge-Selective Catalysis.

Extant proteins exploit thermodynamically activated negatively charged coenzymes and hydrotropes to temporally access mechanistically important conformations that regulate vital biological functions, from metabolic reactions to expression modulation. Herein, we show that a short amyloid peptide can bind to a small molecular coenzyme by exploiting reversible covalent linkage to polymerize and access catalytically proficient nonequilibrium amyloid microphases. Subsequent hydrolysis of the activated coenzyme leads to depolymerization, realizing a variance of the surface charge of the assembly as a function of time. Such temporal change of surface charge dynamically modulates catalytic activities of the transient assemblies as observed in highly evolved modern-day biocatalysts.

Exact Distribution of Threshold Crossing Times for Protein Concentrations: Implication for Biological Timekeeping.

Stochastic protein accumulation up to some concentration threshold sets the timing of many cellular physiological processes. Here we obtain the exact distribution of first threshold crossing times of protein concentration, in either Laplace or time domain, and its associated cumulants: mean, variance, and skewness. The distribution is asymmetric, and its skewness nonmonotonically varies with the threshold. We study lysis times of E. coli cells for holin gene mutants of bacteriophage-λ and find a good match with theory. Mutants requiring higher holin thresholds show more skewed lysis time distributions as predicted. The theory also predicts a linear relationship between infection delay time and host doubling time for lytic viruses, that has recently been experimentally observed.

Fluorescent Microswimmers Based on Cross-ß Amyloid Nanotubes and Divergent Cascade Networks.

Shaped through millions of years of evolution, the spatial localization of multiple enzymes in living cells employs extensive cascade reactions to enable highly coordinated multimodal functions. Herein, by utilizing a complex divergent cascade, we exploit the catalytic potential as well as templating abilities of streamlined cross-ß amyloid nanotubes to yield two orthogonal roles simultaneously. The short peptide based paracrystalline nanotube surfaces demonstrated the generation of fluorescence signals within entangled networks loaded with alcohol dehydrogenase (ADH). The nanotubular morphologies were further used to generate cascade-driven microscopic motility through surface entrapment of sarcosine oxidase (SOX) and catalase (Cat). Moreover, a divergent cascade network was initiated by upstream catalysis of the substrate molecules through the surface mutation of catalytic moieties. Notably, the resultant downstream products led to the generation of motile fluorescent microswimmers by utilizing the two sets of orthogonal properties and, thus, mimicked the complex cascade-mediated functionalities of extant biology.

Cross ß Amyloid Nanotubes Demonstrate Promiscuous Catalysis in a Chemical Reaction Network via Co-option.

In Darwin's warm pond rich with nutrients, lesser number of early catalytic machineries with modest capabilities were able to demonstrate promiscuity by catalyzing diverse biochemical transformations important for protometabolism. Herein, we report catalytically promiscuous amyloid-based short peptide assemblies that could concomitantly catalyse three metabolically important yet orthogonal reactions. The surface exposed catalytic dyads featuring lysines and imidazoles were utilized for C=N condensation via dynamic covalent linkages and modulation of protonation events, respectively. Further, the peptide assemblies could promiscuously catalyse hydrolysis as well as retro-aldol reactions, that could be co-opted to facilitate C=N bond formation, either by a feedforward-driven reaction network or by replenishing depleted substrates. The catalytic diversity of short peptide based promiscuous ß-sheet folds suggests their possible role in promoting the protometabolic network in early earth.