Jerome M. Fox

Initial- and processive-cut products reveal cellobiohydrolase rate limitations and the role of companion enzymes.

Efforts to improve the activity of cellulases, which catalyze the hydrolysis of insoluble cellulose, have been hindered by uncertainty surrounding the mechanistic origins of rate-limiting phenomena and by an incomplete understanding of complementary enzyme function. In particular, direct kinetic measurements of individual steps occurring after enzymes adsorb to the cellulose surface have proven to be experimentally elusive. This work describes an experimental and analytical approach, derived from a detailed mechanistic model of cellobiohydrolase action, for determining rates of initial- and processive-cut product generation by Trichoderma longibrachiatum cellobiohydrolase I (TlCel7A) as it catalyzes the hydrolysis of bacterial microcrystalline cellulose (BMCC) alone and in the presence of Talaromyces emersonii endoglucanase II (TemGH5). This analysis revealed that the rate of TlCel7A-catalyzed hydrolysis of crystalline cellulose is limited by the rate of enzyme complexation with glycan chains, which is shown to be equivalent to the rate of initial-cut product generation. This rate is enhanced in the presence of endoglucanase enzymes. The results confirm recent reports about the role of morphological obstacles in enzyme processivity and also provide the first direct evidence that processive length may be increased by the presence of companion enzymes, including small amounts of TemGH5. The findings of this work indicate that efforts to improve cellobiohydrolase activity should focus on enhancing the enzymes ability to complex with cellulose chains, and the analysis employed provides a new technique for investigating the mechanism by which companion enzymes influence cellobiohydrolase activity.

Autocatalytic, bistable, oscillatory networks of biologically relevant organic reactions

Networks of organic chemical reactions are important in life and probably played a central part in its origin. Network dynamics regulate cell division, circadian rhythms, nerve impulses and chemotaxis, and guide the development of organisms. Although out-of-equilibrium networks of chemical reactions have the potential to display emergent network dynamics such as spontaneous pattern formation, bistability and periodic oscillations, the principles that enable networks of organic reactions to develop complex behaviours are incompletely understood. Here we describe a network of biologically relevant organic reactions (amide formation, thiolate–thioester exchange, thiolate–disulfide interchange and conjugate addition) that displays bistability and oscillations in the concentrations of organic thiols and amides. Oscillations arise from the interaction between three subcomponents of the network: an autocatalytic cycle that generates thiols and amides from thioesters and dialkyl disulfides; a trigger that controls autocatalytic growth; and inhibitory processes that remove activating thiol species that are produced during the autocatalytic cycle. In contrast to previous studies that have demonstrated oscillations and bistability using highly evolved biomolecules (enzymes and DNA) or inorganic molecules of questionable biochemical relevance (for example, those used in Belousov–Zhabotinskii-type reactions), the organic molecules we use are relevant to metabolism and similar to those that might have existed on the early Earth. By using small organic molecules to build a network of organic reactions with autocatalytic, bistable and oscillatory behaviour, we identify principles that explain the ways in which dynamic networks relevant to life could have developed. Modifications of this network will clarify the influence of molecular structure on the dynamics of reaction networks, and may enable the design of biomimetic networks and of synthetic self-regulating and evolving chemical systems.

A single-molecule analysis reveals morphological targets for cellulase synergy

The mechanisms of enzyme activity on solid substrates are not well understood. Unlike enzyme catalysis in aqueous solutions, enzyme activity on surfaces is complicated by adsorption steps and structural heterogeneities that make enzyme-substrate interactions difficult to characterize. Cellulase enzymes, which catalyze the depolymerization of cellulose, show binding specificities for different cellulose surface morphologies, but the influence of these specificities on the activity of multienzyme mixtures has remained unclear. We developed a metric to quantify binding-target arrangements determined by photoactivated localization microscopy, and we used that metric to show that combinations of cellulases designed to bind within similar but nonidentical morphologies can have synergistic activity. This phenomenon cannot be explained with the binary crystalline or amorphous classifications commonly used to characterize cellulase-binding targets. Our results reveal a strategy for improving the activity of cellulolytic mixtures and demonstrate a versatile method for investigating protein organization on heterogeneous surfaces.

Engineering shadows to fabricate optical metasurfaces.

Optical metasurfaces-patterned arrays of plasmonic nanoantennas that enable the precise manipulation of light-matter interactions-are emerging as critical components in many nanophotonic materials, including planar metamaterials, chemical and biological sensors, and photovoltaics. The development of these materials has been slowed by the difficulty of efficiently fabricating patterns with the required combinations of intricate nanoscale structure, high areal density, and/or heterogeneous composition. One convenient strategy that enables parallel fabrication of periodic nanopatterns uses self-assembled colloidal monolayers as shadow masks; this method has, however, not been extended beyond a small set of simple patterns and, thus, has remained incompatible with the broad design requirements of metasurfaces. This paper demonstrates a technique-shadow-sphere lithography (SSL)-that uses sequential deposition from multiple angles through plasma-etched microspheres to expand the variety and complexity of structures accessible by colloidal masks. SSL harnesses the entire, relatively unexplored, space of shadow-derived shapes and-with custom software to guide multiangled deposition-contains sufficient degrees of freedom to (i) design and fabricate a wide variety of metasurfaces that incorporate complex structures with small feature sizes and multiple materials and (ii) generate, in parallel, thousands of variations of structures for high-throughput screening of new patterns that may yield unexpected optical spectra. This generalized approach to engineering shadows of spheres provides a new strategy for efficient prototyping and discovery of periodic metasurfaces.

Warning signals for eruptive events in spreading fires

Significance As flames spread through forests, buildings, or other complex environments, they can erupt, unexpectedly, into fast-moving conflagrations. This study presents evidence that characteristic patterns in the behavior of spreading flames may indicate when such eruptions are likely to occur. Our results rely on the detection of a phenomenon termed “critical slowing down”—the slowed recovery of multistable systems from perturbations as those systems approach tipping points. Using a bistable combustion system in which flames propagate either as small, slowly moving flames, or as large, rapidly moving flames stabilized by feedback between wind and fire, we provide evidence that slowing responses of spreading flames to sudden changes in environment (e.g., wind, terrain) may anticipate the onset of intense, feedback-stabilized modes of propagation. Spreading fires are noisy (and potentially chaotic) systems in which transitions in dynamics are notoriously difficult to predict. As flames move through spatially heterogeneous environments, sudden shifts in temperature, wind, or topography can generate combustion instabilities, or trigger self-stabilizing feedback loops, that dramatically amplify the intensities and rates with which fires propagate. Such transitions are rarely captured by predictive models of fire behavior and, thus, complicate efforts in fire suppression. This paper describes a simple, remarkably instructive physical model for examining the eruption of small flames into intense, rapidly moving flames stabilized by feedback between wind and fire (i.e., “wind–fire coupling”—a mechanism of feedback particularly relevant to forest fires), and it presents evidence that characteristic patterns in the dynamics of spreading flames indicate when such transitions are likely to occur. In this model system, flames propagate along strips of nitrocellulose with one of two possible modes of propagation: a slow, structured mode, and a fast, unstructured mode sustained by wind–fire coupling. Experimental examination of patterns in dynamics that emerge near bifurcation points suggests that symptoms of critical slowing down (i.e., the slowed recovery of the system from perturbations as it approaches tipping points) warn of impending transitions to the unstructured mode. Findings suggest that slowing responses of spreading flames to sudden changes in environment (e.g., wind, terrain, temperature) may anticipate the onset of intense, feedback-stabilized modes of propagation (e.g., “blowup fires” in forests).

Water-Restructuring Mutations Can Reverse the Thermodynamic Signature of Ligand Binding to Human Carbonic Anhydrase.

This study uses mutants of human carbonic anhydrase (HCAII) to examine how changes in the organization of water within a binding pocket can alter the thermodynamics of protein-ligand association. Results from calorimetric, crystallographic, and theoretical analyses suggest that most mutations strengthen networks of water-mediated hydrogen bonds and reduce binding affinity by increasing the enthalpic cost and, to a lesser extent, the entropic benefit of rearranging those networks during binding. The organization of water within a binding pocket can thus determine whether the hydrophobic interactions in which it engages are enthalpy-driven or entropy-driven. Our findings highlight a possible asymmetry in protein-ligand association by suggesting that, within the confines of the binding pocket of HCAII, binding events associated with enthalpically favorable rearrangements of water are stronger than those associated with entropically favorable ones.

Abietane-Type Diterpenoids Inhibit Protein Tyrosine Phosphatases by Stabilizing an Inactive Enzyme Conformation

Protein tyrosine phosphatases (PTPs) contribute to a striking variety of human diseases, yet they remain vexingly difficult to inhibit with uncharged, cell-permeable molecules; no inhibitors of PTPs have been approved for clinical use. This study uses a broad set of biophysical analyses to evaluate the use of abietane-type diterpenoids, a biologically active class of phytometabolites with largely nonpolar structures, for the development of pharmaceutically relevant PTP inhibitors. Results of nuclear magnetic resonance analyses, mutational studies, and molecular dynamics simulations indicate that abietic acid can inhibit protein tyrosine phosphatase 1B, a negative regulator of insulin signaling and an elusive drug target, by binding to its active site in a non-substrate-like manner that stabilizes the catalytically essential WPD loop in an inactive conformation; detailed kinetic studies, in turn, show that minor changes in the structures of abietane-type diterpenoids (e.g., the addition of hydrogens) can improve potency (i.e., lower IC50) by 7-fold. These findings elucidate a previously uncharacterized mechanism of diterpenoid-mediated inhibition and suggest, more broadly, that abietane-type diterpenoids are a promising source of structurally diverse-and, intriguingly, microbially synthesizable-molecules on which to base the design of new PTP-inhibiting therapeutics.

Evolutionarily Conserved Allosteric Communication in Protein Tyrosine Phosphatases

Protein tyrosine phosphatases (PTPs) are an important class of regulatory enzymes that exhibit aberrant activities in a wide range of diseases. A detailed mapping of allosteric communication in these enzymes could, thus, reveal the structural basis of physiologically relevant-and, perhaps, therapeutically informative-perturbations (i.e., mutations, post-translational modifications, or binding events) that influence their catalytic states. This study combines detailed biophysical studies of protein tyrosine phosphatase 1B (PTP1B) with bioinformatic analyses of the PTP family to examine allosteric communication in this class of enzymes. Results of X-ray crystallography, molecular dynamics simulations, and sequence-based statistical analyses indicate that PTP1B possesses a broadly distributed allosteric network that is evolutionarily conserved across the PTP family, and findings from both kinetic studies and mutational analyses show that this network is functionally intact in sequence-diverse PTPs. The allosteric network resolved in this study reveals new sites for targeting allosteric inhibitors of PTPs and helps explain the functional influence of a diverse set of disease-associated mutations.

The Molecular Origin of Enthalpy/Entropy Compensation in Biomolecular Recognition

Biomolecular recognition can be stubborn; changes in the structures of associating molecules, or the environments in which they associate, often yield compensating changes in enthalpies and entropies of binding and no net change in affinities. This phenomenon-termed enthalpy/entropy (H/S) compensation-hinders efforts in biomolecular design, and its incidence-often a surprise to experimentalists-makes interactions between biomolecules difficult to predict. Although characterizing H/S compensation requires experimental care, it is unquestionably a real phenomenon that has, from an engineering perspective, useful physical origins. Studying H/S compensation can help illuminate the still-murky roles of water and dynamics in biomolecular recognition and self-assembly. This review summarizes known sources of H/ S compensation (real and perceived) and lays out a conceptual framework for understanding and dissecting-and, perhaps, avoiding or exploiting-this phenomenon in biophysical systems.

Acetylation of Surface Lysine Groups of a Protein Alters the Organization and Composition of Its Crystal Contacts

This paper uses crystals of bovine carbonic anhydrase (CA) and its acetylated variant to examine (i) how a large negative formal charge can be accommodated in protein-protein interfaces, (ii) why lysine residues are often excluded from them, and (iii) how changes in the surface charge of a protein can alter the structure and organization of protein-protein interfaces. It demonstrates that acetylation of lysine residues on the surface of CA increases the participation of polar residues (particularly acetylated lysine) in protein-protein interfaces, and decreases the participation of nonpolar residues in those interfaces. Negatively charged residues are accommodated in protein-protein interfaces via (i) hydrogen bonds or van der Waals interactions with polar residues or (ii) salt bridges with other charged residues. The participation of acetylated lysine in protein-protein interfaces suggests that unacetylated lysine tends to be excluded from interfaces because of its positive charge, and not because of a loss in conformational entropy. Results also indicate that crystal contacts in acetylated CA become less constrained geometrically and, as a result, more closely packed (i.e., more tightly clustered spatially) than those of native CA. This study demonstrates a physical-organic approach-and a well-defined model system-for studying the role of charges in protein-protein interactions.