Browsing by Author "Toone, Eric J"

Molecular recognition is arguably the most elementary physical process essential for life that arises at the molecular scale. Molecular recognition drives events across virtually all length scales, from the folding of proteins and binding of ligands, to the organization of membranes and the function of muscles. Understanding such events at the molecular level is massively complicated by the unique medium in which life occurs: water. In contrast to recognition in non-aqueous solvents, which are driven largely by attractive interactions between binding partners, binding reactions in water are driven in large measure by the properties of the medium itself. Aqueous binding involves the loss of solute-solvent interactions (desolvation) and the concomitant formation of solute-solute interactions. Despite decades of research, aqueous binding remains poorly understood, a deficit that profoundly limits our ability to design effective pharmaceuticals and new enzymes. Particularly problematic is understanding the energetic consequences of aqueous desolvation, an area the Toone and Beratan groups have considered for many years.

In this dissertation, we embark on a quest to shed new light on aqueous desolvation from two perspectives. In one component of this research, we improve current computational tools to study aqueous desolvation, employing quantum mechanics (QM), molecular dynamics (MD) and Monte Carlo (MC) simulations to better understand the behavior of water near molecular surfaces. In the other, we use a synthetic host, cucurbit[7]uril (CB[7]), in conjunction with a de novo series of ligands to study the structure and thermodynamics of aqueous desolvation in the context of ligand binding with atomic precision, a feat hitherto impossible. A simple and rigid macrocycle, CB[7] alleviates the drawbacks of protein systems for the study of aqueous ligand binding, that arise from conformational heterogeneity and prohibitive computational costs to model.

We first constructed a novel host-guest system that facilitates internalization of the trimethylammonium (methonium) group from bulk water to the hydrophobic cavity of CB[7] with precise (atomic-scale) control over the position of the ligand with respect to the cavity. The process of internalization was investigated energetically using isothermal titration microcalorimetry and structurally by nuclear magnetic resonance (NMR) spectroscopy. We show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by an unfavorable desolvation enthalpy of just 0.49±0.27 kcal*mol-1, a value significantly less endothermic than those values suggested from previous gas-phase model studies. Our results offer a rationale for the wide distribution of methonium in biology and demonstrate important limitations to computational estimates of binding affinities based on simple solvent-accessible surface area approaches.

To better understand our experimental results, we developed a two-dimensional lattice model of water based on random cluster structures that successfully reproduces the temperature-density anomaly of water with minimum computational cost. Using reported well-characterized ligands of CB[7], we probed water structure within the CB[7] cavity and identified an energetically perturbed cluster of water. We offer both experimental and computational evidence that this unstable water cluster provides a significant portion of the driving force for encapsulation of hydrophobic guests.

The studies reported herein shed important light on the thermodynamic and structural nature of aqueous desolvation, and bring our previous understanding of the hydrophobic effect based on ordered water and buried surface area into question. Our approach provides new tools to quantify the thermodynamics of functional group desolvation in the context of ligand binding, which will be of tremendous value for future research on ligand/drug design.

Modern patterning and fabrication techniques provide powerful opportunities for the preparation of micro- and nanostructured objects with applications in fields ranging from drug delivery and bioimaging to organic based electronic devices and real time biochemical sensors. In this thesis we report a systematic study focused on the development of new unconventional patterning and fabrication techniques with applications in the preparation of functional micro- and nanostructured devices.

Catalytic microcontact printing is a powerful technique that offers a simple and effective methodology for patterning chemically-functionalized surfaces with sub-100 nm accuracy. By avoiding diffusive mechanisms of pattern replication it effectively obviates the most significant limitation of traditional microcontact printing - lateral molecular ink diffusion. Moreover, catalytic microcontact printing significantly expands the diversity of patternable surfaces by using prefunctionalized substrates and gives rapid facile access to chemically discriminated surfaces that can be further functionalized with organic and biological molecules. We have developed several catalytic microcontact printing techniques that transfer pattern from an elastomeric stamp bearing an immobilized catalyst to a preformed functionalized self-assembled monolayer. By avoiding diffusive pattern transfer we were able to replicate features with sub-50 nm edge resolution. We also demonstrated that catalytic printing can be expanded to technologically important substrates not accessible through conventional soft lithography, by patterning reactive organic monolayers grafted to chemically passivated silicon.

The non-symmetric structure of Janus particles produces novel physical properties and unusual aggregation behavior that makes these materials attractive candidates for drug delivery and as nano-sensors and nano-probes, SERS and PEF imaging agents, small molecules carriers, and switchable devices. We have developed a new protocol for preparation of non-spherical inorganic Janus particles comprising metallic and semiconductor layers. The method allows for precise control over the composition, shape and size and permits fabrication of non-symmetrical particles, the opposite sides of which can be orthogonally functionalized using well-established organosilane and thiol chemistries.

Monomolecular hybrid organic-inorganic interfaces provide opportunities for applications in fields ranging from sensors to electronics. In this thesis, we report our efforts towards (1) developing a universal method for the modification and soft-lithographic patterning of inorganic materials with stable and functional organic systems; and (2) apply our surface fabrication techniques to advance our understanding of molecular recognition force microscopy.

We report the development of a novel bi-layered molecular system that, in conjunction with an inkless catalytic microcontact printing technique, can be used to accurately replicate micro- and nano-scale patterns of chemically distinctive reactive functionalities on virtually any surface, including inorganic semiconductors. Catalytic printing alleviates problems associated with ink diffusion and enables high resolution replication of patterns through specific chemical or biochemical reaction between a functional surface and a stamp-immobilized catalyst. The methodology provides precise control over shape and size of pattern features and provides access to chemically discriminated patterns that can be further functionalized with organic and biological molecules. We demonstrate catalytic printing on both oxide-free silicon and germanium, substrates that do not react readily with organic molecules and have not heretofore been patterned through traditional approaches. Our approach we relies on a stable highly ordered bilayered molecular system that both affords complete protection of all surface-exposed inorganic atoms with stable covalent bonds and supports covalent immobilization of a reactive overlayer, yielding stability and functionality to the surface. A catalytic acidic stamp was used to achieve pattern-specific hydrolysis of N-hydroxysuccinimide-activated acids immobilized on Si and Ge. Further modification of the chemically discriminated patterns enables chemoselective anchoring of organic molecules and protein.

We demonstrated the utility of the strategy towards a variety of inorganic oxides, including ITO. Utilizing the functionalized bi-layered system on ITO, a single molecular system in combination with different printing approaches can be used to immobilize multiple organic functionalities with exquisite spatial control. The system was used to investigate structure - function relationships of the ordered and functional molecular system on ITO to vertically and laterally control charge injection in organic light emitting diodes (OLEDs).

Finally, we report fabrication of functional hybrid organic-inorganic interfaces for the study of immobilized binding partners, lactose-g3 and complementary ssDNA, in molecular recognition force microscopy (MRFM). We use our system to evaluate the effect of contact force on specific interactions and the effect of dwell time and tether length on the probability of ligand-receptor binding. The methodologies developed enable a reliable evaluation of thermodynamic parameters using MRFM.

The intricate interplay of biomolecules acting together, rather than alone, provides insight into the most basic of cellular functions, such as cell signaling, metabolism, defense, and, ultimately, the creation of life. Inherent in each of these processes is an evolutionary tendency towards increased efficiency by means of biolgocial synergy-- the ability of individual elements of a system to produce a combined effect that is different and often greater than the sum of the effects of the parts. Modern biochemists are challenged to find model systems to characterize biological synergy.

We discuss the multicomponent, enzyme complex the cellulosome as a model system of biological synergy. Native cellulosomes comprise numerous carbohydrate-active binding proteins and enzymes designed for the efficient degradation of plant cell wall matrix polysaccharides, namely cellulose. Cellulosomes are modular enzyme complexes, comparable to enzyme "legos" that may be readily constructed into multiple geometries by synthetic design. Cellulosomal enzymes provide means to measure protein efficiency with altered complex geometry through assay of enzymatic activity as a function of geometry.

Cellulosomes are known to be highly efficient at cellulose depolymerization, and current debates on the molecular origins of this efficiency suggest two related effects provide this efficiency: i) substrate targeting, which argues that the localization of the enzyme complex at the interface of insoluble cell wall polysaccharides facilitates substrate depolymerization; and ii) proximity effects, which describe the implicit benefit for co-localizing multiple enzymes with divergent substrate preferences on the activity of the whole complex.

Substrate targeting can be traced to the activity of a single protein, the cellulosomal scaffoldin cellulose binding module CBM3a that is thought to uniquely bind highly crystalline, insoluble cellulose. We introduce methods to develop a molecular understanding of the substrate preferences for CBM3a on soluble and insoluble cellulosic substrates. Using pivaloylysis of cellulose triacetate, we obtain multiple soluble cello-oligosaccharides with increasing degree of glucose polymerization (DP) from glucose (DP1) to cellodecaose (DP10) in high yield. Using calorimetry and centrifugal titrations, cello-oligosacharides were shown to not bind Clostridial cellulolyticum CMB3a. We developed AFM cantilever functionalization protocols to immobilize CBM3a and then probe the interfacial binding between CBM3a and a cellulose nanocrystal thin film using force spectroscopy. Specific binding at the interface was demonstrated in reference to a control protein that does not bind cellulose. The results indicate that i) CBM3a specifically binds nanocrystalline cellulose and ii) specific interfacial binding may be probed by force spectroscopy with the proper introduction of controls and blocking agents.

The question of enzyme proximity effects in the cellulosome must be answered by assaying the activity of cellulosomal cellulases in response to cellulosome geometry. The kinetic characterization of cellulases requires robust and reproducible assays to quantify functional cellulase content of from recombinant enzyme preparations. To facilitate the real-time routine assay of cellulase activity, we developed a custom synthesis of a fluorogenic cellulase substrate based on the cellohexaoside of Driguez and co-workers (vide infra). Two routes to synthesize a key thiophenyl glycoside building block were presented, with the more concise route providing the disaccharide in four steps from a commercial starting material. The disaccharide building blocks were coupled by chemical activation to yield the fully protected cellohexaoside over additional six steps. Future work will include the elaboration of this compound into an underivatized FRET-paired hexasaccharide and its subsequent use in cellulase activity assays.

This dissertation also covers an experimental system for the evaluation of methonium desolvation thermodynamics. Methonium (-N+Me3, Am) is an organic cation widely distributed in biological systems. The appearance of methonium in biological transmitters and receptors seems at odds with the large unfavorable desolvation free energy reported for tetramethylammonium (TMA+), a frequently utilized surrogate of methonium. We report an experimental system that facilitates incremental internalization of methonium within the molecular cavity of cucurbit[7]uril (CB[7]).

Using a combination of experimental and computational studies we show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by a remarkably small desolvation enthalpy of just 0.5±0.3 kcal*mol-1, a value significantly less endothermic than those values suggested from gas-phase model studies (+49.3 kcal*mol-1). More surprisingly, the incremental withdrawal of methonium surface from water produces a non- monotonic response in desolvation enthalpy. A partially desolvated state exists, in which a portion of the methonium group remains exposed to solvent. This structure incurs an increased enthalpic penalty of ~3 kcal*mol-1 compared to other solvation states. We attribute this observation to the pre- encapsulation de-wetting of the methonium surface. Together, our results offer a rationale for the wide biological distribution of methonium and suggest limitations to computational estimates of binding affinities based on simple parameterization of solvent-accessible surface area.

During the past two decades, soft lithographic techniques that circumvent the limitations of photolithography have emerged as important tools for the transfer of patterns with sub-micron dimensions. Among these techniques, microcontact printing (uCP) has shown special promise. In uCP, an elastomeric stamp is first inked with surface-reactive molecules and placed in contact with an ink-reactive surface, resulting in pattern transfer in the form of self-assembled monolayers in regions of conformal contact. The resolution in uCP is ultimately limited to the diffusion of ink and the elastomechanical properties of the bulk stamping material.

One way to improve resolution is to eliminate diffusion by using inkless methods for pattern transfer. Inkless catalytic-uCP uses a chemical reaction between a stamp-immobilized catalyst and surface bearing cognate substrate to transfer pattern in the areas of conformal contact. By using pre-assembled cognate surfaces, the approach extends the range of surfaces readily amenable to patterning while obviating diffusive resolution limits imposed by traditional uCP.

In this thesis, we report two methods using inkless catalytic uCP: biocatalytic-uCP utilizes an immobilized enzyme as a catalyst whereas catalytic-uCP utilizes an immobilized small molecule as a catalyst, such as an acid or base. Both catalytic techniques demonstrate pattern transfer at the microscale while using unconventional, acrylate-based stamp materials. Previous results produced with catalytic-uCP have shown pattern transfer with sub-50 nm edge resolution. In this demonstration of catalytic-uCP, we use the technique to demonstrate a bi-layered patterning technique for H-terminated silicon, the foremost material in semi-conductor fabrication. This technique simultaneously protects the underlying silicon surface from degradation while a highly-reactive organic overlayer remains patternable by acidic-functionalized PU stamps. Lines bearing widths as small as 150 nm were reproduced on the reactive SAM overlayer, which would not be possible without circumvention of diffusion. Before and after patterning, no oxidation of the underlying silicon was observed, preserving desired electronic properties throughout the whole process. This bi-patterning technique could be extended to other technologically-relevant surfaces for further application in organic-based electronic devices and other related technologies.

Virtually all biologically relevant functions and processes are mediated by non-covalent, molecular recognition events, demonstrating astonishingly diverse affinities and specificities. Despite extensive research, the origin of affinity and specificity in aqueous solution - specifically the relationship between ligand binding thermodynamics and structure - remains remarkably obscure and is further complicated in the context of multivalent interactions. Multivalency describes the combinatorial interaction of multiple discrete epitopes across multiple binding surfaces where the association is considered as the sum of contributions from each epitope and the consequences of multivalent ligand assembly. Gaining the insight necessary to predictably influence biological processes with novel therapeutics begins with an understanding of the molecular basis of solution-phase interactions, and the thermodynamic parameters that follow from those interactions. Here we continue our efforts to understand the basis of aqueous affinity and the nature of multivalent additivity.

Multivalent additivity is the foundation of fragment-based drug discovery, where small, low affinity ligands are covalently assembled into a single high affinity inhibitor. Such systems are ideally suited for investigating the thermodynamic consequences of multivalent ligand assembly. In the first part of this work, we report the design and synthesis of a fragment-based ligand series for the Grb2-SH2 protein and thermodynamic evaluation of the low affinity ligand fragments compared to the intact, high affinity inhibitor by single and double displacement isothermal titration calorimetry (ITC). Interestingly, our investigations reveal positively cooperative multivalent additivity - a binding free energy of the full ligand greater than the sum of its constituent fragments - that is largely enthalpic in origin. These results contradict the most common theory of multivalent affinity enhancement arising from a "savings" in translational and rotational entropy. The Grb2-SH2 system reported here is the third distinct molecular system in which we have observed enthalpically driven multivalent enhancement of affinity.

Previous research by our group into similar multivalent affinity enhancements in protein-carbohydrate systems - the so-called "cluster glycoside effect" - revealed that evaluation of multivalent interactions in the solution-phase is not straightforward due to the accessibility of two disparate binding motifs: intramolecular, chelate-type binding and intermolecular, aggregative binding. Although a number of powerful techniques for evaluation of solution-phase multivalent interactions have been reported, these bulk techniques are often unable to differentiate between binding modes, obscuring thermodynamic interpretation. In the second part of this work, we report a competitive equilibrium approach to Molecular Recognition Force Microscopy (MRFM) for evaluation of ligand binding at the single-molecule level with potential to preclude aggregative associations. We have optimized surface functionalization strategies and MRFM experimental protocols to evaluate the binding constant of surface- and tip-immobilized single stranded DNA epitopes. Surprisingly, the monovalent affinity of an immobilized species is in remarkable agreement with the solution-phase affinity, suggesting the competitive equilibrium MRFM approach presents a unique opportunity to investigate the nature of multivalent additivity at the single molecule level.

The goal of this work is to develop novel molecular systems, functionalization techniques, and data collection routines with which to study the binding of immobilized cognate binding partners. Our ultimate goal is the routine evaluation of thermodynamic parameters for immobilized systems through interpretation of the variation of the binary probability of binding as a function of soluble ligand concentration. The development of both data collection routines that minimize non-specific binding and functionalization techniques that produce stable ordered molecular systems on surfaces are of paramount importance towards achievement of this goal. Methodologies developed here will be applied to investigating the thermodynamics of multivalent systems.

In the first part of this work, the effect of contact force on molecular recognition force microscopy experiments was investigated. Increased contact forces (>250 pN) resulted in increased probabilities of binding and decreased blocking efficiencies for the cognate ligand-receptor pair lactose-G3. Increased contact force applied to two control systems with no known affinity, mannose-G3 and lactose-KDPG aldolase resulted in non-specific ruptures that were indistinguishable from those of specific lactose-G3 interactions. Thus, it is essential to design data collections routines that minimize contact forces to ensure that ruptures originate from specific, blockable interactions.

In the second part of this work we report the first example of the preparation of stable self assembled monolayers through hydrosilylation of a protected aminoalkene onto hydrogen-terminated silicon nitride AFM probes and subsequent conjugation with biomolecules for force microscopy studies. Our technique can be used as a general attachment technique for other molecular systems.

In the third part of this work we develop novel molecular systems for tethering oriented vancomycin and its cognate binding partner L-Lys-D-Ala-D-Ala to surfaces and AFM tips. Unbinding experiments demonstrated that traditional methods for forming low surface density amine layers (silanization with APTMS and etherification with ethanolamine) provided molecular constructs which displayed probabilities of binding that were too low and showed overall variability too high to use for probabilistic evaluation of thermodynamics parameters. Instability and heat-induced polymerization of APTMS layers on tips and surfaces also prohibited their utility. Formation of Alkyl SAMs on silicon provides a more reliable, stable molecular system anchored by Si-C bonds that facilitates attachment of vancomycin and is capable of withstanding prolonged exposure to heated organic and aqueous environments. It follows that covalent immobilization of KDADA to silicon nitride AFM tips via Si-C bonds using hydrosilylation chemistry will be similarly advantageous. These methods offer great promise for probabilistic evaluation of thermodynamic parameters characterizing immobilized binding partners and will permit unambiguous determination of the role of multivalency in ligand binding, using an experimental configuration in which intermolecular binding and aggregation are precluded.

Nearly every aspect of biology is controlled by non-covalent association events, but the diversity of these events is astounding. Association constants can vary over at least a dozen orders of magnitude, and timescales for these interactions range from microseconds to years. Molecular recognition events control the affinity and specificity of virtually all of these biological interactions. Remarkably, the relationships between structure and thermodynamics, and thus the basis of selectivity and affinity in these events remains poorly understood. An overarching goal of the Toone laboratory is to better understand the molecular facets of non-covalent association events in aqueous solution. In this dissertation, three model systems are explored to elucidate aspects of non-covalent binding events.

Camelid-derived single domain antibodies demonstrate significant stability and unique binding properties versus conventional antibodies, and the work described herein helps rationalize the observation that these antibody domains function effectively as antigen binding agents. Early attempts to develop a model system with either lysozyme or biotin as the antigen of interest were not successful, but a model system using methotrexate as the antigen was successfully developed. High affinity binding interactions were observed at neutral pH, and interestingly these antibodies demonstrated some binding affinity for methotrexate even at more extreme pH levels. The observed changes in heat capacity and the effect of salt concentration were in accord with established literature precedents. In addition, no evidence of pH-induced destabilization of secondary single domain antibody structure was observed, even when accompanied by variation in temperature.

The Toone laboratory is also working to elucidate the nature and origins of additivity in multivalent protein - ligand binding events in a number of intermolecular and intramolecular systems. The cluster glycoside effect (enhancements in binding affinity beyond what would be expected with increases in valency) has been demonstrated in a large number of protein - carbohydrate binding systems, but the molecular basis for the apparent enhancements in affinity remains unclear. The monomeric carbohydrate - binding protein Galectin-3 (G3) was studied as a model system for conditional multivalency to explore the origins of this effect. We completed a thorough investigation of the thermodynamics of binding between multivalent dendrimeric ligands and both the full-length and truncated proteins (carbohydrate recognition domain, CRD). Enhanced affinities were observed for multivalent interactions with full-length G3 but not for interactions with the CRD. In addition, aggregation and precipitation was observed only in multivalent binding events with full-length G3. Although multivalent binding can occur through an intramolecular (chelate-type) binding mode, these results are consistent with the notion that the cluster glycoside effect is driven through intermolecular bindng motifs, characterized by aggregation and precipitation.

Traditionally, intramolecular (chelate-type) binding pathways are thought to be driven entropically; however, work from the Toone laboratory has demonstrated that multivalent association in several intramolecular multivalent binding systems is enthalpically favored and entropically opposed. General conclusions regarding the origins of intramolecular additivity in ligand binding are lacking, and the Src SH2 domain was developed as a model system for exploring these concepts. The Src SH2 domain was successfully expressed and purified, and a peptidic series of ligands was synthesized and advanced to crystallographic trials. Extensive apo-crystallization attempts were unsuccessful in producing an atomic-resolution structure, and despite optimization trials, co-crystallization of the peptidic ligands with the Src SH2 domain also did not achieve the necessary resolution to provide structural data.