The cellulosome project


The cellulosome (Fig. 1) is a modular, multi-enzyme complex used by some anaerobic organisms, namely chlostridia, to degrade cellulose, a water-insoluble, b(1®4)-linked polymer of glucose, into soluble sugars, which can be used by the organism as fuel. Interestingly, the cellulosome provides a fermentative organism the ability to degrade lignocellulose, the particularly recalcitrant form of cellulose in biomass, in a single step cellulolysis. Thus, it has become an interesting target of study in both renewable energy technology and enzymology communities. The current understanding of the cellulosome portrays a remarkable cellulose-degrading machine that hydrolyzes recalcitrant lignocellulose with apparent synergistic improvement over free cellulase systems. Text Box: Figure 1. Snapshot of the Cellulosome. (Karpol, A.; Barak, Y.; Lamed, R.; Shoham, Y.; Byer, E. A. Functional asymmetry in cohesin binding belies inherent symmetry of the dockerin module: insight into cellulosome assembly revealed by systematic mutagenesis. Biochem. J., 410, 331-338 (2008).)This is to say the apparent extent of cellulose hydrolysis into soluble sugars for the fully-complexed cellulosome is greater than that for the sum of the free cellulases constituting the enzymatic subunits within the cellulosome. The origin of this synergy is still unknown, for our knowledge of the cellulosome mechanism on cellulose is limited to a qualitative description of its physico-chemical characteristics.

The Toone lab at Duke University has historically shown interest in enzyme kinetics and the thermodynamic nature of protein-carbohydrate interactions. We are interested in the thermodynamics and kinetics of cellulosome-driven cellulolysis as it relates to cellulosome assembly and substrate complexity. We hypothesize that a more quantitative physical description of the cellulosome may illuminate the inherent efficiency in its design. We will use various techniques in physical chemistry, organic synthesis, and enzymology to probe this hypothesis.


Self-assembly driven by non-covalent bonding


The aggregate interactions each individually much weaker than covalent bonds, including ionic, dipolar, hydrogen bonding, and van der Waals interactions -- lies at the heart of myriad biological and abiological phenomena.  From the bulk properties of synthetic polymers to the response of biological systems to external stimuli, non-covalent interactions determine the structure and behavior of an astonishingly large component of our world. Remarkably, the molecular basis of affinity and selectivity in non-covalent complexes remains extraordinarily opaque, and our ability to design ligand-macromolecule complexes with predetermined behaviors is virtually non-existent.  Our overarching goal is to better understand the structural basis of affinity and specificity in the formation of non-covalent complexes, especially in water.  We are currently exploring several model systems for additivity in ligand binding derived from previous fragment-based drug design studies.  Additionally, we are exploring the unique binding of haptens and macromolecules to camelid-derived single domain antibodies.  Although our laboratory considers this problem in the context of ligand-macromolecule binding, the conclusions of the work are widely applicable.


Probing the role of water in non-covalent bonding


2w.bmpWater is so important to human kind that tremendous efforts have been put into the research of this liquid, for nearly a century. On the other hand, water is such a mysterious species that we have not fully understood. In biological systems, non-covalent bonding almost always involves water, where water molecules are either excluded from the surfaces of molecules, or acting as intermolecular bridges. It is believed that the change of local water structure upon non-covalent bonding is responsible for a considerable portion of the overall molecular driving forces for the association process. Despite extensive and consistent studies, we have not yet completely elucidated waters role in non-covalent association in aqueous solutions. In order to CB7.pngcontribute to the quest for a full understanding of water in ligand-receptor binding process, we have set up a synthetic host-guest system for experimental and computational studies of water. The "guests" in this system are bicyclo(2.2.2)octane (BCO) derivatives and the "host" is cucurbit[7]uril (CB[7]). This system is able to mimic the molecular association in biological systems, without suffering from interference from too many uncertainties. We are currently measuring the binding free energies of CB[7]-BCO complexes using isothermal titration calorimetry (ITC). We will be attempting to reproduce the binding processes in silico, with state-of-the-art water models. The combination of experimental and computational approaches will not only evaluate the performances of current water modeling techniques, but also provide dynamic descriptions of how water drives the binding reaction forward, with atomic resolution.



Study of multivalent binding with atomic force microscopy (AFM)


Micro-contact printing (mCP)


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 (mCP) has shown special promise.  In mCP, 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 mCP 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-mCP 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 CP. 


We developed two methods using inkless catalytic CP:   biocatalytic-CP utilizes an immobilized enzyme as a catalyst whereas catalytic-CP 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-CP have shown pattern transfer with sub-50 nm edge resolution.  In this demonstration of catalytic-CP, 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.


Anti-bacterial drug design

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