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The production of biofuels from lignocellulosic biomass using carbohydrate-active enzymes like cellulases is key to a sustainable energy production. Understanding the adsorption mechanism of cellulases and associated binding domain proteins down to the molecular level details will help in the rational design of improved cellulases. In nature, carbohydrate-binding modules (CBMs) from families 17 and 28 often appear in tandem appended to the C-terminus of several endocellulases. Both CBMs are known to bind to the amorphous regions of cellulose non-competitively and show similar binding affinity towards soluble cello-oligosaccharides. Based on the available crystal structures, these CBMs may display a uni-directional binding preference towards cello-oligosaccharides (based on how the oligosaccharide was bound within the CBM binding cleft). However, molecular dynamics (MD) simulations have indicated no such clear preference. Considering that most soluble oligosaccharides are not always an ideal substrate surrogate to study the binding of CBMs to the native cell wall or cell surface displayed glycans, it is critical to use alternative reagents or substrates. To better understand the binding of type B CBMs towards smaller cello-oligosaccharides, we have developed a simple solid-state depletion or pull-down binding assay. Here, we specifically orient azido-labeled carbohydrates from the reducing end to alkyne-labeled micron-sized bead surfaces, using click chemistry, to mimic insoluble cell wall surface-displayed glycans. Our results reveal that both family 17 and 28 CBMs displayed a similar binding affinity towards cellohexaose-modified beads, but not cellopentaose-modified beads, which helps rationalize previously reported crystal structure and MD data. This may indicate a preferred uni-directional binding of specific CBMs and could explain their co-evolution as tandem constructs appended to endocellulases to increase amorphous cellulose substrate targeting efficiency. Overall, our proposed workflow can be easily translated to measure the affinity of glycan-binding proteins to click-chemistry based immobilized surface-displayed carbohydrates or antigens.
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Role of the Supporting Surface in the Thermodynamics and Cooperativity of Axial Ligand Binding to Metalloporphyrins at Interfaces
Abstract: : Metalloporphyrins have been shown to bind axial ligands in a variety of environments including the vacuum/solid and solution/solid interfaces. Understanding the dynamics of such interactions is a desideratum for the design and implementation of next generation molecular devices which draw inspiration from biological systems to accomplish diverse tasks such as molecular sensing, electron transport, and catalysis to name a few. In this article, we review the current literature of axial ligand coordination to surface-supported porphyrin receptors. We will focus on the coordination process as monitored by scanning tunneling microscopy (STM) that can yield qualitative and quantitative information on the dynamics and binding affinity at the single molecule level. In particular, we will address the role of the substrate and intermolecular interactions in influencing cooperative effects (positive or negative) in the binding affinity of adjacent molecules based on experimental evidence and theoretical calculations.
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- PAR ID:
- 10326382
- Date Published:
- Journal Name:
- Current Organic Chemistry
- Volume:
- 26
- ISSN:
- 1385-2728
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.2174/1385272826666220209122508
