Enthalpic and Entropic Controls on 2D Self-Assembly of Proteins on Substrates

Enthalpic and entropic forces are two knobs that can be used to control the assembly of colloidal particles. Strong bonds, such as those provide by DNA ligands, can drive the assembly enthalpically. Entropy, associated with shape complementarity, can also drive assembly. Particles can spontaneously align and assemble into structures to increase the configurational space available to the system, leading to dense packing to maximize the free volume of solvent. Although colloidal particle assembly has been extensively studied from the perspective of entropic and enthalpic drivers, the assembly of biomacromolecules, like proteins, is a rapidly growing field in which such efforts have been limited.
Herein, we create patchy proteins, L-rhamnulose-1-phosphate aldolase (RhuA), with well-defined 3D shapes and tunable bonding interactions and size of ligands by varying the appended functional groups to manipulate both enthalpic and entropic drivers of assembly. In this project, we used in-situ atomic force microscopy (AFM) to observe the 2D assembly of distinct phases of specially designed β-cyclodextrin and azobenzene modified RhuA (^CDRhuA and ^AzoRhuA) at mica-water interfaces, as well as the transition between them. In the case of ^AzoRhuA, the presence of suitable long functional groups and weak inter-protein interactions leads to the formation of various polymorphs and between which phase transitions occur via two distinct pathways. However, when functional groups become excessively long or large, as with ^CDRhuA, densely packed configurations are obtained via an unusual assembly pathway involving protein adsorption and reconfiguration. The entropic interaction, driven by shape complementarity, becomes the dominant factor in determining the outcome of ^CDRhuA assembly. We applied coarse grain simulations in which we varied the relative contributions of protein-substrate interaction and protein-protein interactions, which include both enthalpic interactions and entropic forces, to study the competition between different forces. Our findings indicate that adjusting the strength and flexibility of the inter-molecular bonds can modulate the entropic forces associated with shape complementarity and the enthalpic forces to enable polymorph formation and phase transition. This insight suggests fundamental design principles for the synthesis of novel 2D macromolecular materials.