Tuning self-assembled nanostructures using solvent polarity

Title: Residue-Specific Solvation-Directed Thermodynamic and Kinetic Control over Peptide Self-Assembly with 1D/2D Structure Selection

Authors: Yiyang Lin, Matthew Penna, Michael R. Thomas, Jonathan P. Wojciechowski, Vincent Leonardo, Ye Wang, E. Thomas Pashuck, Irene Yarovsky, Molly M. Stevens

DOI: 10.1021/acsnano.8b08117

Journal: ACS Nano

During winter, after a frosty night, we all have noticed snow-flakes in the morning. While it has its own aesthetic beauty to appreciate, but have we ever thought about the underlying process of formation of the snow-flakes? Or what about the formation of table-salt crystals from its constituents? Intrigued by so many other natural and real-life examples, researchers delved into these assembly processes in great details coining a new term ‘Self-assembly’.

‘Self-assembly’ is the process of spontaneous organization of components without external intervention. Self-assembled structures usually have a higher range of order than isolated components and form through interaction between constituents. Self-assembled structures are prevalent in many natural and biological systems. For example, one of the fundamental constituents of living systems, proteins, are higher order structures made up of interactions between smaller peptides. Peptides are formed when multiple amino acids covalently bond to each other, forming a long amino acid chain. Short amino acid chains are known as peptides, however when they exceed a certain length their classification changes into that of a protein. Proteins also rearrange among themselves to form complex structures using non-covalent interactions[1], a process known as protein folding. Misfolding of proteins happens through different interactions in proteins as compared to interactions in the original state and results in disruption of the exclusive function of proteins. Hence, over the years researchers tried to investigate the overall mechanism of these self-assembly processes and tune the response to external stimuli.  

Based on this, a research team led by Molly Stevens and Irene Yarovsky looked into the self-assembly process of peptides in different solvents both experimentally and computationally. This study helps to elucidate the effect of solvent polarity on the structure of a self-assembling peptide-based nanostructure. For example, in one type of solvent, the same nanomaterial might make a 1D architecture (nanofibrils), whereas in presence of another solvent it might form a 2D structure (nanosheets).

To investigate this solvent-dependent assembly process, researchers created a custom polypeptide by synthetically linking several different amino acids, such as phenylalanine (yellow) and glutamine (blue and pink) (Figure 1). These peptides form 2D nanosheet structures in water due to different secondary interactions[1] such as H-bonding and aromatic interactions. H-bonding operates in between carboxylic acid (-COOH) and amine (-NH) groups in between two peptide strands, whereas aromatic interaction can happen in between two aromatic groups in adjacent peptides forming complex structure. In here, H-bonding is working in the x-axial direction in between the carboxylic acid groups (Figure 1). Similarly, due to the presence of aromatic groups in the peptide, aromatic interaction is also operating in between adjacent peptide but in a different direction. They already observed that replacing the aromatic interactions chemically resulted in interaction through only x-direction and thus forming 1D nanofibrils.


Figure 1. Peptide amphiphile synthesized and different structure formation from water and propanol. Adapted with permission from ref 2. Copyright 2019 American Chemical Society.

Furthermore, they wanted to explore how the 2D network can be disrupted using external stimuli such as solvent polarity without changing the chemical structure. Addition of methanol, which is less polar compared to water, to the water resulted in the nanostructure elongating in length. The reason for this is that the polarity of the solution disrupts the aromatic interactions and thus assembly in the y-direction is less favored. This forces the formation of self-assembly increasingly in the x-direction, resulting in longer, nanobelt-type structures (Figure 2). The rigidity of the peptide nanostructure was investigated using Circular dichroism (CD) spectroscopy, which is a method to investigate higher order structure of proteins. The intensity of CD signals decreased upon addition of methanol, which again confirmed formation of less ordered structures indicating 2D to 1D transition. Relatively less polar alcohols, such as ethanol, propanol, butanol also efficiently transform the peptide nanostructure from predominantly 2D to 1D.

Figure 2: Morphological change of self-assembled structure from nanosheets to nanofibrils. Adapted with permission from ref 2. Copyright 2019 American Chemical Society.

Next, researchers looked into the solvent-peptide interaction computationally. The distribution of solvent molecules in growth solution (solution of the peptide and solvent where the self-assembled structure is forming) showed that solvent molecules tend to interact with the peptide in phenylalanine amino acid region (highlighted as yellow in figure 1) and hydrophobic alkyl tail region (highlighted as grey in figure 1). It was also observed that with decreasing polarity solvent molecules cover more surface area near phenylalanine amino acid region. Interaction of the solvent molecules around phenylalanine amino acid region disrupted the interaction in between corresponding peptide molecules which is necessary for formation of 2D nanosheets. Disruption of interaction hindered the growth of peptide molecules along y-direction (Figure 1) and thus 1D nanofibrils were formed.

The hypothesis that peptide self-assembled structures can be modulated by the polarity of the solvent molecules was confirmed using additional high and low polarity solvents. For example, it was observed that upon going from formamide to N, N-dimethylformamide (which has two -CH3 group excess to formamide and hence less polar), a transition from nanosheet to nanofibril structure was observed. This again reaffirms the idea that less polar solvents tend towards a 1D network. Researchers also explored how the order of solvent addition affected the self-assembly process. They approached this idea using two different pathways. In pathway I, co-solvent is added first and then the peptide solution is added to growth solution. In pathway II, the peptide forms the self-assembly structure and then co-solvent is added (Figure 3). Following pathway II, the addition of low polarity solvents to the pre-formed nanosheet does not disrupt the nanosheet even for more than 2 months whereas growth of the same amount of peptide using pathway I favor nanofibril formation.

Figure 3. a) Different pathways to self-assembly process b) Energy diagram of the pathways to self-assembly. Adapted with permission from ref 2. Copyright 2019 American Chemical Society.

These results are exciting from a fundamental point of view as it sheds light on the effect of solvents on peptide self-assembly processes. This study can serve as a benchmark for designing biomaterials and possibly can inform on structure-functional relationships of protein macromolecules. In the future, maybe similar types of research will bring forward detailed mechanisms of the self-assembly biological macromolecules inside the body.

  1. https://en.wikipedia.org/wiki/Non-covalent_interactions
  2. Y. Lin, M. Penna, M. R. Thomas, J. P. Wojciechowski, V. Leonardo, Y. Wang, E. T. Pashuck, I. Yarovsky and M. M. Stevens. ACS Nano, 2019, 10.1021/acsnano.8b08117

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