Stephen J. Klawa

Living systems transform their shapes via reversible formation of macromolecular structural complexes, leading to deformations at localized sites. Here we report DNA–inorganic flower-shaped crystals with inscribed deformation modes that enable flowers to shrink and bend reversibly. Template-independent DNA polymerization of pH-responsive and inert blocks tune the hierarchical assembly and spatial localization of DNA within flowers. Experiments and simulations demonstrate that reversible, pH-triggered folding of intraflower DNA strands drives reconfiguration of flowers. By contrast, the subflower localization of these contractile DNA motifs dictates the mode of shape change. As microscale flowers close and open, their nanoscale crystal organization changes reversibly, suggesting that mechanical metamorphosis of flowers is transduced across multiple organizational length scales. The adaptability of flowers to environmental changes activates cascaded biocatalytic reactions and reveals gel-encrypted information. Further variation of the DNA polymer sequence, its subcrystal localization and its reversible folding advances a new class of organic–inorganic shape-shifters.

Septins are a class of cytoskeletal proteins that preferentially bind to domains of micron-scale curvature on the cell membrane. Studies have shown that amphipathic helix (AH) domains in septin oligomers are essential for septin curvature sensing. Yet, the underlying mechanochemical interactions that modulate this curvature sensing remain ambiguous. Here we use all-atom molecular dynamics alongside a metadynamics enhanced sampling approach to bridge the gap between time and length scales required to optimize and validate experimental design of amphipathic helices. Simulations revealed that the local charge on the termini of an 18-amino-acid AH peptide impacts its helical content and positioning within lipid membranes. These computational observations are confirmed with experiments measuring the binding of synthetic AH constructs with variable helical content and charged termini to lipid vesicles. Taken together, these results identify the helical content of amphipathic helices as a regulator of septin binding affinity to lipid membranes. Additionally, we examined an extended AH sequence including 8 amino acids upstream and downstream of the minimal 18-amino-acid-long AH domain to more closely mimic the native protein in simulations and experiments. Simulations and experiments show that the extended peptide sequence adopts a strong alpha-helical conformation when free in solution, giving rise to a higher affinity to lipid membranes than that of the shorter AH sequence. Together, these results provide insight into how the native septin proteins interact with membranes, and establish general design principles that can guide the interaction of future synthetic materials with lipid membranes in a programmable manner. STATEMENT OF SIGNIFICANCE Understanding how cells sense and react to their shape is necessary for numerous biological processes. Here we explore the interactions between amphipathic helices, a curvature sensing protein motif, and lipid membranes. Using molecular dynamics simulations, enhanced simulation sampling techniques, and experiments, we find that increasing the helical content of the amphipathic helix or adding charged capping sequences yields higher membrane binding affinity. Understanding these parameters for membrane-binding could enable us to interface and regulate native protein functions, as well as guide the design of synthetic curvature-sensing materials that can interact with and deform lipid membranes.

The bottom-up engineering of artificial cells requires a reconfigurable cytoskeleton that can organize at distinct locations and dynamically modulate its structural and mechanical properties. Here, inspired by the vast array of actin-binding proteins and their ability to reversibly crosslink or bundle filaments, we have designed a library of peptide–DNA crosslinkers varying in length, valency and geometry. Peptide filaments conjoint through DNA hybridization give rise to tactoid-shaped bundles with tunable aspect ratios and mechanics. When confined in cell-sized water-in-oil droplets, the DNA crosslinker design guides the localization of cytoskeletal structures at the cortex or within the lumen of the synthetic cells. The tunable spatial arrangement regulates the passive diffusion of payloads within the droplets and complementary DNA handles allow for the reversible recruitment and release of payloads on and off the cytoskeleton. Heat-induced reconfiguration of peptide–DNA architectures triggers shape deformations of droplets, regulated by DNA melting temperatures. Altogether, the modular design of peptide–DNA architectures is a powerful strategy towards the bottom-up assembly of synthetic cells.

In neurodegenerative diseases, polymorphism and supramolecular assembly of β-sheet amyloids are implicated in many different etiologies and may adopt either a left- or right-handed supramolecular chirality. Yet, the underlying principles of how sequence regulates supramolecular chirality remains unknown. Here, we characterize the sequence specificity of the central core of amyloid-β 42 and design derivatives which enable chirality inversion at biologically relevant temperatures. We further find that C-terminal modifications can tune the energy barrier of a left-to-right chiral inversion. Leveraging this design principle, we demonstrate how temperature-triggered chiral inversion of peptides hosting therapeutic payloads modulates the dosed release of an anticancer drug. These results suggest a generalizable approach for fine-tuning supramolecular chirality that can be applied in developing treatments to regulate amyloid morphology in neurodegeneration as well as in other disease states.

This work employs peptide-modified gold nanoparticles to develop a fieldable SERS sensor for direct detection of viral antigens using a handheld platform.

Cytoskeletal assemblies produce functionality through multiscale structural reconfiguration by exploiting a family of protein crosslinkers. Inspired by these principles, we designed an array of complementary peptide-DNA monomers able to form multistranded junctions with different geometries and stabilities and tested their effect on the structural organization of co-polymerized filament-forming peptides. In striking similarity to their biological counterparts, the peptide-DNA crosslinkers organized fibers into bundled architectures with base-pair tunable aspect ratios. Controlling solvent or temperature drove assembly through distinct pathways to form different structural and mechanical states exhibiting multiple levels of hierarchy from the same set of components. The thermal reversibility of DNA crosslinks drove structures to form, disassemble, and rebuild over multiple cycles. Sequential transitioning between bundles and fibers using orthogonal triggers enabled the dosed release of proteins to modulate catalytic reactions. This programmable peptide-DNA nanotechnology framework will pave the way towards the design of hierarchical materials with reconfigurable properties.

The native extracellular matrix communicates and interacts with cells by dynamically displaying signals to control their behavior. Mimicking this dynamic environment in vitro is essential in order to unravel how cell–matrix interactions guide cell fate. Here, we present a synthetic platform for the temporal display of cell-adhesive signals using coiled-coil peptides. By designing an integrin-engaging coiled-coil pair to have a toehold (unpaired domain), we were able to use a peptide strand displacement reaction to remove the cell cue from the surface. This allowed us to test how the user-defined display of RGDS ligands at variable duration and periodicity of ligand exposure influence cell spreading degree and kinetics. Transient display of α_Vβ₃-selective ligands instructed fibroblast cells to reversibly spread and contract in response to changes in ligand exposure over multiple cycles, exhibiting a universal kinetic response. Also, cells that were triggered to spread and contract repeatedly exhibited greater enrichment of integrins in focal adhesions versus cells cultured on persistent RGDS-displaying surfaces. This dynamic platform will allow us to uncover the molecular code by which cells sense and respond to changes in their environment and will provide insights into ways to program cellular behavior.