Unveiling the nanoscale architectures and dynamics of protein assembly with in situ atomic force microscopy

Proteins play a vital role in different biological processes by forming complexes through precise folding with exclusive inter-and intra-molecular interactions.Understanding the structural and regulatory mechanisms underlying protein complex formation provides insights into biophysical processes.Furthermore,the principle of protein assembly gives guidelines for new biomimetic materials with potential appli-cations in medicine,energy,and nanotechnology.Atomic force microscopy(AFM)is a powerful tool for investigating protein assembly and interactions across spatial scales(single molecules to cells)and temporal scales(milliseconds to days).It has significantly contributed to understanding nanoscale architectures,inter-and intra-molecular interactions,and regulatory elements that determine protein structures,assemblies,and functions.This review describes recent advancements in elucidating protein assemblies with in situ AFM.We discuss the structures,diffusions,interac-tions,and assembly dynamics of proteins captured by conventional and high-speed AFM in near-native environments and recent AFM developments in the multimodal high-resolution imaging,bimodal imaging,live cell imaging,and machine-learning-enhanced data analysis.These approaches show the significance of broadening the horizons of AFM and enable unprecedented explorations of protein assembly for biomaterial design and biomedical research.

In situ assembly of fibrinogen/hyaluronic acid hydrogel via knob-hole interaction for 3D cellular engineering

Hyaluronic acid(HA)-based hydrogels have applied widely for biomedical applications due to its biocompatibility and biodegradability.However,the use of initiators or crosslinkers during the hydrogel formation may cause cytotoxicity and thereby impair the biocompatibility.Inspired by the crosslinking mechanism of fibrin gel,a novel HA-based hydrogel was developed via the in situ supramolecular assembly based on knob-hole interactions between fibrinogen and knob-grafted HA(knob-g-HA)in this study.The knob-grafted HA was synthesized by coupling knob peptides(GPRPAAC,a mimic peptide of fibrin knob A)to HA via Michael addition.Then the translucent fibrinogen/knob-g-HA hydrogels were prepared by simply mixing the solutions of knob-g-HA and fibrinogen at the knob/hole ratio of 1.2.The rheological behaviors of the fibrinogen/knob-g-HA hydrogels with the fibrinogen concentrations of 50,100 and 200 mg/mL were evaluated,and it was found that the dynamic storage moduli(G0)were higher than the loss moduli(G00)over the whole frequency range for all the groups.The SEM results showed that fibrinogen/knob-g-HA hydrogels presented the heterogeneous mesh-like structures which were different from the honeycomb-like structures of fibrinogen/MA-HA hydrogels.Correspondingly,a higher swelling ratio was obtained in the groups of fibrinogen/knob-g-HA hydrogel.Finally,the cytocompatibility of fibrinogen/knob-g-HA hydrogels was proved by live/dead stainings and MTT assays in the 293T cells encapsulation test.All these results highlight the biological potential of the fibrinogen/knob-g-HA hydrogels for 3D cellular engineering.

Ultra-robust stretchable electrode for e-skin:In situ assembly using a nanofiber scaffold and liquid metal to mimic water-to-net interaction

The development of stretchable electronics could enhance novel interface structures to solve the stretchability-conductivity dilemma,which remains a major challenge.Herein,we report a nano-liquid metal(LM)-based highly robust stretchable electrode(NHSE)with a self-adaptable interface that mimics water-tonet interaction.Based on the in situ assembly of electrospun elastic nanofiber scaffolds and electrosprayed LM nanoparticles,the NHSE exhibits an extremely low sheet resistance of 52 mΩsq^(-1).It is not only insensitive to a large degree of mechanical stretching(i.e.,350%electrical resistance change upon 570%elongation)but also immune to cyclic deformation(i.e.,5%electrical resistance increases after 330000 stretching cycles with 100%elongation).These key properties are far superior to those of the state-of-the-art reports.Its robustness and stability are verified under diverse circumstances,including long-term exposure to air(420 days),cyclic submersion(30000 times),and resilience against mechanical damages.The combination of conductivity,stretchability,and durability makes the NHSE a promising conductor/electrode solution for flexible/stretchable electronics for applications such as wearable on-body physiological signal detection,human-machine interaction,and heating e-skin.

Self-Assembled Protein Hybrid Nanofibrils for Photosynthetic Hydrogen Evolution

In artificial photosynthesis systems,synthetic diiron complexes are popular[FeFe]-hydrogenase mimics,which are attractive for the fabrication of photocatalyst-protein hybrid structures to amplify hydrogen(H2)generation capability.However,constructing a highly bionic and efficient catalytic hybrid system is a major challenge.Notably,we designed an ideal hybrid nanofibrils system that incorporates the crucial components:(1)a[FeFe]-H2ase mimic,which has a three-arm architecture(named triFeFe)for more interaction sites and higher catalytic activity and(2)uniform hybrid nanofibrils as the biological environment in which cysteine-catalyst coordination and the hydrogen-bonding network play a vital role in both catalyst binding and hydrogen evolution reaction activity.The assembled hybrid nanofibrils achieve efficient H2 generation with a turnover number of 2.3×103,outperforming previously reported diiron catalyst-protein hybrid systems.Additionally,the hybrid nanofibrils work with photosynthetic thylakoids to produce H2,without extra photosensitizers or electron shuttle proteins,which advances the bioengineering of living systems for solar-driven biofuel production.