Structure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression.

Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.

Single-molecule analysis of osmolyte-mediated nanomechanical unfolding behavior of a protein domain.

The small organic molecules, known as osmolytes being ubiquitously present in different cell types, affect protein folding, stability and aggregation. However, it is unknown how the osmolytes affect the nanomechanical unfolding behavior of protein domain. Here, we show the osmolyte-dependent mechanical unfolding properties of protein titin immunoglobulin-27 (I27) domain using an atomic force microscopy (AFM)-based single-molecule force spectroscopy. We found that amines and methylamines improved the mechanical stability of I27 domain, whereas polyols had no effect. Interestingly, glycine betaine (GB) or trimethylamine-N-oxide (TMAO) increased the average unfolding force of the protein domain. The kinetic parameters analyzed at single-molecule level reveal that stabilizing effect of osmolytes is due to a decrease in the unfolding rate constant of I27, which was confirmed by molecular dynamics simulations. Our study reveals different effects that diverse osmolytes have on the mechanical properties of the protein, and suggests the potential use of osmolytes in modulating the mechanical stability of proteins required for various nano-biotechnological applications.

Sequence-dependent aggregation-prone conformations of islet amyloid polypeptide.

Amyloid proteins, which aggregate to form highly ordered structures, play a crucial role in various disease pathologies. Despite many previous studies on amyloid fibrils, which are an end product of protein aggregation, the structural characteristics of amyloid proteins in the early stage of aggregation and their related aggregation mechanism still remain elusive. The role of the amino acid sequence in the aggregation-prone structures of amyloid proteins at such a stage is not understood. Here, we have studied the sequence-dependent structural characteristics of islet amyloid polypeptide based on atomistic simulations and spectroscopic experiments. We show that the amino acid sequence determines non-bonded interactions that play a leading role in the formation of aggregation-prone conformations. Specifically, a single point mutation critically changes the population of aggregation-prone conformations, resulting in a change of the aggregation mechanism. Our simulation results were supported by experimental results suggesting that mutation affects the kinetics of aggregation and the structural characteristics of amyloid aggregates. Our study provides an insight into the role of sequence-dependent aggregation-prone conformations in the underlying mechanisms of amyloid aggregation.

Different Aggregation Pathways and Structures for Aß40 and Aß42 Peptides.

Self-aggregation of amyloid-ß (Aß) peptides has been known to play a vital role in the onset stage of neurodegenerative diseases, indicating the necessity of understanding the aggregation process of Aß peptides. Despite previous studies on the aggregation process of Aß peptides, the aggregation pathways of Aß isoforms (i.e., Aß40 and Aß42) and their related structures have not been fully understood yet. Here, we study the aggregation pathways of Aß40 and Aß42, and the structures of Aß40 and Aß42 aggregates during the process, based on fluorescence and atomic force microscopy (AFM) experiments. It is shown that in the beginning of aggregation process for both Aß40 and Aß42, a number of particles (i.e., spherical oligomers) are formed. These particles are subsequently self-assembled together, resulting in the formation of different shapes of amyloid fibrils. Our finding suggests that the different aggregation pathways of Aß isoforms lead to the amyloid fibrils with contrasting structure.

Role of physique and physical fitness in the balance of Korean national snowboard athletes.

BACKGROUND: The objective of this study is to understand the relationship between physique, physical fitness, and balance performance for snowboard athletes. METHODS: We considered all Korean national snowboard athletes (n = 9 with 6 males and 3 females, age = 17.44 ± 4.42), who have an experience of competition at continental cup level, to measure their physique, physical fitness, and (both static and dynamic) balance. Static balance was evaluated based on one-legged standing, while dynamic balance was estimated using a stability platform. RESULTS: Static balance is strongly correlated (p < 0.05) with circumference of the left lower leg (34.49 ± 2.42 cm; ρ = 0.68), sit-up (57.56 ± 8.8; ρ = 0.72), sargent jump (50.22 ± 11.78 cm; ρ = 0.67), strength of bench press (39.11 ± 17.73 kg; ρ = 0.67), angle of left ankle dorsiflexion (73.78 ± 7.86°; ρ = 0.77), average extension strength at 180° for left knee (321 ± 63.95 %BW; ρ = 0.77) and right knee (337 ± 60.32 %BW; ρ = 0.77), and right knee peak flexion strength at 60° (148 ± 25.61 %BW; ρ = 0.73). Center dynamic balance is negatively correlated with circumference of the right lower leg (34.63 ± 2.38 cm; ρ = -0.67, p < 0.05), while right dynamic balance is positively correlated with left ankle flexion (148.44 ± 5.20°; ρ = 0.78, p < 0.05). CONCLUSION: Static balance is related to core muscle endurance and power, ankle flexibility, and knee stability, while dynamic balance is negatively correlated with circumference of the most frequently used lower leg (i.e., the leg dominating the snowboarding stance). The relationship between physique, physical fitness, and balance provides an insight into improving the balance performance of elite snowboard athletes through a training program that can affect the physique and physical fitness factors related to balance.

Variable Membrane Dielectric Polarization Characteristic in Individual Live Cells.

Investigation of the dielectric properties of cell membranes plays an important role in understanding the biological activities that sustain cellular life and realize cellular functionalities. Herein, the variable dielectric polarization characteristics of cell membranes are reported. In controlling the dielectric polarization of a cell using dielectrophoresis force spectroscopy, different cellular crossover frequencies were observed by modulating both the direction and sweep rate of the frequency. The crossover frequencies were used for the extraction of the variable capacitance, which is involved in the dielectric polarization across the cell membranes. In addition, this variable phenomenon was investigated by examining cells whose membranes were cholesterol-depleted with methyl-ß-cyclodextrin, which verified a strong correlation between the variable dielectric polarization characteristics and membrane composition changes. This study presented the dielectric polarization properties in live cells' membranes that can be modified by the regulation of external stimuli and provided a powerful platform to explore cellular membrane dielectric polarization.

Ligand-binding alters the mechanical stability of muscle protein titin domains.

Communicated by Ramaswamy H. Sarma.

Atomic force microscopy-based cancer diagnosis by detecting cancer-specific biomolecules and cells.

Atomic force microscopy (AFM) has recently attracted much attention due to its ability to analyze biomolecular interactions and to detect certain biomolecules, which play a crucial role in disease expression. Despite recent studies reporting AFM imaging for the analyses of biomolecules, the application of AFM-based cancer-specific biomolecule/cell detection has remained largely underexplored, especially for the early diagnosis of cancer. In this paper, we review the recent attempts, including our efforts, to analyze and detect cancer-specific biomolecules and cancer cells. We particularly focus on two AFM-based cancer diagnosis techniques: (i) AFM imaging-based biomolecular and cellular detection, (ii) AFM cantilever-based biomolecular sensing and cell analysis. It is shown that AFM-based biomolecular detection has been applied for not only early diagnosing cancer, by measuring the minute amount of cancer-specific proteins, but also monitoring of cancer progression, by correlating the amount of cancer-specific proteins with the progression of cancer. In addition, AFM-based cell imaging and detection have been employed for diagnosing cancer, by detecting cancerous cells in tissue, as well as understanding cancer progression, by characterizing the dynamics of cancer cells. This review, therefore, highlights AFM-based biomolecule/cell detection, which will pave the way for developing a fast and point-of-care diagnostic system for biomedical applications.

Correlation between the hierarchical structures and nanomechanical properties of amyloid fibrils.

Amyloid fibrils have recently been highlighted due to their excellent mechanical properties, which not only play a role in their biological functions but also imply their applications in biomimetic material design. Despite recent efforts to unveil how the excellent mechanical properties of amyloid fibrils originate, it has remained elusive how the anisotropic nanomechanical properties of hierarchically structured amyloid fibrils are determined. Here, we characterize the anisotropic nanomechanical properties of hierarchically structured amyloid fibrils using atomic force microscopy experiments and atomistic simulations. It is shown that the hierarchical structure of amyloid fibrils plays a crucial role in determining their radial elastic property but does not make any effect on their bending elastic property. This is attributed to the role of intermolecular force acting between the filaments (constituting the fibril) on the radial elastic modulus of amyloid fibrils. Our finding illustrates how the hierarchical structure of amyloid fibrils encodes their anisotropic nanomechanical properties. Our study provides key design principles of amyloid fibrils, which endow valuable insight into the underlying mechanisms of amyloid mechanics.

Metal ions affect the formation and stability of amyloid ß aggregates at multiple length scales.

Amyloid ß (Aß) aggregates, which are a hallmark for neurodegenerative disease, are formed through a self-assembly process such as aggregation of Aß peptide chains. This aggregation process depends on the solvent conditions under which the proteins are aggregated. Nevertheless, the underlying mechanism of the ionic effect on the formation and stability of amyloid aggregates has not been fully understood. Here, we report how metal ions play a role in the formation and stability of Aß aggregates at different length scales, i.e. oligomers and fibrils. It is shown that the metal (i.e. zinc or copper) ion increases the stability of Aß oligomers, whereas the metal ion reduces the stability of Aß fibrils. In addition, we found that zinc ions are able to more effectively destabilize fibril structures than copper ions. Metal ion-mediated (de)stabilization of Aß oligomers (or fibrils) is attributed to the critical effect of the metal ion on the ß-sheet rich crystalline structure of the amyloid aggregate and the status of hydrogen bonds within the aggregate. Our study sheds light on the role of the metal ion in stabilizing the amyloid oligomers known as a toxic agent (to functional cells), which is consistent with clinical observation that high concentrations of metal ions are found in patients suffering from neurodegenerative diseases.

Blood Droplet-Based Cancer Diagnosis via Proteolytic Activity Measurement in Cancer Progression.

Matrix metalloproteinase (MMP) is a key marker and target molecule for cancer diagnosis, as MMP is able to cleave peptide chains resulting in degradation of extracellular matrix (ECM), a necessary step for cancer development. In particular, MMP2 has recently been recognized as an important biomarker for lung cancer. Despite the important role of detecting MMP molecules in cancer diagnosis, it is a daunting task to quantitatively understand a correlation between the status of cancer development and the secretion level of MMP in a blood droplet. Here, we demonstrate a nanoscale cancer diagnosis by nanomechanical quantitation of MMP2 molecules under cancer progression with using a blood droplet of lung cancer patients. Specifically, we measured the frequency dynamics of nanomechanical biosensor functionalized with peptide chains mimicking ECM in response to MMP2 secreted from tumors in lung with different metastasis level. It is shown that the frequency shift of the biosensor, which exhibits the detection sensitivity below 1 nM, enables the quantitation of the secretion level of MMP2 molecules during the progression of cancer cells or tumor growth. More importantly, using a blood droplet of lung cancer patients, nanomechanical biosensor is shown to be capable of depicting the correlation between the secretion level of MMP2 molecules and the level of cancer metastasis, which highlights the cantilever-based MMP2 detection for diagnosis of lung cancer. Our finding will broaden the understanding of cancer development activated by MMP and allow for a fast and point-of-care cancer diagnostics.

Nature-Inspired Construction of Two-Dimensionally Self-Assembled Peptide on Pristine Graphene.

Peptide assemblies have received significant attention because of their important role in biology and applications in bionanotechnology. Despite recent efforts to elucidate the principles of peptide self-assembly for developing novel functional devices, peptide self-assembly on two-dimensional nanomaterials has remained challenging. Here, we report nature-inspired two-dimensional peptide self-assembly on pristine graphene via optimization of peptide-peptide and peptide-graphene interactions. Two-dimensional peptide self-assembly was designed based on statistical analyses of >104 protein structures existing in nature and atomistic simulation-based structure predictions. We characterized the structures and surface properties of the self-assembled peptide formed on pristine graphene. Our study provides insights into the formation of peptide assemblies coupled with two-dimensional nanomaterials for further development of nanobiocomposite devices.

Stretchable, Transparent, and Stretch-Unresponsive Capacitive Touch Sensor Array with Selectively Patterned Silver Nanowires/Reduced Graphene Oxide Electrodes.

Stretchable and transparent touch sensors are essential input devices for future stretchable transparent electronics. Capacitive touch sensors with a simple structure of only two electrodes and one dielectric are an established technology in current rigid electronics. However, the development of stretchable and transparent capacitive touch sensors has been limited due to changes in capacitance resulting from dimensional changes in elastomeric dielectrics and difficulty in obtaining stretchable transparent electrodes that are stable under large strains. Herein, a stretch-unresponsive stretchable and transparent capacitive touch sensor array was demonstrated by employing stretchable and transparent electrodes with a simple selective-patterning process and by carefully selecting dielectric and substrate materials with low strain responsivity. A selective-patterning process was used to embed a stretchable and transparent silver nanowires/reduced graphene oxide (AgNWs/rGO) electrode line into a polyurethane (PU) dielectric layer on a polydimethylsiloxane (PDMS) substrate using oxygen plasma treatment. This method provides the ability to directly fabricate thin film electrode lines on elastomeric substrates and can be used in conventional processes employed in stretchable electronics. We used a dielectric (PU) with a Poisson's ratio smaller than that of the substrate (PDMS), which prevented changes in the capacitance resulting from stretching of the sensor. The stretch-unresponsive touch sensing capability of our transparent and stretchable capacitive touch sensor has great potential in wearable electronics and human-machine interfaces.

Mechanical Deformation Mechanisms and Properties of Prion Fibrils Probed by Atomistic Simulations.

Prion fibrils, which are a hallmark for neurodegenerative diseases, have recently been found to exhibit the structural diversity that governs disease pathology. Despite our recent finding concerning the role of the disease-specific structure of prion fibrils in determining their elastic properties, the mechanical deformation mechanisms and fracture properties of prion fibrils depending on their structures have not been fully characterized. In this work, we have studied the tensile deformation mechanisms of prion and non-prion amyloid fibrils by using steered molecular dynamics simulations. Our simulation results show that the elastic modulus of prion fibril, which is formed based on left-handed ß-helical structure, is larger than that of non-prion fibril constructed based on right-handed ß-helix. However, the mechanical toughness of prion fibril is found to be less than that of non-prion fibril, which indicates that infectious prion fibril is more fragile than non-infectious (non-prion) fibril. Our study sheds light on the role of the helical structure of amyloid fibrils, which is related to prion infectivity, in determining their mechanical deformation mechanisms and properties.

Finite-size effect on the dynamic and sensing performances of graphene resonators: the role of edge stress.

We have studied the finite-size effect on the dynamic behavior of graphene resonators and their applications in atomic mass detection using a continuum elastic model such as modified plate theory. In particular, we developed a model based on von Karman plate theory with including the edge stress, which arises from the imbalance between the coordination numbers of bulk atoms and edge atoms of graphene. It is shown that as the size of a graphene resonator decreases, the edge stress depending on the edge structure of a graphene resonator plays a critical role on both its dynamic and sensing performances. We found that the resonance behavior of graphene can be tuned not only through edge stress but also through nonlinear vibration, and that the detection sensitivity of a graphene resonator can be controlled by using the edge stress. Our study sheds light on the important role of the finite-size effect in the effective design of graphene resonators for their mass sensing applications.

Biaxial Dielectrophoresis Force Spectroscopy: A Stoichiometric Approach for Examining Intermolecular Weak Binding Interactions.

The direct quantification of weak intermolecular binding interactions is very important for many applications in biology and medicine. Techniques that can be used to investigate such interactions under a controlled environment, while varying different parameters such as loading rate, pulling direction, rupture event measurements, and the use of different functionalized probes, are still lacking. Herein, we demonstrate a biaxial dielectrophoresis force spectroscopy (BDFS) method that can be used to investigate weak unbinding events in a high-throughput manner under controlled environments and by varying the pulling direction (i.e., transverse and/or vertical axes) as well as the loading rate. With the BDFS system, we can quantitatively analyze binding interactions related to hydrogen bonding or ionic attractions between functionalized microbeads and a surface within a microfluidic device. Our BDFS system allowed for the characterization of the number of bonds involved in an interaction, bond affinity, kinetic rates, and energy barrier heights and widths from different regimes of the energy landscape.

Morphology and mechanical properties of multi-stranded amyloid fibrils probed by atomistic and coarse-grained simulations.

Amyloid fibrils are responsible for pathogenesis of various diseases and exhibit the structural feature of an ordered, hierarchical structure such as multi-stranded helical structure. As the multi-strandedness of amyloid fibrils has recently been found to be highly correlated with their toxicity and infectivity, it is necessary to study how the hierarchical (i.e. multi-stranded) structure of amyloid fibril is formed. Moreover, although it has recently been reported that the nanomechanics of amyloid proteins plays a key role on the amyloid-induced pathogenesis, a critical role that the multi-stranded helical structure of the fibrils plays in their nanomechanical properties has not fully characterized. In this work, we characterize the morphology and mechanical properties of multi-stranded amyloid fibrils by using equilibrium molecular dynamics simulation and elastic network model. It is shown that the helical pitch of multi-stranded amyloid fibril is linearly proportional to the number of filaments comprising the amyloid fibril, and that multi-strandedness gives rise to improving the bending rigidity of the fibril. Moreover, we have also studied the morphology and mechanical properties of a single protofilament (filament) in order to understand the effect of cross-ß structure and mutation on the structures and mechanical properties of amyloid fibrils. Our study sheds light on the underlying design principles showing how the multi-stranded amyloid fibril is formed and how the structure of amyloid fibrils governs their nanomechanical properties.

Self-assembled amyloid fibrils with controllable conformational heterogeneity.

Amyloid fibrils are a hallmark of neurodegenerative diseases and exhibit a conformational diversity that governs their pathological functions. Despite recent findings concerning the pathological role of their conformational diversity, the way in which the heterogeneous conformations of amyloid fibrils can be formed has remained elusive. Here, we show that microwave-assisted chemistry affects the self-assembly process of amyloid fibril formation, which results in their conformational heterogeneity. In particular, microwave-assisted chemistry allows for delicate control of the thermodynamics of the self-assembly process, which enabled us to tune the molecular structure of ß-lactoglobulin amyloid fibrils. The heterogeneous conformations of amyloid fibrils, which can be tuned with microwave-assisted chemistry, are attributed to the microwave-driven thermal energy affecting the electrostatic interaction during the self-assembly process. Our study demonstrates how microwave-assisted chemistry can be used to gain insight into the origin of conformational heterogeneity of amyloid fibrils as well as the design principles showing how the molecular structures of amyloid fibrils can be controlled.

ß-sheet-like formation during the mechanical unfolding of prion protein.

Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrP(C), whose misfolded form PrP(Sc) can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded ß-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the ß-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.

The role of binding site on the mechanical unfolding mechanism of ubiquitin.

We apply novel atomistic simulations based on potential energy surface exploration to investigate the constant force-induced unfolding of ubiquitin. At the experimentally-studied force clamping level of 100 pN, we find a new unfolding mechanism starting with the detachment between ß5 and ß3 involving the binding site of ubiquitin, the Ile44 residue. This new unfolding pathway leads to the discovery of new intermediate configurations, which correspond to the end-to-end extensions previously seen experimentally. More importantly, it demonstrates the novel finding that the binding site of ubiquitin can be responsible not only for its biological functions, but also its unfolding dynamics. We also report in contrast to previous single molecule constant force experiments that when the clamping force becomes smaller than about 300 pN, the number of intermediate configurations increases dramatically, where almost all unfolding events at 100 pN involve an intermediate configuration. By directly calculating the life times of the intermediate configurations from the height of the barriers that were crossed on the potential energy surface, we demonstrate that these intermediate states were likely not observed experimentally due to their lifetimes typically being about two orders of magnitude smaller than the experimental temporal resolution.