Biomolecular Function Research Articles

Screening of structural and functional alterations in duckweed (Lemna minor) induced by per- and polyfluoroalkyl substances (PFASs) with FTIR spectroscopy

As a class of common emerging pollutants, per- and polyfluoroalkyl substances (PFASs) and their alternatives have been widely detected in various environmental matrices, exhibiting a great threat to the ecological environment and human health. Nevertheless, changes in biomolecular structure and function of duckweed caused by PFASs and their alternatives remain unknown thus far. Herein, the effects of four PFASs, including two common legacy PFASs (perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA)) and two PFASs alternatives (perfluorobutane sulfonic acid (PFBS) and 1H,1H,2H, 2H-perfluorooctane sulfonic acid (6:2 FTS)) on duckweed (Lemna minor) at biochemical level were investigated with Fourier transform infrared spectroscopy (FTIR). Although no obvious inhibitions were observed in the growth of L. minor with PFASs exposure at three levels of 1 μg L−1, 100 μg L−1, and 10 mg L−1, significant structural and functional alterations were induced at the biochemical level. In response to PFASs exposure, lipid peroxidation, proteins aggregation and α-helix to β-sheet transformation of the protein conformation, as well as changes of DNA conformations were detected. Moreover, alterations in lipid, protein, and DNA were proved to be concentration-related and compound-specific. Compared to the two legacy PFASs (PFOS and PFOA), alternative ones exhibited greater effects on the biological macromolecules of L. minor. The findings of this study firstly reveal structural and functional alterations in L. minor induced by PFASs exposure, providing further understanding of their toxicity effects.

Interactive Flexible-Receptor Molecular Docking in Virtual Reality Using DockIT.

Interactive docking enables the user to guide and control the docking of two biomolecules into a binding pose. It is of particular use when the binding site is known and is thought to be applicable to structure-based drug design (SBDD) and educating students about biomolecular interactions. For SBDD, it enables expertise and intuition to be brought to bear in the drug design process. In education, it can teach students about the most basic level of biomolecular function. Here, we introduce DockIT for virtual reality (VR) that uses a VR headset and hand-held controllers. Using the method of linear response on explicit solvent molecular dynamics simulations, DockIT can model both global and local conformational changes within the receptor due to forces of interaction with the ligand. It has real-time flexible molecular surface rendering and can show the real-time formation and breaking of hydrogen bonds, both between the ligand and receptor and within the receptor itself as it smoothly changes conformation.

Biomolecular Topology: Modelling and Analysis.

With the great advancement of experimental tools, a tremendous amount of biomolecular data has been generated and accumulated in various databases. The high dimensionality, structural complexity, the nonlinearity, and entanglements of biomolecular data, ranging from DNA knots, RNA secondary structures, protein folding configurations, chromosomes, DNA origami, molecular assembly, to others at the macromolecular level, pose a severe challenge in their analysis and characterization. In the past few decades, mathematical concepts, models, algorithms, and tools from algebraic topology, combinatorial topology, computational topology, and topological data analysis, have demonstrated great power and begun to play an essential role in tackling the biomolecular data challenge. In this work, we introduce biomolecular topology, which concerns the topological problems and models originated from the biomolecular systems. More specifically, the biomolecular topology encompasses topological structures, properties and relations that are emerged from biomolecular structures, dynamics, interactions, and functions. We discuss the various types of biomolecular topology from structures (of proteins, DNAs, and RNAs), protein folding, and protein assembly. A brief discussion of databanks (and databases), theoretical models, and computational algorithms, is presented. Further, we systematically review related topological models, including graphs, simplicial complexes, persistent homology, persistent Laplacians, de Rham-Hodge theory, Yau-Hausdorff distance, and the topology-based machine learning models.

Connecting Non-Gaussian Water Density Fluctuations to the Lengthscale Dependent Crossover in Hydrophobic Hydration.

We connect density fluctuations in liquid water to lengthscale dependent crossover in hydrophobic hydration. Specifically, we employ indirect umbrella sampling (INDUS) simulations to characterize density fluctuations in observation volumes of various sizes and shapes in water and as a function of temperature and salt concentration. Consistent with previous observations, density fluctuations are Gaussian in small molecular scale volumes, but they display non-Gaussian "low-density fat tails" in larger volumes. These non-Gaussian tails are indicative of the proximity of water to its liquid to vapor phase transition and have implications on biomolecular interactions and function. We show that the onset of non-Gaussian fluctuations in large volumes is accompanied by the formation of a cavity in the observation volume. We develop a model that uses the physics of cavity-water interface formation as a key ingredient and show that it captures the nature of non-Gaussian density fluctuations over a broad region in water and in salt solutions. We discuss the limitations of this model in the very low density region of the distribution. Our calculations provide new insights into the origins of non-Gaussian density fluctuations in water and their connections to lengthscale dependent crossover in hydrophobic hydration.

Molecular Mechanisms Underlying Qi-Invigorating Effects in Traditional Medicine: Network Pharmacology-Based Study on the Unique Functions of Qi-Invigorating Herb Group.

Qi-invigorating herbs (QIHs) are a group of herbs that invigorate Qi, the most vital force for maintaining the physiological functions of the human body in traditional medicine. However, the mechanism underlying the Qi-invigorating effects remains unclear. This study aimed to elucidate the unique mechanisms of QIHs based on unique compounds, using a network pharmacology approach. QIHs and their compounds were identified using existing literature and the TCMSP database, respectively. Subsequently, a method was proposed to screen for unique compounds that are common in QIHs but rare in other traditional herbs. Unique compounds’ targets were predicted using the TCMSP, BATMAN-TCM, and SwissTargetPrediction databases. Finally, enriched GO and KEGG pathways were obtained using DAVID to uncover the biomolecular functions and mechanisms. Thirteen unique compounds, mainly including amino acids and vitamins that participate in energy metabolism and improve Qi deficiency syndrome, were identified among the eight QIHs. GO and KEGG pathway analyses revealed that these compounds commonly participate in neuroactive ligand–receptor interaction and the metabolism of amino acids, and are related to the components of mitochondria and neuronal cells. Our results appropriately reflect the characteristics of traditional Qi-invigorating effects; therefore, this study facilitates the scientific interpretation of Qi functions and provides evidence regarding the treatment effectiveness of QIHs.

The Performance of Different Water Models on the Structure and Function of Cytochrome P450 Enzymes.

Modeling approaches and modern simulations to investigate the biomolecular structure and function rely on various methods. Since water molecules play a crucial role in all sorts of chemistry, the accurate modeling of water molecules is vital for such simulations. In cytochrome P450 (CYP450), in particular, water molecules play a key role in forming active oxidant that ultimately performs oxidation and metabolism. In the present study, we have highlighted the behavior of the three most widely used water models─TIP3P, SPC/E, and OPC─for three different CYP450 enzymes─CYP450BM3, CYP450OleT, and CYP450BSβ─during MD simulations and QM/MM calculations. We studied the various properties, such as RMSD, RMSF, H-bond, water occupancy, and hydrogen atom transfer (HAT), using QM/MM calculations and compared them for all three water models. Our study shows that the stabilities of the enzyme complexes are well maintained in all three water models. However, the OPC water model performs well for the polar active sites, that is, in CYP450OleT and CYP450BSβ, while the TIP3P water model is superior for the hydrophobic site, such as CYP450BM3.

Urea-Bond Scission Induced by Therapeutic Ultrasound for Biofunctional Molecule Release.

Ultrasound-triggered remote control of biomolecular functions in cells provides unique advantages for us to interrogate nature. However, strategies to design therapeutic ultrasound-responsive functional molecules remain elusive, and rare ultrasound-cleavable chemical bonds have been developed to date. Herein, therapeutic ultrasound (1 MHz)-induced scission of urea bonds for drug release is demonstrated for the first time. Such a transformation has been verified to be initiated by hydroxyl radicals generated in the interior of cavitation bubbles, occurring specifically at the cavitation bubble-liquid interface. A series of urea-bond-containing prodrugs based on methylene blue (MB), namely MBUs, are designed. Upon sonication with low-intensity therapeutic ultrasound, the urea bonds linked with primary amines can be selectively cleaved, and free MB is released in a physiologically relevant environment, accompanied by recovered absorbance, fluorescence, and photosensitivity. Moreover, an FDA-approved alkylating agent (i.e., melphalan) bearing urea bond is also developed (MBU-Mel), successfully achieving ultrasound-triggered drug release in deep-seated cancer cells (mimic with 1 cm pigskin), showing the scalability of our ultrasound-responsive molecule platform in bioactive molecules release. This may set the starting point for therapeutic ultrasound-induced drug release, making a forward step in "sonopharmacology".

A Liquid Chromatography-Mass Spectrometry Method to Study the Interaction between Membrane Proteins and Low-Molecular-Weight Compound Mixtures.

Molecular interaction analysis is an essential technique for the study of biomolecular functions and the development of new drugs. Most current methods generally require manipulation to immobilize or label molecules, and require advance identification of at least one of the two molecules in the reaction. In this study, we succeeded in detecting the interaction of low-molecular-weight (LMW) compounds with a membrane protein mixture derived from cultured cells expressing target membrane proteins by using the size exclusion chromatography-mass spectrometry (SEC-MS) method under the condition of 0.001% lauryl maltose neopentyl glycol as detergent and atmospheric pressure chemical ionization. This method allowed us to analyze the interaction of a mixture of medicinal herbal ingredients with a mixture of membrane proteins to identify the two interacting ingredients. As it does not require specialized equipment (e.g., a two-dimensional liquid chromatography system), this SEC-MS method enables the analysis of interactions between LMW compounds and relatively high-expressed membrane proteins without immobilization or derivatization of the molecules.

419 Glucose controls protein-protein interactions and epidermal differentiation

Glucose serves as a universal energy currency in living organisms, however, its potential non-energetic biomolecular functions are less well defined. Glucose was among the most increased analytes among >14,000 assessed across epidermal differentiation, an elevation verified in tissue engineered with fluorescent glucose sensors. Free glucose accumulation, but not its increased metabolism, was essential for epidermal differentiation and required GLUT1, GLUT3, and SGLT1 transporters. Consistent with this, decreasing cellular glucose levels, by restricting available glucose or by increasing intracellular glucose catabolizing enzymes, HK1/2 and G6PD, blocked differentiation and differentiation was also rescued with a non-metabolizable glucose analog.

Multiscale simulation unravels the light-regulated reversible inhibition of dihydrofolate reductase by phototrexate.

Molecular photoswitches are widely used in photopharmacology, where the biomolecular functions are photo-controlled reversibly with high spatiotemporal precision. Despite the success of this field, it remains elusive how the protein environment modulates the photochemical properties of photoswitches. Understanding this fundamental question is critical for designing more effective light-regulated drugs with mitigated side effects. In our recent work, we employed first-principles non-adiabatic dynamics simulations to probe the effects of protein on the trans to cis photoisomerization of phototrexate (PTX), a photochromic analog of the anticancer therapeutic methotrexate that inhibits the target enzyme dihydrofolate reductase (DHFR). Building upon this study, in this work, we employ multiscale simulations to unravel the full photocycle underlying the light-regulated reversible inhibition of DHFR by PTX, which remains elusive until now. First-principles non-adiabatic dynamics simulations reveal that the cis to trans photoisomerization quantum yield is hindered in the protein due to backward isomerization on the ground-state following non-adiabatic transition, which arises from the favorable binding of the cis isomer with the protein. However, free energy simulations indicate that cis to trans photoisomerization significantly decreases the binding affinity of the PTX. Thus, the cis to trans photoisomerization most likely precedes the ligand unbinding from the protein. We propose the most probable photocycle of the PTX-DHFR system. Our comprehensive simulations highlight the trade-offs among the binding affinity, photoisomerization quantum yield, and the thermal stability of the ligand's different isomeric forms. As such, our work reveals new design principles of light-regulated drugs in photopharmacology.

Parallel Field-Effect Nanosensors Detect Trace Biomarkers Rapidly at Physiological High-Ionic-Strength Conditions.

Sensitivity and speed of detection are contradicting demands that profoundly impact the electrical sensing of molecular biomarkers. Although single-molecule sensitivity can now be achieved with single-nanotube field-effect transistors, these tiny sensors, with a diameter less than 1 nm, may take hours to days to capture the molecular target at trace concentrations. Here, we show that this sensitivity-speed challenge can be addressed using covalently functionalized double-wall CNTs that form many individualized, parallel pathways between two electrodes. Each carrier that travels across the electrodes is forced to take one of these pathways that are fully gated chemically by the target-probe binding events. This sensor design allows us to electrically detect Lyme disease oligonucleotide biomarkers directly at the physiological high-salt concentrations, simultaneously achieving both ultrahigh sensitivity (as low as 1 fM) and detection speed (<15 s). This unexpectedly simple strategy may open opportunities for sensor designs to broadly achieve instant detection of trace biomarkers and real-time probing of biomolecular functions directly at their physiological states.

Hydrophobic-cationic peptides modulate RNA polymerase ribozyme activity by accretion

Accretion and the resulting increase in local concentration is a widespread mechanism in biology to enhance biomolecular functions (for example, in liquid-liquid demixing phases). Such macromolecular aggregation phases (e.g., coacervates, amyloids) may also have played a role in the origin of life. Here, we report that a hydrophobic-cationic RNA binding peptide selected by phage display (P43: AKKVWIIMGGS) forms insoluble amyloid-containing aggregates, which reversibly accrete RNA on their surfaces in an RNA-length and Mg2+-concentration dependent manner. The aggregates formed by P43 or its sequence-simplified version (K2V6: KKVVVVVV) inhibited RNA polymerase ribozyme (RPR) activity at 25 mM MgCl2, while enhancing it significantly at 400 mM MgCl2. Our work shows that such hydrophobic-cationic peptide aggregates can reversibly concentrate RNA and enhance the RPR activity, and suggests that they could have aided the emergence and evolution of longer and functional RNAs in the fluctuating environments of the prebiotic earth.

Lung Biomolecular Profile and Function of Grafts from Donors after Cardiocirculatory Death with Prolonged Donor Warm Ischemia Time.

The acceptable duration of donor warm ischemia time (DWIT) after cardiocirculatory death (DCD) is still debated. We analyzed the biomolecular profile and function during ex vivo lung perfusion (EVLP) of DCD lungs and their correlation with lung transplantation (LuTx) outcomes. Donor data, procurement times, recipient outcomes, and graft function up to 1 year after LuTx were collected. During EVLP, the parameters of graft function and metabolism, perfusate samples to quantify inflammation, glycocalyx breakdown products, coagulation, and endothelial activation markers were obtained. Data were compared to a cohort of extended-criteria donors after brain death (EC-DBD). Eight DBD and seven DCD grafts transplanted after EVLP were analyzed. DCD’s DWIT was 201 [188;247] minutes. Donors differed only regarding the duration of mechanical ventilation that was longer in the EC-DBD group. No difference was observed in lung graft function during EVLP. At reperfusion, “wash-out” of inflammatory cells and microthrombi was predominant in DCD grafts. Perfusate biomolecular profile demonstrated marked endothelial activation, characterized by the presence of inflammatory mediators and glycocalyx breakdown products both in DCD and EC-DBD grafts. Early graft function after LuTx was similar between DCD and EC-DBD. DCD lungs exposed to prolonged DWIT represent a potential resource for donation if properly preserved and evaluated.

Fluctuation in the Sliding Movement of Kinesin-Driven Microtubules Is Regulated Using the Deep-Sea Osmolyte Trimethylamine N-Oxide.

Nowadays, biomolecular motor-based miniaturized lab-on-a-chip devices have been attracting much attention for their wide range of nanotechnological applications. Most of the applications are dependent on the motor-driven active transportation of their associated filamentous proteins as shuttles. Fluctuation in the movement of the shuttles is a major contributor to the dispersion in motor-driven active transportation, which limits the efficiency of the miniaturized devices. In this work, by employing the biomolecular motor kinesin and its associated protein filament microtubule as a model active transport system, we demonstrate that the deep-sea osmolyte trimethylamine N-oxide (TMAO) is useful in regulating the fluctuation in the motility of microtubule shuttles. We show that the motional diffusion coefficient, a measure of the fluctuation in the movement of the kinesin-propelled microtubules, gradually decreases upon increasing the concentration of TMAO in the transportation system. We have been able to reduce the motional diffusion coefficient of microtubules more than 200 times by employing TMAO at a concentration of 2 M. We also show that upon elimination of TMAO, the motional diffusion coefficient of microtubules can be restored, which confirms that TMAO can be used as a tool to reversibly regulate the fluctuation in the sliding movement of kinesin-propelled microtubule shuttles. Such reversible regulation of the dynamic behavior of the shuttles does not require sacrificing the concentration of fuel used for transportation. Our results confirm the ability to manipulate the nanoscale motion of biomolecular motor-driven active transporters in an artificial environment. This work is expected to further enhance the tunability of biomolecular motor functions, which, in turn, will foster their nanotechnological applications based on active transportation.

Current Advances in Aptamer-based Biomolecular Recognition and Biological Process Regulation.

The interaction between biomolecules with their target ligands plays a great role in regulating biological functions. Aptamers are short oligonucleotide sequences that can specifically recognize target biomolecules via structural complementarity and thus regulate related biological functions. In the past ten years, aptamers have made great progress in target biomolecule recognition, becoming a powerful tool to regulate biological functions. At present, there are many reviews on aptamers applied in biomolecular recognition, but few reviews pay attention to aptamer-based regulation of biological functions. Here, we summarize the approaches to enhancing aptamer affinity and the advancements of aptamers in regulating enzymatic activity, cellular immunity and cellular behaviors. Furthermore, this review discusses the challenges and future perspectives of aptamers in target recognition and biological functions regulation, aiming to provide some promising ideas for future regulation of biomolecular functions in a complex biological environment.

Development of Photoresponsive Probes to Investigate Cellular Functions

Technologies for the optical control of biomolecular functions have recently attracted considerable attention because they can be combined with advanced laser and microscopic techniques for diverse applications at the cellular and intravital levels. In this account, I review the summary of optical control technologies for biomolecular functions based on organic chemistry or protein science, and then introduce our recent studies on the development of small molecule-based photoregulation techniques. The first is the development of a photoactivatable protein labeling method based on a caged ligand. This method was applied to the photocontrol of intracellular protein dimerization and localization. The second is the development of a reversibly photoswitchable enzyme inhibitor, which was designed from the conformation of the inhibitor in the crystal structure of the enzyme-inhibitor complex. Based on our research strategies and results, I have also outlined the respective advantages and disadvantages of these two technologies: caged compounds and photoswitchable compounds.

PDB‐101: Molecular Explorations through Biology and Medicine

PDB‐101 is an online portal for teachers, students, and the general public to promote exploration of the structural biology of proteins and nucleic acids (pdb101.rcsb.org). Learning about the diverse shapes and functions of these biological macromolecules helps to understand all aspects of biomedicine and agriculture, from protein synthesis to health and disease to biological energy.Why PDB‐101? Researchers around the world are studying these molecules at the atomic level. These 3D structures are freely available at the Protein Data Bank (PDB), the central storehouse of biomolecular structures. This website builds introductory materials to help beginners get started in the basics of biomolecular structure and function (“101”, as in an entry level course) as well as resources for extended learning.Since 2011, PDB‐101 has been developed by the RCSB PDB, a global resource for the advancement of research and education in biology and medicine. Along with our Worldwide PDB collaborators, RCSB PDB curates, annotates, and makes publicly available the PDB data deposited by scientists around the globe. The RCSB PDB then provides a window to these data through a rich online resource with powerful searching, reporting, and visualization tools for researchers. This information is then streamlined for students and teachers at PDB‐101.Features include the ongoing Molecule of the Month series, educational materials such as paper models, posters, molecular animations, educational curricula and more. The section “Guide to Understanding PDB Data” is a primer for detailed PDB‐specific information: PDB Data, Visualizing Structures, Reading Coordinate Files, scientific methods for structure determination, and more.PDB‐101 also runs annual Video Challenges for high school students. Participants create short videos that tell molecular stories that connect structural biology and medicine. Previous topics have included HIV/AIDS, diabetes, and antimicrobial resistance. The 2022 challenge will focus on Molecular Mechanisms of Cancer.PDB‐101 activities are evaluated using user surveys, feedback from in‐person activities, and website analytics. In 2020, PDB‐101 hosted &gt;850,000 users and &gt;2.6 million page views.

The role of externally-modulated electrostatic interactions in amplifying charge transport across lysine-doped peptide junctions

Many evolved biomolecular functions such as ion pumping or redox catalysis rely on controlled charge transport through the polypeptide matrix, which can be regulated by shifts in molecular protonation states and dependent supramolecular packing modes in response to environmental cues. However, the exact roles of such dynamic, non-covalent interactions in peptide charge transport have remained elusive. To tackle this challenge, here we report the modulation of charge transport in a series of lysine (Lys)-substituted hepta-glycine (Gly) peptide self-assembled monolayers (SAMs) on template-striped gold (AuTS) bottom electrodes with eutectic gallium-indium (EGaIn) liquid metal top electrodes. We demonstrate systematic modulation of hydrogen bonding and more general electrostatic interactions by shifting the position of the charged Lys-residue and creating different protonation patterns by changing the environmental pH in the AuTS/peptide//GaOx/EGaIn junctions. The effective modulation is evidenced by current density–voltage (J-V) measurements combined with SAM characterization using ultraviolet photoelectron spectroscopy (UPS) and angle-resolved X-ray photoelectron spectroscopy (ARXPS), polarization modulation–infrared reflection-absorption spectroscopy (PM-IRRAS), and molecular dynamics (MD) simulations. Decreasing the hydrogen bonding inside the peptide SAMs and increasing the electrostatic interactions by environmental counterions amplifies the charge transport differently with Lys-position, which means that the sensitive electrical response of peptide SAMs can be tuned by the peptide sequence. Our results provide insights into the relationship between molecular design and in situ modulation of charge transport properties for the development of bionanoelectronics.

Aggregation Reduces Subcellular Localization and Cytotoxicity of Single-Walled Carbon Nanotubes.

The non-covalent biomolecular functionalization of fluorescent single-walled carbon nanotubes (SWCNTs) has resulted in numerous in vitro and in vivo sensing and imaging applications due to many desirable optical properties. In these applications, it is generally presumed that pristine, singly dispersed SWCNTs interact with and enter live cells at the so-called nano-biointerface, for example, the cell membrane. Despite numerous fundamental studies published on this presumption, it is known that nanomaterials have the propensity to aggregate in protein-containing environments before ever contacting the nano-biointerface. Here, using DNA-functionalized SWCNTs with defined degrees of aggregation as well as near-infrared hyperspectral microscopy and toxicological assays, we show that despite equal rates of internalization, initially aggregated SWCNTs do not further accumulate within individual subcellular locations. In addition to subcellular accumulations, SWCNTs initially with a low degree of aggregation can induce significant deleterious effects in various long-term cytotoxicity and real-time proliferation assays, which are markedly different when compared to those of SWCNTs that are initially aggregated. These findings suggest the importance of the aggregation state as a critical component related to intracellular processing and toxicological response of engineered nanomaterials.

Biomolecular Surface Functionalization and Stabilization Method to Fabricate Quantum Dots Nanobeads for Accurate Biosensing Detection.

The surface functionalization of quantum dots (QDs) is essential for their application as a label material in a biological field. Here, a protein surface functionalization approach was introduced to combine with silica encapsulation for the sustainable and stable synthesis of QDs nanobeads for biomarker detection. The formation of QDs nanobeads was achieved by multiple mercapto groups in bovine serum albumin (BSA) macromolecules as multidentate ligands to replace hydrophobic ligands on the surface of QDs and decompression. The resulting QDs nanobeads exhibited 20 times more photoluminescence than the corresponding hydrophobic QDs and presented excellent stability under physiological conditions due to the protection of BSA and silica. The nanobeads served as a robust signal-generating reagent to construct the lateral flow immunoassay (LFIA) biosensor for the detection of glycosylated hemoglobin (HbA1c). The concentration of HbA1c was determined within 10 min with high specificity using only 60 μL of whole blood samples collected clinically. The nanobeads-based LFIA biosensor exhibited linear detection of HbA1c from 4.2% to 13.6%. The accuracy and stability of this approach in clinical utility was demonstrated by the detection of HbA1c after a long-term storage of test strips. This protein surface modification technology provides a new way for improving the biological properties of QDs in clinical diagnosis.