Native State Research Articles

Inhibition of primary and secondary nucleation alongwith disruption of amyloid fibrils and alleviation of associated cytotoxicity: A biophysical insight of a novel property of Chlorpropamide (an anti-diabetic drug)

Aggregation of physiologically synthesized soluble proteins to insoluble, cytotoxic fibrils is a pre-requisite for pathogenesis of amyloid associated disorders including Alzheimer's disease, non-systemic amyloidosis, Parkinson's disease, etc. Considerable advancement has been made to understand the mechanism behind aggregation process but till date we have no efficient cure and preventive therapy for associated diseases. Strategies to prevent protein aggregation are nevertheless many which have been proved promisingly successful in vitro. One of those is repurposing already approved drugs that saves time and money too and has been employed in this study. Here, for the first time we are reporting the effectiveness of an anti-diabetic drug chlorpropamide (CHL) under dosage conditions, a novel property to inhibit aggregation in human lysozyme (HL) in vitro. Spectroscopic (Turbidity, RLS, ThT, DLS, ANS) and microscopic (CLSM) results demonstrates that CHL has the potency to suppress aggregation in HL up to 70 %. CHL is shown to affect the elongation of fibrils with IC50 value of 88.5 μM as clear from the kinetics results, may be by interacting near/with aggregation prone regions of HL. Hemolytic assay also revealed the reduced cytotoxicity in the presence of CHL. Disruption of amyloid fibrils and inhibition of secondary nucleation in the presence of CHL was also evidenced by ThT, CD and CLSM results with reduced cytotoxicity as confirmed by hemolytic assay. We also performed preliminary studies on α-synuclein fibrillation inhibition and surprisingly found that CHL is not just inhibiting the fibrillation but also stabilizing the protein in its native state. These findings insinuate that CHL (anti-diabetic) possess multiple roles and can be a promising drug for developing therapeutic against non-systemic amyloidosis, Parkinson's disease and other amyloid associated disorders.

Understanding Lithium-Ion Dynamics in Single-Ion and Salt-in-Polymer Perfluoropolyethers and Polyethyleneglycol Electrolytes Using Solid-State NMR

Lithium metal batteries with high energy densities can enable a revolution in energy storage and accelerate shifts in electric transportation and electricity generation. However, several morphological and electro-chemo-mechanical challenges impede their development. Solid-state electrolytes such as those based on polymers show great promise in replacing liquid electrolytes in lithium metal batteries. Polyether-based polymer electrolytes are the most investigated but are plagued by low room-temperature ionic conductivity and poor oxidative stability. Hence, there is great need for the development and understanding of ion transport in new classes of polymer electrolytes. Perfluoropolyether (PFPE)-based electrolytes have shown improved oxidative stability, but little is understood about their lithium solvation and transport mechanism. In this work, we use multinuclear solid-state magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy to investigate the lithium cation environment and mobility in crosslinked single-ion and salt-in-polymer PFPE electrolytes and compare it directly to that of well-known polyethylene glycol (PEG) electrolytes. We show that the interaction of the lithium cation with the polymer backbone is weaker in PFPE systems compared to PEG, likely resulting in stronger ion pairing in the PFPE systems. Line shape analyses show lower lithium mobility in PFPE electrolytes despite lower activation energies being derived from spin-lattice relaxation (T1) measurements as compared to those for the PEG systems. The rapid relaxation is instead ascribed to the local fluctuations caused by polymer backbone mobility. By studying different modes of ion binding (single-ion vs salt-in-polymer), we show that differences across polymer backbones (PFPE vs PEG) have a greater effect on mobility than differences in ion binding modes within each polymer class (especially when single-ion conducting site density is not high). Our ability to use MAS NMR to study polymer electrolytes in their native state opens new opportunities to develop and understand novel polymer or hybrid solid-state electrolytes for next-generation lithium metal batteries.

Characterization of Monoamine Oxidase-A in tropical liver fluke, Fasciola gigantica.

Fasciola gigantica, responsible for the zoonotic disease fasciolosis, pose a great threat to the livestock and human health worldwide. The triclabendazole (TCBZ) has been used for decades as a broad spectrum anthelmintic to control this perilous disease but the emergence of resistance in flukes against TCBZ has prompted researchers across the world to explore for new drugs and antigenic targets. World Health Organization has strongly recommended the utilization of neurobiologically significant biomolecules as new drug/antigenic targets because of their significant role in the physiology of parasites. Monoamine Oxidase (MAO) is an important neurobiological enzyme which catabolizes aminergic neurotransmitters thus preventing prolonged excitation of neurons and in non-neuronal cells it prevents cellular toxicity due to accumulation of toxic monoamines. Owing to the important role of MAO in the survival and perpetuation of parasites, multipronged approaches were undertaken for the characterization of MAO-A in F. gigantica. The activity of MAO was found to be 1.5 times higher in the mitochondrial samples than the whole homogenate samples. The adult worms of the F. gigantica appeared to possess both the isoforms of MAO i.e., MAO-A and MAO-B. The zymographic studies revealed strong enzyme activity in its native state as assessed through prominent dark bands at 250KDa in the zymogram. The enzyme was also found to be highly immunogenic as revealed by high antibody titer at 1:6400 dilution. The immunogenicity of MAO-A enzyme was further established in the Western Blots in which a strong band of 50KDa was distinctly evident. Despite ubiquitous presence of MAO in F. gigantica some regions like tegumental surface and intestinal caecae displayed strong immunofluorescence as compared to other regions. The detection of MAO-A in the F. gigantica samples in Dot-Blot assay indicate a great potential of this molecule for the immunodiagnostics of fasciolosis, particularly in the field conditions. The enzyme activity was sensitive to the specific inhibitor clorgyline in a concentration dependant manner, particularly in the late incubation period. The zymographic results also exhibited similar trend. The strong intensity of spots in Dot-blots indicate high immunogenicity of the MAO protein. The intensity of bands/spots in the samples of worms treated with clorgyline also declined, clearly indicating that the tropical liver fluke possesses prominent MAO-A activity.

Applications of ambient ionization mass spectrometry in 2022: An annual review

AbstractThe development of ambient ionization mass spectrometry (AIMS) has transformed analytical science, providing the means of performing rapid analysis of samples in their native state, both in and out of the laboratory. The capacity to eliminate sample preparation and pre‐MS separation techniques, leading to true real‐time analysis, has led to AIMS naturally gaining a broad interest across the scientific community. Since the introduction of the first AIMS techniques in the mid‐2000s, the field has exploded with dozens of novel ion sources, an array of intriguing applications, and an evident growing interest across diverse areas of study. As the field continues to surge forward each year, ambient ionization techniques are increasingly becoming commonplace in laboratories around the world. This annual review provides an overview of AIMS techniques and applications throughout 2022, with a specific focus on some of the major fields of research, including forensic science, disease diagnostics, pharmaceuticals and food sciences. New techniques and methods are introduced, demonstrating the unwavering drive of the analytical community to further advance this exciting field and push the boundaries of what analytical chemistry can achieve.

Recent Advances in NMR Protein Structure Prediction with ROSETTA.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for studying the structure and dynamics of proteins in their native state. For high-resolution NMR structure determination, the collection of a rich restraint dataset is necessary. This can be difficult to achieve for proteins with high molecular weight or a complex architecture. Computational modeling techniques can complement sparse NMR datasets (<1 restraint per residue) with additional structural information to elucidate protein structures in these difficult cases. The Rosetta software for protein structure modeling and design is used by structural biologists for structure determination tasks in which limited experimental data is available. This review gives an overview of the computational protocols available in the Rosetta framework for modeling protein structures from NMR data. We explain the computational algorithms used for the integration of different NMR data types in Rosetta. We also highlight new developments, including modeling tools for data from paramagnetic NMR and hydrogen-deuterium exchange, as well as chemical shifts in CS-Rosetta. Furthermore, strategies are discussed to complement and improve structure predictions made by the current state-of-the-art AlphaFold2 program using NMR-guided Rosetta modeling.

Peptide Bond of Aqueous Dipeptides Is Resilient to Deep Ultraviolet Irradiation

The susceptibility of aqueous dipeptides to photodissociation by deep ultraviolet irradiation is studied by femtosecond spectroscopy supported by density functional theory calculations. The primary photodynamics of the aqueous dipeptides of glycyl-glycine (gly-gly), alalyl-alanine (ala-ala), and glycyl-alanine (gly-ala) show that upon photoexcitation at a wavelength of 200 nm, about 10% of the excited dipeptides dissociate by decarboxylation within 100 ps, while the rest of the dipeptides return to their native ground state. Accordingly, the vast majority of the excited dipeptides withstand the deep ultraviolet excitation. In those relatively few cases, where excitation leads to dissociation, the measurements show that deep ultraviolet irradiation breaks the Cα-C bond rather than the peptide bond. The peptide bond is thereby left intact, and the decarboxylated dipeptide moiety is open to subsequent reactions. The experiments indicate that the low photodissociation yield and in particular the resilience of the peptide bond to dissociation are due to rapid internal conversion from the excited state to the ground state, followed by efficient vibrational relaxation facilitated by intramolecular coupling among the carbonate and amide modes. Thus, the entire process of internal conversion and vibrational relaxation to thermal equilibrium on the dipeptide ground state occurs on a time scale of less than 2 ps.

EVALUACIÓN DE LOS PARÁMETROS ZOOTÉCNICOS PARA ALEVINES DE Brycon cephalus SÁBALO COLA ROJA, EN LA ZONA COSTERA DEL SUR DEL PERÚ

In order to evaluate the zootechnical parameters for red-tailed tarpon Brycon cephalus fingerlings, in a region other than Amazonia, the evaluation of growth parameters in temperate waters was carried out for 60 days in the port of Ilo, Department of Moquegua. 84 fingerlings were used in 70 liters of water and two biometrics were performed, fluctuating in size from 3.5 cm to 9 cm at the end of the trial. Fingerlings were fed commercial trout feed of 45% protein, 8% fat and 25% carbohydrate, a feeding rate of 4.5% of biomass, distributed 3 times per day. The results reported as an average water temperature of 20.6°C, survival rate of 2.4%, growth determined by y=0.0155 X 2.8284 and a coefficient of determination of R²=0.975, average condition factor (K): 0.04393±0.0126 SD, Relative Growth Rate (TCR) of 9.5%, Specific Growth Rate (TCE) of 3.9% and Apparent Feed Conversion (CAA) of 1.4. With which it is observed that the parameters evaluated in the coastal zone of southern Peru are close to the native cultivation conditions.

Chiral and Structural Polymorphism of Fibril Architectures of Homologous Lysozymes.

Amyloid fibrils are fascinating and complex structures with the multilayered chiral organization. Using the multimodal methodology, including VCD, ECD, cryo-EM, and TEM, we characterized in detail different levels of organization (secondary structure/protofilament/mesoscopic structure) of amyloid fibrils prepared from proteins highly homologous in the structure (hen egg white and human lysozymes). Our results demonstrate that small changes in the native protein structure or preparation conditions translate into significant differences in the handedness and architecture of the formed fibrils at various levels of their complexity. In particular, fibrils of hen egg white and human lysozymes obtained in vitro at the same preparation conditions, possess different secondary structure, protofilament twist and ultrastructure. Yet, formed fibrils adopted a relatively similar mesoscopic structure, as observed in high-resolution 3D cryo-EM, scarcely used up to now for fibrils obtained in vitro in denaturing condition. Our results add to other puzzling experiments implicating the indeterministic nature of fibril formation.

DOES KINEMATICALLY ALIGNED TOTAL KNEE ARTHROPLASTY RESTORE NATIVE JOINT LAXITY BETTER THAN MECHANICAL ALIGNMENT? A CADAVERIC STUDY

Mechanical alignment (MA) in total knee arthroplasty (TKA), although considered the gold standard, reportedly has up to 25% of patients expressing post-operative dissatisfaction. Biomechanical outcomes following kinematic alignment (KA) in TKA, developed to restore native joint alignment, remain unclear. Without a clear consensus for the optimal alignment strategy during TKA, the purpose of this study was to conduct a paired biomechanical comparison of MA and KA in TKA by experimentally quantifying joint laxity and medial collateral ligament (MCL) strain.14 bilateral native fresh-frozen cadaveric lower limbs underwent medially-stabilised TKA (GMK Sphere, Medacta, Switzerland) using computed CT-based subject-specific guides, with KA and MA performed on left and right legs, respectively. Each specimen was subjected to sensor-controlled mediolateral laxity tests. A handheld force sensor (Mark-10, USA) was used to generate an abduction-adduction moment of 10Nm at the knee at fixed flexion angles (0°, 30°, 60°, 90°). A digital image correlation system was used to compute the strain on the superficial medial collateral ligament. A six-camera optical motion capture system (Vicon MX+, UK) was used to acquire kinematics using a pre-defined CT-based anatomical coordinate system. A linear mixed model and Tukey's posthoc test were performed to compare native, KA and MA conditions (p&lt;0.05).Unlike MA, medial joint laxity in KA was similar to the native condition; however, no significant difference was found at any flexion angle (p&gt;0.08). Likewise, KA was comparable with the native condition for lateral joint laxity, except at 30°, and no statistical difference was observed. Although joint laxity in MA seemed lower than the native condition, this difference was significant only for 30° flexion (p=0.01). Both KA and MA exhibited smaller MCL strain at 0° and 30°; however, all conditions were similar at 60° and 90°.Medial and lateral joint laxity seemed to have been restored better following KA than MA; however, KA did not outperform MA in MCL strain, especially after mid-flexion. Although this study provides only preliminary indications regarding the optimal alignment strategy to restore native kinematics following TKA, further research in postoperative joint biomechanics for load bearing conditions is warranted.

Enhanced stability of a disaggregated Aβ fibril on removal of ligand inhibits refibrillation: An all atom Molecular Dynamics simulation study

The extraneuronally deposited senile plaques, composed of neurotoxic aggregates of Aβ fibril, define Alzheimer's disease (AD). Natural compounds have been tested for their destabilization potential on Aβ fibril, thereby curing AD. However, the resultant destabilized Aβ fibril, needs to be checked for its irreversibility to the native organized state after removal of the ligand. Herein, we assessed the stability of a destabilized fibril after the ligand (ellagic acid represented as REF) is removed from the complex. The study has been conducted via Molecular Dynamics (MD) simulation of 1 μs for both Aβ-Water (control) and Aβ-REF″ (test or REF removed) system. The increased value of RMSD, Rg, SASA, lower β-sheet content and reduced number of H-bonds explains enhanced destabilization observed in Aβ-REF″ system. The increased inter-chain distance demonstrates breaking of the residual contacts, testifying the drift of terminal chains from the pentamer. The increased SASA along with the ∆Gps(polar solvation energy) accounts for the reduced interaction amongst residues, and more with solvent molecules, governing irreversibility to native state. The higher Gibb's free energy of the misaligned structure of Aβ-REF″ ensures irreversibility to the organized structure due to its inability to cross such high energy barrier. The observed stability of the disaggregated structure, despite ligand elimination, establishes the effectiveness of the destabilization technique as a promising therapeutic approach towards treating AD.

How a highly acidic SH3 domain folds in the absence of its charged peptide target.

Charged residues on the surface of proteins are critical for both protein stability and interactions. However, many proteins contain binding regions with a high net charge that may destabilize the protein but are useful for binding to oppositely charged targets. We hypothesized that these domains would be marginally stable, as electrostatic repulsion would compete with favorable hydrophobic collapse during folding. Furthermore, by increasing the salt concentration, we predict that these protein folds would be stabilized by mimicking some of the favorable electrostatic interactions that take place during target binding. We varied the salt and urea concentrations to probe the contributions of electrostatic and hydrophobic interactions for the folding of the yeast SH3 domain found in Abp1p. The SH3 domain was significantly stabilized with increased salt concentrations due to Debye-Huckel screening and a nonspecific territorial ion-binding effect. Molecular dynamics and NMR show that sodium ions interact with all 15 acidic residues but do little to change backbone dynamics or overall structure. Folding kinetics experiments show that the addition of urea or salt primarily affects the folding rate, indicating that almost all the hydrophobic collapse and electrostatic repulsion occur in the transition state. After the transition state formation, modest yet favorable short-range salt bridges are formed along with hydrogen bonds, as the native state fully folds. Thus, hydrophobic collapse offsets electrostatic repulsion to ensure this highly charged binding domain can still fold and be ready to bind to its charged peptide targets, a property that is likely evolutionarily conserved over 1 billion years.

Knee kinematics during staircase descent

The goal was to evaluate tibiofemoral knee joint kinematics during stair descent, by simulating the full stair descent motion in vitro. The knee joint kinematics were evaluated for two types of knee implants: bi-cruciate retaining and bi-cruciate stabilized. It was hypothesized that the bi-cruciate retaining implant better approximates native kinematics. The in vitro study included 20 specimens which were tested during a full stair descent with physiological muscle forces in a dynamic knee rig. Laxity envelopes were measured by applying external loading conditions in varus/valgus and internal/external direction. The laxity results show that both implants are capable of mimicking the native internal/external-laxity during the controlled lowering phase. The kinematic results show that the bi-cruciate retaining implant tends to approximate the native condition better compared to bi-cruciate stabilized implant. This is valid for the internal/external rotation and the anteroposterior translation during all phases of the stair descent, and for the compression-distraction of the knee joint during swing and controlled lowering phase. The results show a better approximation of the native kinematics by the bi-cruciate retaining knee implant compared to the bi-cruciate stabilized knee implant for internal/external rotation and anteroposterior translation. Whether this will result in better patient outcomes remains to be investigated.

Possibilities of Studying Biological Objects on a Pulsed Reactor

Small-angle scattering makes it possible to solve structural biology problems without specific sample preparation, which is typical for methods such as X-ray diffraction of protein crystals or cryo-electron microscopy of proteins. In our review, it is shown how to use small-angle scattering to address biological problems. The use of small-angle scattering is suggested for applications as a tool to control the quality of the assembly of proteins and protein complexes and to test the identity of the structural organization of biological objects in the native state and in prepared samples before measurements by X-ray diffraction or cryo-electron microscopy. This work demonstrates the possibilities of the small-angle neutron scattering spectrometer YuMO based on the IBR-2 pulsed reactor (Laboratory of neutron physics, Joint Institute for Nuclear Research, Dubna, Russia) to solve a whole array of problems, with an eye toward applying these in biophysics, structural biology, and biotechnology. This review presents and discusses the main findings of the studies of various biological systems obtained by using the setup small-angle scattering of neutrons YuMO. The possibilities of development of structural biology methods with the help of small-angle scattering, including protein crystallization, are shown.

Metabolomic profiling and antianginal activity of the bark of Sterculia setigera from Mali

The present work focuses on the phytochemical characterization and evaluation of antianginal activity of the bark of Sterculia setigera. It was collected and authenticated in the African region of Mali, where the local population largely employs this plant for the treatment of several diseases.In the context of traditional or folk medicine and recent progresses in alternative medicine practices, it is essential to expand the knowledge about the chemical composition of such medicinal plants. In this research, a direct-Mass Spectrometry (MS) technique, known as Rapid Evaporative Ionization Mass Spectrometry (REIMS) was used for the identification of the main constituents of the Sterculia setigera bark. The REIMS source is here coupled with an electroknife as sampling device, so that the dried and pulverized bark was directly cut through the electroknife to generate a vapor, which was online transferred to the source via a Venture tube. In this way, an ambient MS approach was realized, which avoids any sample preparation procedure or pretreatment; the sample was analyzed in its native state according to a time-saving analytical process. A quadrupole-time of flight MS/MS analyzer was exploited for the identification process, based on mass accuracy data and MS/MS experiments for structure elucidation purposes. Lipids, including triterpenes, fatty acids, γ-sitosterol and α-tocopherol, and phenolic compounds were identified, some of them reported for the first time in a plant of the Sterculia genus and further confirmed through a gas chromatography-mass spectrometry analysis. The obtained metabolomic profile was successfully correlated to the antianginal activity of this plant.

Cellular Proteomic Profiling Using Proximity Labeling by TurboID-NES in Microglial and Neuronal Cell Lines

Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, invivo proteomic labeling with proximity-dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without the need for cellular isolation. To complement invivo proximity labeling approaches, invitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole-cell proteome and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID labeled endolysosome, translation, vesicle, and signaling proteins in BV2 microglia and synaptic, neuron projection, and microtubule proteins in N2A neurons. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2Acells and did not affect lipopolysaccharide-mediated cytokine production or resting cellular respiration in BV2 cells. MS analysis of the microglial biotin-labeled proteins captured the impact of lipopolysaccharide treatment (>500 differentially abundant proteins) including increased canonical proinflammatory proteins (Il1a, Irg1, and Oasl1) and decreased anti-inflammatory proteins (Arg1 and Mgl2).

Novel differential calcium regulation of hematopoietic stem and progenitor cells under physiological low oxygen conditions.

Low oxygen bone marrow (BM) niches (~1%-4% low O2 ) provide critical signals for hematopoietic stem/progenitor cells (HSC/HSPCs). Our presented data are the first to investigate live, sorted HSC/HSPCs in their native low O2 conditions. Transcriptional and proteomic analysis uncovered differential Ca2+ regulation that correlated with overlapping phenotypic populations consisting of robust increases of cytosolic and mitochondrial Ca2+ , ABC transporter (ABCG2) expression and sodium/hydrogen exchanger (NHE1) expression in live, HSC/HSPCs remaining in constant low O2. We identified a novel Ca2+ high population in HSPCs predominantly detected in low O2 that displayed enhanced frequency of phenotypic LSK/LSKCD150 in low O2 replating assays compared to Ca2+ low populations. Inhibition of the Ca2+ regulator NHE1 (Cariporide) resulted in attenuation of both the low O2 induced Ca2+ high population and subsequent enhanced maintenance of phenotypic LSK and LSKCD150 during low O2 replating. These data reveal multiple levels of differential Ca2+ regulation in low O2 resulting in phenotypic, signaling, and functional consequences in HSC/HSPCs.

Thermodynamic modulation of folding and aggregation energy landscape by DNA binding of functional domains of TDP-43

TDP-43 is a vital nucleic acid binding protein which forms stress-induced aberrant aggregates in around 97% cases of ALS, a fatal neurodegenerative disease. The functional tandem RRM domain of the protein (TDP-43tRRM) has been shown to undergo amyloid-like aggregation under stress in a pH-dependent fashion. However, the underlying thermodynamic and molecular basis of aggregation and how the energy landscape of folding, stability, and aggregation are coupled and modulated by nucleic acid binding is poorly understood. Here, we show that the pH stress thermodynamically destabilizes the native protein and systematically populates the unfolded-like aggregation-prone molecules which leads to amyloid-like aggregation. We observed that specific DNA binding inhibits aggregation and populates native-like compact monomeric state even under low-pH stress as measured by circular dichroism, ANS binding, size exclusion chromatography, and transmission electron microscopy. We show that DNA-binding thermodynamically stabilizes and populates the native state even under stress and reduces the population of unfolded-like aggregation-prone molecules which leads to systematic aggregation inhibition. Our results suggest that thermodynamic modulation of the folding and aggregation energy landscape by nucleic-acid-like molecules could be a promising approach for effective therapeutic intervention in TDP-43-associated proteinopathies.

Bioactivated and PEG-Protected Circa 2nm Gold Nanoparticles for in Cell Labelling and Cryo-Electron Microscopy.

Advances in cryo-electron microscopy (EM) enable imaging of protein assemblies within mammalian cells in a near native state when samples are preserved by cryogenic vitrification. To accompany this progress, specialized EM labelling protocols must be developed. Gold nanoparticles (AuNPs) of 2nm are synthesized and functionalized to bind selected intracellular targets inside living human cells and to be detected in vitreous sections. As a proof of concept, thioaminobenzoate-, thionitrobenzoate-coordinated gold nanoparticles are functionalized on their surface with SV40 Nuclear Localization Signal (NLS)-containing peptides and 2kDa polyethyleneglycols (PEG) by thiolate exchange to target the importin-mediated nuclear machinery and facilitate cytosolic diffusion by shielding the AuNP surface from non-specific binding to cell components, respectively. After delivery by electroporation into the cytoplasm of living human cells, the PEG-coated AuNPs diffuse freely in the cytoplasm but do not enter the nucleus. Incorporation of NLS within the PEG coverage promotes a quick nuclear import of the nanoparticles in relation to the density of NLS onto the AuNPs. Cryo-EM of vitreous cell sections demonstrate the presence of 2nm AuNPs as single entities in the nucleus. Biofunctionalized AuNPs combined with live-cell electroporation procedures are thus potent labeling tools for the identification of macromolecules in cellular cryo-EM.

Recent advances in the application of atomic force microscopy to structural biology

The application of atomic force microscopy (AFM) for functional imaging and manipulating biomolecules at all levels of organization has enabled great progress in the structural biology field over the last decades, contributing to the discovery of novel structural entities of biological significance across many disciplines ranging from biochemistry, biomedicine and biophysics to molecular and cell biology, up to food systems and beyond. AFM has the capability to generate high-resolution topographic images spanning from the submolecular to the (sub)cellular range and can probe biochemical and biophysical sample properties in close to native conditions with excellent temporal resolution. Instrumental developments in the past decade enable dynamical structural and conformational studies of single biomolecules and new techniques for structural and chemical modification of the AFM probe have converted the cantilever into a versatile tool to study different biological phenomena, such as the mechanical stability of biomolecular complexes or the force induced dynamic changes of mechanically stressed proteins at the nanoscopic level. To improve the functionality of AFM and approach dynamic processes of complex biological systems ex vivo, AFM is combined with complementary microscopy, nanoscopy and spectroscopy tools. These multimethodological approaches provide unprecedented possibilities of probing physical, chemical and biological properties of complex cellular systems with high spatio-temporal resolution, leading to novel applications that correlate structural results with functional biochemical, biophysical, immunological, or genetic data of the system under study.

Shieldin complex assembly kinetics and DNA binding by SHLD3

The Shieldin complex represses end resection at DNA double-strand breaks (DSBs) and thereby serves as a pro-non homologous end joining (NHEJ) factor. The molecular details of the assembly of Shieldin and its recruitment to DSBs are unclear. Shieldin contains two REV7 molecules, which have the rare ability to slowly switch between multiple distinct native states and thereby could dynamically control the assembly of Shieldin. Here, we report the identification of a promiscuous DNA binding domain in SHLD3. At the N-terminus, SHLD3 interacts with a dimer of REV7 molecules. We show that the interaction between SHLD3 and the first REV7 is remarkably slow, while in contrast the interaction between SHLD3 and SHLD2 with a second REV7 molecule is fast and does not require structural remodeling. Overall, these results provide insights into the rate-limiting step of the molecular assembly of the Shieldin complex and its recruitment at DNA DSBs.