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Localized surface plasmon resonance (LSPR) detection offers highly sensitive label-free detection of biomolecular interactions. Simple and robust surface architectures compatible with real-time detection in a flow-through system are required for broad application in quantitative interaction analysis. Here, we established self-assembly of a functionalized gold nanoparticle (AuNP) monolayer on a glass substrate for stable, yet reversible immobilization of Histidine-tagged proteins. To this end, one-step coating of glass substrates with poly-L-lysine graft poly(ethylene glycol) functionalized with ortho-pyridyl disulfide (PLL-PEG-OPSS) was employed as a reactive, yet biocompatible monolayer to self-assemble AuNP into a LSPR active monolayer. Site-specific, reversible immobilization of His-tagged proteins was accomplished by coating the AuNP monolayer with tris-nitrilotriacetic acid (trisNTA) PEG disulfide. LSPR spectroscopy detection of protein binding on these biocompatible functionalized AuNP monolayers confirms high stability under various harsh analytical conditions. These features were successfully employed to demonstrate unbiased kinetic analysis of cytokine-receptor interactions.

Insights into the conformational organization and dynamics of proteins complexes at membranes is essential for our mechanistic understanding of numerous key biological processes. Here, we introduce graphene-induced energy transfer (GIET) to probe axial orientation of arrested macromolecules at lipid monolayers. Based on a calibrated distance-dependent efficiency within a dynamic range of 25 nm, we analyzed the conformational organization of proteins and complexes involved in tethering and fusion at the lysosome-like yeast vacuole. We observed that the membrane-anchored Rab7-like GTPase Ypt7 shows conformational reorganization upon interactions with effector proteins. Ensemble and time-resolved single-molecule GIET experiments revealed that the HOPS tethering complex, when recruited via Ypt7 to membranes, is dynamically alternating between a ‘closed’ and an ‘open’ conformation, with the latter possibly interacting with incoming vesicles. Our work highlights GIET as a unique spectroscopic ruler to reveal the axial orientation and dynamics of macromolecular complexes at biological membranes with sub-nanometer resolution. Proteins are part of the building blocks of life and are essential for structure, function and regulation of every cell, tissue and organ of the body. Proteins adopt different conformations to work efficiently within the various environments of a cell. They can also switch between shapes. One way to monitor how proteins change their shapes involves energy transfer. This approach can measure how close two proteins, or two parts of the same protein, are, by using dye labels that respond to each other when they are close together. For example, in a method called FRET, one dye label absorbs light and transfers the energy to the other label, which emits it as a different color of light. However, FRET only works over short distances (less than 10nm apart or 1/100,000th of a millimeter), so it is not useful for larger proteins. Here, Füllbrunn, Li et al. developed a method called GIET that uses graphene to analyze the dynamic structures of proteins on membrane surfaces. Graphene is a type of carbon nanomaterial that can absorb energy from dye labels and could provide a way to study protein interactions over longer distances. Graphene was deposited on a glass surface where it was coated with single layer of membrane, which could then be used to capture specific proteins. The results showed that GIET worked over longer distances (up to 30 nm) than FRET and could be used to study proteins attached to the membrane around graphene. Füllbrunn, Li et al. used it to examine a specific complex of proteins called HOPS, which is linked to multiple diseases, including Ebola, measuring distances between the head or tail of HOPS and the membrane to understand protein shapes. This revealed that HOPS adopts an upright position on membranes and alternates between open and closed shapes. The study of Füllbrunn, Li et al. highlights the ability of GIET to address unanswered questions about the function of protein complexes on membrane surfaces and sheds new light on the structural dynamics of HOPS in living cells. As it allows protein interactions to be studied over much greater distances, GIET could be a powerful new tool for cell biology research. Moreover, graphene is also useful in electron microscopy and both approaches combined could achieve a detailed structural picture of proteins in action.

MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization of intermolecular interactions with several advantages over other technologies. One of these is its capability to determine equilibrium constants in solution including complex biological matrices such as cell lysates. MST requires one binding partner to be fluorescent, which is typically achieved by labeling target proteins with a suitable fluorophore. Here, we present a near-native, site-specific in situ labeling strategy for MST experiments that enables reliable measurements in cell lysates and that has distinct advantages over routine covalent labeling techniques. To this end, we exploited the high-affinity interaction of tris-NTA with oligohistidine-tags, which are popular for purification, immobilization or detection of recombinant proteins. We used various DYE-tris-NTA conjugates to successfully label His-tagged proteins that were either purified or a component of cell lysate. The RED-tris-NTA was identified as the optimal dye conjugate with a high affinity towards oligohistidine-tags, a high fluorescence signal and an optimal signal-to-noise ratio in MST binding experiments. Owing to its emission in the red region of the spectrum, it also enables reliable measurements in complex biological matrices such as cell lysates allowing a more physiologically realistic assessment and eliminating the need for protein purification.

The permeability barrier of nuclear pore complexes (NPCs) controls bulk nucleocytoplasmic exchange. It consists of nucleoporin domains rich in phenylalanine-glycine motifs (FG domains). As a bottom-up nanoscale model for the permeability barrier, we have used planar films produced with three different end-grafted FG domains, and quantitatively analyzed the binding of two different nuclear transport receptors (NTRs), NTF2 and Importin β, together with the concomitant film thickness changes. NTR binding caused only moderate changes in film thickness; the binding isotherms showed negative cooperativity and could all be mapped onto a single master curve. This universal NTR binding behavior – a key element for the transport selectivity of the NPC – was quantitatively reproduced by a physical model that treats FG domains as regular, flexible polymers, and NTRs as spherical colloids with a homogeneous surface, ignoring the detailed arrangement of interaction sites along FG domains and on the NTR surface. The cells of animals, plants and other eukaryotic organisms contain a compartment called the nucleus that contains most of the cell's genetic material. Proteins and other molecules – collectively known as cargos – can enter and exit the nucleus via tiny channels in the membrane that surrounds and protects it. Receptor proteins – called nuclear transport receptors – bind to potential cargos and shuttle them through the channels. This selective transport process relies on the nuclear transport receptors being attracted to flexible, spaghetti-like proteins that are anchored to the walls on the inside of each channel. However, because of their flexible and disordered nature, these so-called FG domains are difficult to study, and the details of the transport process are poorly understood. Zahn, Osmanović et al. decided to study how the FG domains behave and what happens when they interact with nuclear transport receptors by using ultrathin films made of just the FG domains. This is a good model system because the films are easier to study than the whole channels, but are likely to retain the essential properties of the real barrier formed in the nuclear envelope. Zahn, Osmanović et al. compared the binding of two nuclear transport receptors of different sizes, taken from humans and yeast, to FG domain films made from one of three different FG domains. The experiments showed that the different nuclear transport receptors bind to the different FG domains in very similar ways. Zahn, Osmanović et al. then used a computational model that essentially represented the FG domains as sticky spaghetti and the nuclear transport receptors as perfectly round meatballs. This sticky-spaghetti-with-meatballs model reproduced the experimental data, implying that the exact chemical make-up and structure of the molecules may not be critical for controlling the transport of cargo across the nuclear envelope. Future studies will test whether the generic physical features of nuclear transport receptors and FG domains can indeed explain how the cargo molecules pass through the nuclear envelope.

Polycaprolactone (PCL) has been widely used as a scaffold material for tissue engineering. Reliable applications of the PCL scaffolds require overcoming their native hydrophobicity and obtaining the sustained release of signaling factors to modulate cell growth and differentiation. Here, we report a surface modification strategy for electrospun PCL nanofibers using an azide-terminated amphiphilic graft polymer. With multiple alkylation and pegylation on the side chains of poly-L-lysine, stable coating of the graft polymer on the PCL nanofibers was achieved in one step. Using the azide-alkyne “click chemistry”, we functionalized the azide-pegylated PCL nanofibers with dibenzocyclooctyne-modified nanocapsules containing growth factor, which rendered the nanofiber scaffold with satisfied cell adhesion and growth property. Moreover, by specific immobilization of pH-responsive nanocapsules containing bone morphogenetic protein 2 (BMP-2), controlled release of active BMP-2 from the PCL nanofibers was achieved within 21 days. When bone mesenchyme stem cells were cultured on this nanofiber scaffold, enhanced ossification was observed in correlation with the time-dependent release of BMP-2. The established surface modification can be extended as a generic approach to hydrophobic nanomaterials for longtime sustainable release of multiplex signaling proteins for tissue engineering.

Protein immobilization into micro and nanoscaled patterns opens exciting possibilities in fundamental and applied research. Developing efficient capturing techniques while preserving the structural and functional integrity of the proteins on surfaces is a key challenge for surface scientists. In this paper, current techniques for site-specific protein immobilization into engineered surface architectures are reviewed. Fundamental principles for functional protein immobilization on solid supports are discussed and popular affinity-based recognition pairs and their application for capturing proteins into nano and microstructures are presented.

Photobleaching‐induced protein binding (PiPB) is a light‐based molecular patterning technique that is introduced as Protein Bitmaps. This technique has significant applications in immunoassays and cell‐substrate interactions. However, commonly used active surfaces, bovine serum albumin (BSA) coatings prepared via physical adsorption, are prone to desorption or displacement by biomolecules with higher substrate affinities, limiting their stability under complex conditions over several days. To address this, covalently bound high‐density antifouling polyethylene glycol (PEG) monolayers are developed by solvent‐free coupling of cost‐effective homo‐bifunctional PEGs (acrylate‐PEG‐acrylate, PEGDA) to glass/glass‐type surfaces silanized with (3‐mercaptopropyl) trimethoxysilane (MPTMS) using thiol‐acrylate conjugate addition reactions at room temperature, resulting in stable antifouling PEGDA surfaces with terminal‐acrylates as free radical acceptors for PiPB. Non‐specific protein binding on PEGDA‐modified surfaces is evaluated using reflectometric interference spectroscopy (RIfS), showing superior antifouling performance compared to BSA‐coated surfaces against avidin and comparable performance against streptavidin and BSA. Furthermore, PiPB with fluorescein‐5‐biotin conjugate (F5B) is carried out on PEGDA‐modified surfaces, performed using a custom‐built digital mirror device (DMD)‐based lithography system, confirming the suitability of PEGDA‐modified surfaces for biomolecule immobilization. The method presented for PEGDA coating preparation has the potential to broaden the applicability of PiPB, particularly using DMD‐based devices, in biomedical and surface engineering fields. Homo‐bifunctional PEG, acrylate‐PEG‐acrylate (PEGDA), coatings are prepared on glass and glass‐type surfaces using solvent‐free thiol‐acrylate conjugate addition reactions at room temperature. The prepared PEGDA coatings are stable, antifouling, and provide feature terminal acrylates that can act as free radical acceptors for photobleaching‐induced protein binding (PiPB), performed using a custom‐built digital mirror device (DMD)‐based lithography system.

Site-specific protein modification—e.g. for immobilization or labelling—is a key prerequisite for numerous bioanalytical applications. Although modification by use of short peptide tags is particularly attractive, efficient and bio-orthogonal systems are still lacking. Here, we review the application of multivalent chelators (MCH) for high-affinity yet reversible recognition of oligohistidine (His)-tagged proteins. MCH are based on multiple nitrilotriacetic acid (NTA) moieties grafted on to molecular scaffolds suitable for conjugation to surfaces, probes or other biomolecules. Reversible interaction with the His-tag is mediated via transition metal ions chelated by the NTA moieties. The small size and biochemical compatibility of these recognition units and the possibility of rapid dissociation of the interaction with His-tagged proteins despite sub-nanomolar binding affinity, enable distinct and versatile handling and modification of recombinant proteins. In this review, we briefly introduce the key principles and features of MCH–His-tag interactions and recapitulate the broad spectrum of bioanalytical applications with a focus on quantitative protein interaction analysis on micro or nano-patterned solid surfaces and specific protein labelling in living cells. Figure 1 ᅟ

The assumption of a static environment is a prerequisite for most of the traditional visual simultaneous localization and mapping (v-SLAM) algorithms, which limits their widespread application in a dynamic environment. Furthermore, in many applications such as autonomous driving, robot collaboration and AR/VR, it is necessary to track the moving objects in the environment. In this work, we propose a v-SLAM method that can effectively track multiple objects in dynamic environments by integrating a 3D object detection thread into the ORB-SLAM2 framework. The dynamic objects were detected and tracked in three steps. Firstly, 3D object detection was performed on the current frame, and the 3D bounding box was projected into a bird's-eye view. Secondly, an association for the object is made based on the motion state of the object and the bounding box in the bird’s-eye view. Thirdly, we track the object and remove feature points corresponding to the dynamic region. In addition, we set up a multi-view constraint adjustment for static objects to jointly optimize the pose of the camera, object, and map point. Experiments have been conducted on the KITTI-odom and KITTI-raw datasets. The performance of our method was verified in challenging scenarios. We demonstrate that dynamic object tracking not only provides useful information for scene understanding, but also help to improve camera tracking.

Sparse arrays can increase the array aperture, thereby enhancing angular resolution. However, this also introduces additional computational complexity. This letter proposes a symmetric sparse array structure, where subarrays with different inter‐element spacings sample distinct spatial domain signals, analogous to the use of mother wavelets at different scales in wavelet theory to process various frequency components of a signal. The root‐MUSIC method can be directly applied to the proposed method, and the simulations demonstrate that it achieves direction‐of‐arrival estimation performance comparable to that of super‐nested arrays while maintaining lower computational complexity. In this letter, a simple but effective sparse array configuration is proposed, in which the array element spacings are arranged from dense to sparse, achieving multi‐resolution in the spatial domain. Moreover, the traditional root‐MUSIC method can be straightforwardly applied to such array. The proposed array outperforms conventional ULAs and achieves performance comparable to other sparse array configurations with lower computational complexity.