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Protein immobilization is an essential method for both basic and applied research for protein, and covalent, site-specific attachment is the most desirable strategy. Classic methods typically rely ...

Cation-π interaction is an electrostatic interaction between a cation and an electron-rich arene. It plays an essential role in many biological systems as a vital driving force for protein folding, stability, and receptor-ligand interaction/recognition. To date, the discovery of most cation-π interactions in proteins relies on the statistical analyses of available three-dimensional (3D) protein structures and corresponding computational calculations. However, their experimental verification and quantification remain sparse at the molecular level, mainly due to the limited methods to dynamically measure such a weak non-covalent interaction in proteins. Here, we use atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the stability of protein neutrophil gelatinase-associated lipocalin (also known as NGAL, siderocalin, lipocalin 2) that can bind iron through the cation-π interactions between its three cationic residues and the iron-binding tri-catechols. Based on a site-specific cysteine engineering and anchoring method, we first characterized the stability and unfolding pathways of apo-NGAL. Then, the same NGAL but bound with the iron-catechol complexes through the cation-π interactions as a holo-form was characterized. AFM measurements demonstrated stronger stabilities and kinetics of the holo-NGAL from two pulling sites, F122 and F133. Here, NGAL is stretched from the designed cysteine close to the cationic residues for a maximum unfolding effect. Thus, our work demonstrates high-precision detection of the weak cation-π interaction in NGAL.Supplementary material (additional SDS-PAGE, UV-vis, protein sequences, and more experimental methods) is available in the online version of this article at 10.1007/s12274-021-4065-9.

Enzymatic protein ligation has become the most powerful and widely used method for high-precision atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) study of protein mechanics.

Enzymes are widely used for protein ligation because of their efficient and site-specific connections under mild conditions. However, many enzymatic ligations are restricted to connections between protein termini while protein-protein conjugation at a specific internal site is limited. Previous work has found that Sortase A (SrtA) conjugates small molecules/peptides to a pilin protein at an internal lysine site via an isopeptide bond. Herein, we apply this noncanonical ligation property of SrtA for protein-protein conjugation at a designed YPKH site. Both a small protein domain, I27, and a large protein, GFP, were ligated at the designed internal site. Moreover, besides characterization by classic methods at the ensemble level, the specific ligation site at the interior YPKH motif is unambiguously verified by atomic force microscopy-based single-molecule force spectroscopy, showing the characteristic unfolding signature at the single-molecule level. Finally, steered molecular dynamics simulations also agreed with the results.

Cobalt is a trace transition metal. Although it is not abundant on earth, tens of cobalt-containing proteins exist in life. Moreover, the characteristic spectrum of Co(II) ion makes it a powerful probe for the characterization of metal-binding proteins through the formation of cobalt-ligand bonds. Since most of these natural and artificial cobalt-containing proteins are stable, we believe that these cobalt-ligand bonds in the protein system are also mechanically stable. To prove this, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to directly measure the rupture force of Co(II)-thiolate bond in Co-substituted rubredoxin (CoRD). By combining the chemical denature/renature method for building metalloprotein and cysteine coupling-based polyprotein construction strategy, we successfully prepared the polyprotein sample (CoRD)

Kraits of the genus Bungarus Daudin 1803 are widely known venomous snakes distributed from Iran to China and Indonesia. Here, we use a combination of mitochondrial DNA sequence data and morphological data to describe a new species from Yingjiang County, Yunnan Province, China: Bungarus suzhenaesp. nov. Phylogenetically, this species forms a monophyletic lineage sister to the Bungarus candidus/multicinctus/wanghaotingi complex based on cyt b and ND4 genes but forms a sister species pair with the species B. magnimaculatus Wall & Evans, 1901 based on COI gene fragments. Morphologically, B. suzhenaesp. nov. is similar to the B. candidus/multicinctus/wanghaotingi complex but differs from these taxa by a combination of dental morphology, squamation, coloration pattern, as well as hemipenial morphology. A detailed description of the cranial osteology of the new species is given based on micro-CT tomography images. We revised the morphological characters of B. candidus/multicinctus/wanghaotingi complex and verified the validity of three species in this complex. The distribution of these species was revised; the records of B. candidus in China should be attributed to B. wanghaotingi. We also provide an updated key to species of Bungarus.

Multiple disciplines can help to discover cryptic species and resolve taxonomic confusions. The Asian horned toad genusMegophryssensu latoas a diverse group was proposed to contain dozens of cryptic species. Based on molecular phylogenetics, morphology, osteology, and bioacoustics data, the species profiles ofMegophrystoads in the eastern corner of Himalayas in Medog County, Tibet Autonomous Region, China was investigated. The results indicated that this small area harbored at least fourMegophrysspecies, i.e.,M. medogensis,M. pachyproctus,Megophrys zhouisp. nov., andMegophrys yeaesp. nov., the latter two being described in this study. Additionally, the mitochondrial DNA trees nested the low-middle-elevation and high-elevation groups ofM. medogensisinto a monophyletic group, being in discordance with the paraphyletic relationship between them revealed in the nuclear DNA trees. The findings highlighted the underestimated biodiversity in Himalayas, and further indicated that theMegophrystoads here have been probably experienced complicated evolutionary history, for example, introgression between clades or incomplete lineage sorting and niche divergences in microhabitats. Anyway, it is urgent for us to explore the problems because these toads are suffering from increasing threats from human activities and climatic changes.

Confused geographical-structure of population and mito-nuclear discordance have been shaped by a combination of rapid population demographic changes and ecological shifts. In this study, we generated a time calibrated phylogeny of Scutiger boulengeri, an endemic Tibetan alpine toad occurring in mountain streams in Qinghai-Tibet Plateau, based on three mitochondria (mtDNA) genes, in which eight clades were assigned into three deeply divergent lineages. While nuclear DNA (nuDNA) revealed three distinct clusters without geographical structure indicating significantly high rates of gene flow by population structure analysis. The coalescent theory framework (approximate Bayesian computation model DIYABC and Migrate-N) suggested that the intraspecific main clusters divergence was the result of hybridization after secondary contact in Holocene around 0.59 Ma, with population size change. The ratio of mtDNA divergence to nuDNA divergence was 2.3, less than 4, failed to test a sex-biased dispersal. Geographic cline analysis showing a wide hybrid zone initially established in southwestern China, without significant reproductive isolation but a strong influence of introgression in S. boulengeri, suggested high fitness of hybrids. Moreover, mtDNA exhibited isolation by distance (IBD) while nuDNA showed significant isolation by environment (IBE). Our results suggested such mito-nuclear discordance might have been first caused by geographic isolations, followed by hybridization mediated by precipitation, and produced a wide hybrid zone, which contributed to confused geographical-structure of the nuDNA in S. boulengeri. This study unveiled the complicated historical process that might have led to specific genetic pattern and specific climate factor facilitating phenotypic diversity in this system.

Having the ability to provide seamless coverage and alleviate the frequency scarcity, the cognitive satellite terrestrial network becomes a promising candidate for future communication networks. In the cognitive network, spectrum sensing plays an important role in detecting the channel state for opportunistic utilization, where cooperative spectrum sensing is employed to improve the sensing performance. Additionally, it is critical for battery-powered satellite mobile terminals to diminish energy consumption costs. In this regard, this paper proposes a novel sensing-based cognitive satellite terrestrial network (SCSTN), which integrates the cognitive satellite terrestrial network with the distributed cooperative spectrum sensing network. Specifically, we focus on energy-efficient cooperative sensing in the SCSTN, which maximizes the energy efficiency (EE) of the cognitive satellite network by a tradeoff between the average throughput and the average energy consumption. In the SCSTN, the energy detection threshold of the sensing node and the rule threshold of fusion affect the average throughput and the average energy consumption. Hence, the objective of this paper is to identify the energy detection threshold of the sensing node and the rule threshold of fusion to achieve the maximum EE. We first study the EE formulation of the rule threshold of fusion when the energy detection threshold of the sensing node is given, and transform the ratio-type objective function of EE into a parametric formulation. Subsequently, by exploring the relationship between the two formulations and making use of the monotonicity of the parametric formulation, an algorithm to obtain the optimal rule threshold of fusion for the original problem is developed. Furthermore, we study the optimal formulation of the energy sensing threshold of the sensing node and discuss the effect of the sensing duration and the number of distributed cooperative terminals on the EE. Lastly, the performance of the proposed method is evaluated through numerical simulation results.

SARS-CoV-2 has been spreading around the world for the past year. Recently, several variants such as B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma), which share a key mutation N501Y on the receptor-binding domain (RBD), appear to be more infectious to humans. To understand the underlying mechanism, we used a cell surface-binding assay, a kinetics study, a single-molecule technique, and a computational method to investigate the interaction between these RBD (mutations) and ACE2. Remarkably, RBD with the N501Y mutation exhibited a considerably stronger interaction, with a faster association rate and a slower dissociation rate. Atomic force microscopy (AFM)-based single-molecule force microscopy (SMFS) consistently quantified the interaction strength of RBD with the mutation as having increased binding probability and requiring increased unbinding force. Molecular dynamics simulations of RBD–ACE2 complexes indicated that the N501Y mutation introduced additional π-π and π-cation interactions that could explain the changes observed by force microscopy. Taken together, these results suggest that the reinforced RBD–ACE2 interaction that results from the N501Y mutation in the RBD should play an essential role in the higher rate of transmission of SARS-CoV-2 variants, and that future mutations in the RBD of the virus should be under surveillance.

Ecologists have generally concluded that species distributions are not random (e.g. aggregate), based on single-species studies that applied single-species-based statistical methods, like the negative binomial model. Although it is common knowledge that some specific species in an ecological community present aggregate distributions, this does not necessarily imply that the entire community presents an aggregate distribution. Studying community-level distributional aggregation patterns requires different statistical methods. Herein, by utilizing a novel conspecific-encounter index derived from a multiple species Markov transition model that accounts for the non-independent sampling of consecutive individuals along line transects, we were able to show that tree assemblages in tropical forest ecosystems can present a strong signal of extensive distributional interspersion. This interesting result is not unexpected, given the fact that neighbouring tree individuals in highly diverse tropical forests are usually of different species, resulting in strong niche packing or interspersed patterns. In contrast, for the amphibian assemblages surveyed from southwestern China and central-south Vietnam, the conspecific-encounter index was found to be consistently high, implying that amphibian communities tend to be highly aggregate in space. Conclusively, using the conspecific-encounter index derived from the Markov non-independent sampling model, we provide a legible definition of community-level distributional aggregation as an interspersed or cluster-like distribution of different species. This definition is not idiosyncratic, as it is coincident with the definition of the contagion index used in landscape ecology. To this end, the model used in this paper establishes a framework explicitly linking community ecology and landscape ecology from a multi-object perspective.

A new species of genus Megophrys from Gaoligong Mountains, Yunnan Province, China is described. Phylogenetic analyses based on mitochondrial DNA and nuclear DNA all clustered the new species as an independent clade nested into the subgenus Panophrys. The smallest genetic distance based on 16S rRNA gene between the new species and its congeners was 3.0%. The new species could be identified from its congeners by a combination of following characters: moderate body size (SVL 31.0–34.8 mm in males); vomerine ridge weak, vomerine teeth absent; dorsal skin relatively smooth; tongue slightly notched behind; tympanum rounded and relatively large, 0.54 times of eye length; a horn-like tubercle on edge of each upper eyelid small; tibio-tarsal articulation reaches middle eye when leg stretched forward; finger tips rounded, not expanded to small pad; toes with narrow fringes and rudimentary webbing; ventral hindlimbs semitransparent purplish with greyish white pigments; ventral body scattered with distinct dark patches in the middle.

SARS-CoV-2 is spreading around the world for the past year. Enormous efforts have been taken to understand its mechanism of transmission. It is well established now that the receptor-binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) as its first step of entry. Being a single-stranded RNA virus, SARS-CoV-2 is evolving rapidly. Recently, several variants such as B.1.1.7, B.1.351, and P.1, with a key mutation N501Y on the RBD, appear to be more infectious to humans. To understand its mechanism, we combined cell surface binding assay, kinetics study, single-molecule technique, and computational method to investigate the interaction between these RBD (mutations) and ACE2. Remarkably, RBD with the N501Y mutation exhibited a considerably stronger interaction characterized from all these methodologies, while the other two mutations from B.1.351 contributed to a less effect. Fluorescence-activated cell scan (FACS) assays found that RBD N501Y mutations are of higher binding affinity to ACE2 than the wild type. Surface plasmon resonance further indicated that N501Y mutation had a faster association rate and slower dissociation rate. Consistent with the kinetics study, atomic force microscopy-based single-molecule force microscopy quantify their strength on living cells, showing a higher binding probability and unbinding force for the mutation. Finally, Steered Molecular Dynamics (SMD) simulations on the dissociation of RBD-ACE2 complexes revealed that the N501Y introduced additional π-π and π-cation interaction for the higher force/interaction. Taken together, we suggested that the reinforced interaction from N501Y mutation in RBD should play an essential role in the higher transmission of COVID-19 variants.

The mitochondrial outer membrane protein, mitoNEET (mNT), is an iron-sulfur protein containing an Fe2S2(His)1(Cys)3 cluster with a unique single Fe-N bond. Previous studies have shown that this Fe(III)-N(His) bond is essential for metal cluster transfer and protein function. To further understand the effect of this unique Fe-N bond on the metal cluster and protein, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to investigate the mechanical unfolding mechanism of an mNT monomer, focusing on the rupture pathway and kinetic stability of the cluster. We found that the Fe-N bond was the weakest point of the cluster, the rupture of which occurred first, and could be independent of the cluster break. Moreover, this Fe-N bond enabled a dynamic and labile iron-sulfur cluster, as multiple unfolding pathways of mNT with a unique Fe2S2(Cys)3 intermediate were observed accordingly.

Sortase is one of the most widely used enzymes for covalent protein conjugation that links protein and protein/small molecules together in a site-specific way. It typically recognizes the "GGG" and "LPXTG" peptide sequences and conjugates them into an "LPXTGGG" linker. As a non-natural linker with several flexible glycine residues, it is unknown whether it affects the properties of the conjugated protein. To verify the use of sortase for protein-protein conjugation, we combined single-molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations to characterize sortase-conjugated polyprotein I27 with three different linkers. We found that the I27 with classic linkers "LPETGGG" and "LPETG" from sortase ligation were of normal stability. However, a protein with a longer artificial linker "LPETGGGG" showed a 15% lower unfolding force. MD simulations revealed that the 4G linker showed a high probability of a closed conformation, in which the adjacent monomer has transient protein-protein interaction. Thus, we verify the use of sortase for protein conjugation, and a longer linker with a higher glycine content should be used with caution.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Genetic adaptation to extremes is a fascinating topic. Nevertheless, few studies have explored the genetic adaptation of closely related species respectively inhabiting distinct extremes. With deep transcriptome sequencing, we attempt to detect the genetic architectures of tadpoles of five closely related toad species adapted to the Tibetan Plateau, middle-altitude mountains and karst caves. Molecular evolution analyses indicated that not only the number of fast evolving genes (FEGs), but also the functioning coverage of FEGs, increased with elevation. Enrichment analyses correspondingly revealed that the highland species had most of the FEGs involved in high-elevation adaptation, for example, amino acid substitutions of XRCC6 in its binding domains might improve the capacity of DNA repair of the toad. Yet, few FEGs and positively selected genes (PSGs) involved in high-elevation adaptation were identified in the cave species, and none of which potentially contributed to cave adaptation. Accordingly, it is speculated that in the closely related toad tadpoles, genetic selection pressures increased with elevation, and cave adaptation was most likely derived from other factors (e.g., gene loss, pseudogenization or deletion), which could not be detected by our analyses. The findings supply a foundation for understanding the genetic adaptations of amphibians inhabiting extremes.

A new species of the genusMegophrysfrom Zhejiang Province, China is described. Molecular phylogenetic analyses supported the new taxon as an independent clade nested into theMegophrysclade and sister toM. lishuiensis. The new species could be distinguished from its congeners by a combination of the following morphological characteristics: (1) small size (SVL 31.0–36.3 mm in male and 41.6 mm in female); (2) vomerine ridge present and vomerine teeth absent; (3) tongue not notched behind; (4) a small horn-like tubercle at the edge of each upper eyelid; (5) tympanum distinctly visible, rounded; (6) two metacarpal tubercles in hand; (7) relative finger lengths: II < I < IV < III; (8) toes with rudimentary webbing at bases; (9) heels overlapping when thighs are positioned at right angles to the body; (10) tibiotarsal articulation reaching tympanum to eye when leg stretched forward; (11) an internal single subgular vocal sac in male; (12) in breeding male, the nuptial pads with black nuptial spines on the dorsal bases of the first and second fingers.

The development of chemical and biological coupling technologies in recent years has made possible of protein polymers engineering. We have developed an enzymatic method for building polyproteins using a protein ligase OaAEP1 (asparagine endopeptidase 1) and protease TEV (tobacco etching virus). Using a mobile TEV protease site compatible with the OaAEP1 ligation, we achieved a stepwise polymerization of the protein on the surface. The produced polyprotein can be verified by protein unfolding scenario using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides an alternative method for polyprotein engineering and immobilization.

This paper presents a cognitive satellite communication based wireless sensor network, which combines the wireless sensor network and the cognitive satellite terrestrial network. To address the conflict between the continuously increasing demand and the spectrum scarcity in the space network, the cognitive satellite terrestrial network becomes a promising candidate for future hybrid wireless networks. With the higher transmit capacity demand in satellite networks, explicit concerns on efficient resource allocation in the cognitive network have gained more attention. In this background, we propose a sensing-based dynamic spectrum sharing scheme for the cognitive satellite user, which is able to maximize the ergodic capacity of the satellite user with the interference of the primary terrestrial user below an acceptable average level. Firstly, the cognitive satellite user monitors the channel allocated to the terrestrial user through the wireless sensor network, then, it adjusts the transmit power based on the sensing results. If a terrestrial user is busy, the satellite user can access the channel with constrained power to avoid deteriorating the communication quality of the terrestrial user. Otherwise, if the terrestrial user is idle, the satellite user allocates the transmit power based on its benefit to enhance the capacity. Since the sensing-based dynamic spectrum sharing optimization problem can be modified into a nonlinear fraction programming problem in perfect/imperfect sensing conditions, respectively, we solve them by the Lagrange duality method. Computer simulations have shown that, compared with the opportunistic spectrum access, the proposed method can increase the channel capacity more than&nbsp, 20% for Pav&nbsp, = 10 dB in a perfect sensing scenario. In an imperfectsensing scenario, Pav = 15 dB and Qav = 5 dB, the optimal sensing time achieving the highest ergodiccapacity is about 2.34 ms when the frame duration is 10 ms.

To address the spectrum scarcity in future satellite communications, employing the cognitive technique in the satellite systems is considered as a promising candidate, which leads to an advanced architecture known as cognitive satellite terrestrial networks. Power control is a significant research challenge in cognitive satellite terrestrial networks, especially when the perfect channel state information (CSI) of satellite or terrestrial links is unavailable. In this context, we investigate the impact of imperfect CSI of both desired satellite link and harmful terrestrial interference link on the power control scheme in cognitive satellite terrestrial networks. By adopting a pilot-based channel estimation of satellite link and a back-off interference power constraint of terrestrial interference link, a novel power control scheme is presented to maximize the outage capacity of the satellite user while guaranteeing the communication quality of primary terrestrial user. Extensive numerical results quantitatively demonstrate the effect of various system parameters on the proposed power control scheme in cognitive satellite terrestrial networks with imperfect CSI.

With the development of satellite communication, the number of satellites in space continuously increases. However, the available spectrum resources are scarce. To address the spectrum scarcity, sharing the spectrum between different communication systems is a promising option. In this paper, a novel cognitive satellite network with geostationary earth orbit (GEO) and low earth orbit (LEO) broadband systems is studied in the downlink case. First, we present a general interference analysis model and simplify it by transforming the spatial dimension into the time dimension according to the satellite motion. Based on the interference analysis, an optimization algorithm with beamhopping and adaptive power control techniques is proposed, which can simultaneously enhance the spectral efficiency and protect the primary system. The system performance of the coexistence scenario is evaluated in terms of the system throughput. Numerical results demonstrate that the spectrum-sharing method between GEO and LEO systems is feasible, and the cognitive network can achieve a high spectral efficiency. Furthermore, the factors that affect the system in the spectral coexistence scenario are analyzed.