人工纤维小体骨架蛋白的分子设计与自组装

纤维小体是厌氧微生物分泌的能够高效降解木质纤维素的一种多酶复合体。其骨架蛋白质的大量重组合成是人工纤维小体构建的关键。为了构建全长的结构复杂的能招募大量酶亚基的骨架蛋白质,本研究对解纤维梭菌Ruminiclostridum cellulolyticum骨架蛋白CipC进行分子模块化设计,并分别与肽-蛋白质共价偶联系统SpyTag/SpyCatcher和SnoopTag/SnoopCatcher融合表达。然后分别通过非固定化和固定化组装策略对人工纤维小体骨架蛋白质进行了初步组装。结果显示,SpyTag和SpyCatcher、SnoopTag和SnoopCatcher之间自发形成稳定的共价异肽键,而且SpyTag和SnoopCatcher,SnoopTag和SpyCacther之间无交叉反应。最终成功组装出含有不同数量黏附域的骨架蛋白质,实现骨架蛋白链的延伸。本研究为构建结构更复杂、活性更高的人工纤维小体提供了新的设计思路并奠定了其技术基础。     

Surface-accessible residues in the monomeric and assembled forms of a bacterial surface layer protein

The S-layer protein SbsB of the thermophilic, Gram-positive organism Bacillus stearothermophilus PV72/p2 forms a crystalline, porous array constituting the outermost component of the cell envelope. SbsB has a molecular mass of 98 kDa, and the corresponding S-layer exhibits an oblique lattice symmetry. To investigate the molecular structure and assembly of SbsB, we replaced 75 residues (mainly serine, threonine, and alanine), located throughout the primary sequence, with cysteine, which is not found in the wild-type protein. As determined by electron microscopy, 72 out of 75 mutants formed regularly-structured self-assembly products identical to wild-type, thereby proving that the replacement of most of the selected amino acids by cysteine does not dramatically alter the structure of the protein. The three defective mutants, which showed a greatly reduced ability to self-assemble, were, however, successfully incorporated into S-layers of wild-type protein. Monomeric SbsB mutants and SbsB mutants assembled into S-layers were subjected to a surface accessibility screen by targeted chemical modification with a 5-kDa hydrophilic cysteine-reactive polyethylene glycol conjugate. In the monomeric form of SbsB, 34 of the examined residues were not surface accessible, while 23 were classified as very accessible, and 18 were of intermediate surface accessibility. By contrast, in the assembled S-layers, 57 of the mutated residues were not accessible, six were very accessible, and 12 of intermediate accessibility. Together with other structural information, the results suggest a model for SbsB in which functional domains are segregated along the length of the polypeptide chain.     

Site-specific attachment of a protein to a carbon nanotube end without loss of protein function

Establishing a nanobiohybrid device largely relies on the availability of various bioconjugation procedures which allow coupling of biomolecules and inorganic materials. Especially, site-specific coupling of a protein to nanomaterials is highly useful and significant, since it can avoid adversely affecting the protein's function. In this study, we demonstrated a covalent coupling of a protein of interest to the end of carbon nanotubes without affecting protein's function. A modified Staudinger-Bertozzi ligation was utilized to couple a carbon nanotube end with an azide group which is site-specifically incorporated into a protein of interest. We demonstrated that Ca(2+)-sensor protein, calmodulin, can be attached to the end of the nanotubes without affecting the ability to bind to the substrate in a calcium-dependent manner. This procedure can be applied not only to nanotubes, but also to other nanomaterials, and therefore provides a fundamental technique for well-controlled protein conjugation.     

Emergent Pseudocapacitance of 2D Nanomaterials

Two dimensional (2D) nanomaterials are very attractive due to their unique structural and surface features for energy storage applications. Motivated by the recent pioneering works demonstrating “the emergent pseudocapacitance of 2D nanomaterials,” the energy storage and nanoscience communities could revisit bulk layered materials though state‐of‐the‐art nanotechnology such as nanostructuring, nanoarchitecturing, and compositional control. However, no review has focused on the fundamentals, recent progress, and outlook on this new mechanism of 2D nanomaterials yet. In this study, the key aspects of emergent pseudocapacitors based on 2D nanomaterials are comprehensively reviewed, which covers the history, classification, thermodynamic and kinetic aspects, electrochemical characteristics, and design guidelines of materials for extrinsically surface redox and intercalation pseudocapacitors. The structural and compositional controls of graphene and other carbon nanosheets, transition metal oxides and hydroxides, transition metal dichalcogenides, and metal carbide/nitride on both microscopic and macroscopic levels will be particularly addressed, emphasizing the important results published since 2010. Finally, perspectives on the current impediments and future directions of this field are offered. Unlimited combinations and modifications of 2D nanomaterials can provide a rational strategy to overcome intrinsic limitations of existing materials, offering a new‐generation energy storage materials toward a high and new position in the Ragone plot.     

Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag

Peptide tagging is a key strategy for observing and isolating proteins. However, the interactions of proteins with peptides are nearly all rapidly reversible. Proteins tagged with the peptide SpyTag form an irreversible covalent bond to the SpyCatcher protein via a spontaneous isopeptide linkage, thereby offering a genetically encoded way to create peptide interactions that resist force and harsh conditions. Here, we determined the crystal structure of the reconstituted covalent complex of SpyTag and SpyCatcher at 2.1 Å resolution. The structure showed the expected reformation of the β-sandwich domain seen in the parental streptococcal adhesin, but flanking sequences at both N- and C-termini of SpyCatcher were disordered. In addition, only 10 out of 13 amino acids of the SpyTag peptide were observed to interact with SpyCatcher, pointing to specific contacts important for rapid split protein reconstitution. Based on these structural insights, we expressed a range of SpyCatcher variants and identified a minimized SpyCatcher, 32 residues shorter, that maintained rapid reaction with SpyTag. Together, these results give insight into split protein β-strand complementation and enhance a distinct approach to ultrastable molecular interaction.     

The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.     

TSGIT: An N‐ and C‐terminal tandem tag system for purification of native and intein‐mediated ligation‐ready proteins

A large variety of fusion tags have been developed to improve protein expression, solubilization, and purification. Nevertheless, these tags have been combined in a rather limited number of composite tags and usually these composite tags have been dictated by traditional commercially‐available expression vectors. Moreover, most commercially‐available expression vectors include either N‐ or C‐terminal fusion tags but not both. Here, we introduce TSGIT, a fusion‐tag system composed of both N‐ and a C‐terminal composite fusion tags. The system includes two affinity tags, two solubilization tags and two cleavable tags distributed at both termini of the protein of interest. Therefore, the N‐ and the C‐terminal composite fusion tags in TSGIT are fully orthogonal in terms of both affinity selection and cleavage. For using TSGIT, we streamlined the cloning, expression, and purification procedures. Each component tag is selected to maximize its benefits toward the final construct. By expressing and partially purifying the protein of interest between the components of the TSGIT fusion, the full‐length protein is selected over truncated forms, which has been a long‐standing problem in protein purification. Moreover, due to the nature of the cleavable tags in TSGIT, the protein of interest is obtained in its native form without any additional undesired N‐ or C‐terminal amino acids. Finally, the resulting purified protein is ready for efficient ligation with other proteins or peptides for downstream applications. We demonstrate the use of this system by purifying a large amount of native fluorescent mRuby3 protein and bacteriophage T7 gp2.5 ssDNA‐binding protein.     

2D Zn‐Hexamine Coordination Frameworks and Their Derived N‐Rich Porous Carbon Nanosheets for Ultrafast Sodium Storage

Metal‐organic coordination frameworks have been widely used as efficient precursors for the preparation of functional carbon‐based materials with various nanostructures. However, to date, the design of 2D carbon nanostructures from single coordination frameworks remains a great challenge. Herein, an efficient strategy for the fabrication of N‐rich porous carbon nanosheets from 2D Zn‐hexamine coordination framework nanosheets is developed. Remarkably, the N‐doping level of carbon nanosheets can attain 16.54 at%. In addition, the thickness of the carbon nanosheets can effectively be tuned by simply adjusting the molar ratio of the starting materials. As a proof‐of‐concept application, the as‐prepared carbon nanosheets as an anode material for sodium‐ion batteries exhibit an ultrafast sodium storage capability of 194 mAh g^?1 even at 10 A g^?1. As far as it is known, such a high‐rate capability has been rarely achieved in previous studies on carbonaceous anode materials for Na‐ion storage. Moreover, this approach is readily controllable and could be extended to prepare a series of 2D N‐doped carbon‐based nanomaterials on a large scale.     

Structural inhibition of the colicin D tRNase by the tRNA-mimicking immunity protein

Colicins are toxins secreted by Escherichia coli in order to kill their competitors. Colicin D is a 75 kDa protein that consists of a translocation domain, a receptor-binding domain and a cytotoxic domain, which specifically cleaves the anticodon loop of all four tRNA(Arg) isoacceptors, thereby inactivating protein synthesis and leading to cell death. Here we report the 2.0 A resolution crystal structure of the complex between the toxic domain and its immunity protein ImmD. Neither component shows structural homology to known RNases or their inhibitors. In contrast to other characterized colicin nuclease-Imm complexes, the colicin D active site pocket is completely blocked by ImmD, which, by bringing a negatively charged cluster in opposition to a positively charged cluster on the surface of colicin D, appears to mimic the tRNA substrate backbone. Site-directed mutations affecting either the catalytic domain or the ImmD protein have led to the identification of the residues vital for catalytic activity and for the tight colicin D/ImmD interaction that inhibits colicin D toxicity and tRNase catalytic activity.     

Unprecedented Synthesis of Holey 2D Layered Double Hydroxide Nanomesh for Enhanced Oxygen Evolution

Creating nanosized pores in 2D materials can increase the edge sites, improve the mass transfer, and contribute to different physical properties, which shows potential applications in many fields including filtration membranes, electronics and energy storage devices, and catalysts. An iconic member of this type of material is porous graphene. Herein, a unique 2D layered double hydroxide (LDH) nanomesh is designed and synthesized as a new class of 2D holey materials. It represents the first case of exfoliation method for preparing 2D holey materials among all published reports. As an oxygen evolution reaction electrocatalyst, the 2D CoCo‐LDH nanomesh has apparently lower onset overpotential (220 mV) than that of compact nanosheets without holes (270 mV) owing to the pores through the plane that offer more highly active edge sites with lower coordination number and promote the mass diffusion. This work opens up a new avenue for designing 2D porous materials for energy conversion and storage.     

The surface location of individual residues in a bacterial S-layer protein

Bacterial surface layer (S-layer) proteins self-assemble into large two-dimensional crystalline lattices that form the outermost cell-wall component of all archaea and many eubacteria. Despite being a large class of self-assembling proteins, little is known about their molecular architecture. We investigated the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 to identify residues located at the subunit-subunit interface and to determine the S-layer's topology. Twenty-three single cysteine mutants, which were previously mapped to the surface of the SbsB monomer, were subjected to a cross-linking screen using the photoactivatable, sulfhydryl-reactive reagent N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide. Gel electrophoretic analysis on the formation of cross-linked dimers indicated that 8 out of the 23 residues were located at the interface. In combination with surface accessibility data for the assembled protein, 10 residues were assigned to positions at the inner, cell-wall-facing lattice surface, while 5 residues were mapped to the outer, ambient-exposed lattice surface. In addition, the cross-linking screen identified six positions of intramolecular cross-linking within the assembled protein but not in the monomeric S-layer protein. Most likely, these intramolecular cross-links result from conformational changes upon self-assembly. The results are an important step toward the further structural elucidation of the S-layer protein via, for example, X-ray crystallography and cryo-electron microscopy. Our approach of identifying the surface location of residues is relevant to other planar supramolecular protein assemblies.     

Iron‐Oxide‐Based Advanced Anode Materials for Lithium‐Ion Batteries

Iron oxides, such as Fe₂O₃ and Fe₃O₄, have recently received increased attention as very promising anode materials for rechargeable lithium‐ion batteries (LIBs) because of their high theoretical capacity, non‐toxicity, low cost, and improved safety. Nanostructure engineering has been demonstrated as an effective approach to improve the electrochemical performance of electrode materials. Here, recent research progress in the rational design and synthesis of diverse iron oxide‐based nanomaterials and their lithium storage performance for LIBs, including 1D nanowires/rods, 2D nanosheets/flakes, 3D porous/hierarchical architectures, various hollow structures, and hybrid nanostructures of iron oxides and carbon (including amorphous carbon, carbon nanotubes, and graphene). By focusing on synthesis strategies for various iron‐oxide‐based nanostructures and the impacts of nanostructuring on their electrochemical performance, novel approaches to the construction of iron‐oxide‐based nanostructures are highlighted and the importance of proper structural and compositional engineering that leads to improved physical/chemical properties of iron oxides for efficient electrochemical energy storage is stressed. Iron‐oxide‐based nanomaterials stand a good chance as negative electrodes for next generation LIBs.     

Site-specific cross-linking of proteins through tyrosine hexahistidine tags

The genetic addition of hexahistidine (H(6)) tags is widely used to isolate recombinant proteins by immobilized metal-affinity chromatography (IMAC). Addition of a tyrosine residue to H(6) tags enabled proteins to be covalently cross-linked under mild conditions in a manner similar to the natural, site-specific cross-linking of tyrosines into dityrosine. A series of seven hexahistidine tags with tyrosines placed in various positions (H(6)Y tags) were added to the amino terminus of the I28 immunoglobulin domain of the human cardiac titin. The H(6)Y-tagged I28 dimerized in the presence of excess Ni(2+) with a K(D) of 200 microM. Treatment of Ni(2+)-dimerized H(6)Y-I28 with an oxidant, monoperoxyphthalic acid (MMPP) or sodium sulfite, resulted in covalent protein multimerization through chelated Ni(2+)-catalyzed cross-linking of the Y residues engineered into the H(6) tag. The protein oligomerization was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The presence of dityrosine in the cross-linked proteins was confirmed by fluorescence emission at 410 nm. Proteins lacking the Y residue in the H(6) tag treated with the same oxidative conditions did not cross-link or exhibit dityrosine fluorescence, despite the presence of an endogenous Y residue. The method may have potential uses in other protein conjugation applications such as protein labeling and interfacial immobilization of proteins on artificial surfaces.     

Transition from Diffusion‐Controlled Intercalation into Extrinsically Pseudocapacitive Charge Storage of MoS2 by Nanoscale Heterostructuring

2D nanomaterials have been found to show surface‐dominant phenomena and understanding this behavior is crucial for establishing a relationship between a material's structure and its properties. Here, the transition of molybdenum disulfide (MoS₂) from a diffusion‐controlled intercalation to an emergent surface redox capacitive behavior is demonstrated. The ultrafast pseudocapacitive behavior of MoS₂ becomes more prominent when the layered MoS₂ is downscaled into nanometric sheets and hybridized with reduced graphene oxide (RGO). This extrinsic behavior of the 2D hybrid is promoted by the fast Faradaic charge‐transfer kinetics at the interface. The heterostructure of the 2D hybrid, as observed via high‐angle annular dark field–scanning transmission electron microscopy and Raman mapping, with a 1T MoS₂ phase at the interface and a 2H phase in the bulk is associated with the synergizing capacitive performance. This 1T phase is stabilized by the interactions with the RGO. These results provide fundamental insights into the surface effects of 2D hetero‐nanosheets on emergent electrochemical properties.     

小鼠cAMP依赖的蛋白激酶催化亚基α的重组表达研究

将小鼠ｃＡＭＰ依赖的蛋白激酶（ｃＡＰＫ）催化亚基α（ｍＣα）分别以成熟、麦芽糖结合蛋白（ＭＢＰ）融合以及Ｎ端连续六个组氨酸（Ｈｉｓ_６）融合的形式在大肠杆菌中得到了高效表达,且成熟及融合的重组ｍＣα均有明显的蛋白激酶活性,表明蛋白激酶催化核心结构具有相对的独立性。其中Ｈｉｓ_６－ｍＣα可利用金属离子（Ｎｉ￣（２＋））配体亲和层析（Ⅰ－ＭＡＣ）一步纯化,所得融合蛋白可通过Ｈｉｓ_６亲合手臂（Ｔａｇ）固相化于金属离子（Ｎｉ￣（２＋））配体亲和树脂上,为进一步利用ＰｈａｇｅＤｉｓｐｌａｙ多肽库筛选ｃＡＰＫ识别的底物序列和专一性抑制剂打下了基础。     

Cleavage of colicin D is necessary for cell killing and requires the inner membrane peptidase LepB

Colicin D is known to kill target cells by cleaving tRNA(Arg). A colicin D-resistant mutant was selected that was altered in the inner membrane leader peptidase, LepB. The substituted residue (Asn274Lys) is located close to the catalytic site. The mutation abolishes colicin D cleavage but not the processing of exported proteins. LepB is required for colicin D cleavage, releasing a small C-terminal fragment that retains full tRNase activity. The immunity protein was found to prevent colicin D processing and furthermore masks tRNase activity, thus protecting colicin D against LepB-mediated cleavage during export. Catalytic colicins share a consensus sequence at their putative processing site. Mutations affecting normal processing of colicin D abolish cytotoxicity without affecting the in vitro tRNase activity.     

Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3.     

Defect Suppression in Oriented 2D Perovskite Solar Cells with Efficiency over 18% via Rerouting Crystallization Pathway

Vertically oriented 2D perovskites exhibit promising optoelectronic properties and intrinsic stability, but their photovoltaic application is still limited by the low power conversion efficiency (PCE) compared to 3D analogs. Here, a new crystallization pathway (RCP) is reported to suppress defects in vertically oriented 2D perovskite caused by its over-rapid self-assembly behavior. By controlling the specific adsorption of an ammonium halide additive on different perovskite crystal planes, the dynamic preferred growth of (111) plane is intentionally restrained, and the minority (202) planes emerge as secondary nucleation sites to stimulate the creation of large grains. As the halogen-regulated deprotonation of ammonium proceeds, the (111) crystal plane gradually recovers its growth dominance, and a vertically oriented 2D perovskite film finally forms with high homogeneity, reduced trap density of states, and desired carrier transport/collection kinetics. Solar cells using RCP-2D films show a highly reproducible and stable PCE reaching 18.5% with a high fill factor of 83.4%. These findings provide critical missing information on simultaneously achieving highly oriented and less defective 2D perovskite films for excellent device performance.     

Fragment complementation of calbindin D28k

Calbindin D28k is a highly conserved Ca2+-binding protein abundant in brain and sensory neurons. The 261-residue protein contains six EF-hands packed into one globular domain. In this study, we have reconstituted calbindin D28k from two fragments containing three EF-hands each (residues 1-132 and 133-261, respectively), and from other combinations of small and large fragments. Complex formation is studied by ion-exchange and size-exclusion chromatography, electrophoresis, surface plasmon resonance, as well as circular dichroism (CD), fluorescence, and NMR spectroscopy. Similar chromatographic behavior to the native protein is observed for reconstituted complexes formed by mixing different sets of complementary fragments, produced by introducing a cut between EF-hands 1, 2, 3, or 4. The C-terminal half (residues 133-261) appears to have a lower intrinsic stability compared to the N-terminal half (residues 1-132). In the presence of Ca2+, NMR spectroscopy reveals a high degree of structural similarity between the intact protein and the protein reconstituted from the 1-132 and 133-261 fragments. The affinity between these two fragments is 2 x 10(7) M(-1), with association and dissociation rate constants of 2.7 x 10(4) M(-1) s(-1) and 1.4 x 10(-3) s(-1), respectively. The complex formed in the presence of Ca2+ is remarkably stable towards unfolding by urea and heat. Both the complex and intact protein display cold and heat denaturation, although residual alpha-helical structure is seen in the urea denatured state at high temperature. In the absence of Ca2+, the fragments do not recombine to yield a complex resembling the intact apo protein. Thus, calbindin D28k is an example of a protein that can only be reconstituted in the presence of bound ligand. The alpha-helical CD signal is increased by 26% after addition of Ca2+ to each half of the protein. This suggests that Ca2+-induced folding of the fragments is important for successful reconstitution of calbindin D28k.     

The regulatory and catalytic subunits of rat liver cyclic AMP-dependent protein kinases respond differently to thyroparathyroidectomy and 1 alpha,25-dihydroxyvitamin D3

The regulatory subunits of cytoplasmic and nuclear cyclic AMP-dependent protein kinases increased, while the holoenzyme activities fell in the livers of thyroparathyroidectomized (TPTX) rats. 1 alpha,25-dihydroxyvitamin D3 reversed this TPTX-induced decline in holoenzyme activities. TPTX also prevented the increase in holoenzyme activities, but not the increase in the concentration of regulatory subunits that normally occurs in partially hepatectomized animals. An injection of 1 alpha, 25-dihydroxyvitamin D3 into these TPTX animals, at the time of partial hepatectomy restored the increases in holoenzyme activities, without affecting the changes in regulatory subunits. Thus, the synthesis of regulatory and catalytic subunits can be uncoupled by TPTX and 1 alpha, 25-dihydroxyvitamin D3 appears to selectively control the activity of C subunits.