Cellular Synthesis and X‐ray Crystal Structure of a Designed Protein Heterocatenane

Herein, we report the biosynthesis of protein heterocatenanes using a programmed sequence of multiple post‐translational processing events including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The approach is general, autonomous, and can obviate the need for any additional enzymes. The catenane topology was convincingly proven using a combination of SDS‐PAGE, LC‐MS, size exclusion chromatography, controlled proteolytic digestion, and protein crystallography. The X‐ray crystal structure clearly shows two mechanically interlocked protein rings with intact folded domains. It opens new avenues in the nascent field of protein‐topology engineering.     

Cellular Synthesis of Protein Catenanes

Direct cellular production of topologically complex proteins is of great interest both in supramolecular chemistry and protein engineering. We describe the first cellular synthesis of protein catenanes through the use of the p53 dimerization domain to guide the intertwining of two protein chains and SpyTag–SpyCatcher chemistry for efficient cyclization. The catenane topology was unambiguously proven by SDS‐PAGE, SEC, and partial digestion experiments and was shown to confer enhanced stability toward trypsin digestion relative to monomeric control mutants. The assembly–reaction synergy enabled by protein folding and genetically encoded protein chemistry offers a convenient yet powerful approach for creating mechanically interlocked, complex protein topologies in vivo.     

Organic Ligand Binding by a Hydrophobic Cavity in a Designed Tetrameric Coiled‐Coil Protein

The design and characterization of a hydrophobic cavity in de novo designed proteins provides a wide range of information about the functions of de novo proteins. We designed a de novo tetrameric coiled‐coil protein with a hydrophobic pocketlike cavity. Tetrameric coiled coils with hydrophobic cavities have previously been reported. By replacing one Leu residue at the a position with Ala, hydrophobic cavities that did not flatten out due to loose peptide chains were reliably created. To perform a detailed examination of the ligand‐binding characteristics of the cavities, we originally designed two other coiled‐coil proteins: AM2, with eight Ala substitutions at the adjacent a and d positions at the center of a bundled structure, and AM2W, with one Trp and seven Ala substitutions at the same positions. To increase the association of the helical peptides, each helical peptide was connected with flexible linkers, which resulted in a single peptide chain. These proteins exhibited CD spectra corresponding to superhelical structures, despite weakened hydrophobic packing. AM2W exhibited binding affinity for size‐complementary organic compounds. The dissociation constants, K_d, of AM2W were 220 nM for adamantane, 81 μM for 1‐adamantanol, and 294 μM for 1‐adamantaneacetic acid, as measured by fluorescence titration analyses. Although it was contrary to expectations, AM2 did not exhibit any binding affinity, probably due to structural defects around the designed hydrophobic cavity. Interestingly, AM2W exhibited incremental structure stability through ligand binding. Plugging of structural defects with organic ligands would be expected to facilitate protein folding.     

Regulating the Coordination State of a Heme Protein by a Designed Distal Hydrogen-Bonding Network

Heme coordination state determines the functional diversity of heme proteins. Using myoglobin as a model protein, we designed a distal hydrogen-bonding network by introducing both distal glutamic acid (Glu29) and histidine (His43) residues and regulated the heme into a bis-His coordination state with native ligands His64 and His93. This resembles the heme site in natural bis-His coordinated heme proteins such as cytoglobin and neuroglobin. A single mutation of L29E or F43H was found to form a distinct hydrogen-bonding network involving distal water molecules, instead of the bis-His heme coordination, which highlights the importance of the combination of multiple hydrogen-bonding interactions to regulate the heme coordination state. Kinetic studies further revealed that direct coordination of distal His64 to the heme iron negatively regulates fluoride binding and hydrogen peroxide activation by competing with the exogenous ligands. The new approach developed in this study can be generally applicable for fine-tuning the structure and function of heme proteins.     

Synthesis and Crystal Structure of a New Sulfur-Palladium-Nitrogen Complex

The reaction of 4-amino-6-methyl-1,2,4-triazine-3(2H)-thione-5-one (AMTTO, 1 ) with palladium(II) chloride in acetonitrile/methanol leads to the N,S-coordinated complex [Pd(η²-AMTTO-N,S)Cl₂] · CH₃OH ( 2 ). 2 has been characterized by IR and MS techniques. The ligand 1 and the complex 2 were also investigated by X-ray structure determinations. 1 crystallizes in the space group P1¯ with the lattice dimensions at –70 °C: a = 419.6(1), b = 598.2(1), c = 1351.3(1) pm, α = 92.23(1), β = 91.20(1), γ = 100.51(1)°, Z = 2, R₁ = 0.0441. 2 crystallizes in the space group P2₁/c with the lattice dimensions at 20 °C: a = 683.3(1), b = 1323.0(1), c = 1254.2(1) pm, β = 92.61(1)°, Z = 4, R₁ = 0.0361. According to the structure analysis 1 consists of planar C,N-heterocycles connected by hydrogen bridges forming an infinite chain along [110]. The basic heterocyclic skeleton of 2 is essentially planar and linked three-dimensionally through hydrogen bridges.     

Synthesis and X-ray Crystal Structure of a New Molecular Clip

The synthesis and X-ray crystal structure of a new molecular clip 2 was reported. It （C24H24N4O2, Mr = 400.47） crystallizes in the space group C2/c with a = 15.587（2）, b = 8.5805（12）, c = 15.259（2） A, β = 102.448（3）°, V= 1992.9 （5）A63, Z = 4, Dc = 1.335 g/cm63,μ = 0.087 mm^-1 and F（000） = 848. It remains monomeric in the crystal and a tape-like structure is formed in the crystal structure of molecular clip. The most unusual structural feature of 2 is the boat conformation of its cyclohexyl ring imposed by the ring fusion at C（9）-C（9a）.     

Synthesis and X-ray Crystal Structure of a Methylene-bridged Glycoluril Dimer

The synthesis and X-ray crystal structure of a methylene-bridged glycoluril dimer 2 was reported.The methylene-bridged glycoluril dimer 2(C38H36Br4N8O12,Mr=1116.35) crystallizes in space group P1 with a=10.5802(6),b=16.8469(9),c=24.7673(14) ,α=98.00,β=96.263(1),γ=101.606(1)o,V=4239.3(4)3,Z=4,Dc=1.749 g/cm3,μ=3.869 mm-1 and F(000)=2224.It crystallizes in an S-shaped conformation that displays two ethoxycarbonyl groups on each face of the molecule.     

Crystal Structure Analysis of the Repair of Iron Centers Protein YtfE and Its Interaction with NO

Molecular mechanisms underlying the repair of nitrosylated [Fe–S] clusters by the microbial protein YtfE remain poorly understood. The X‐ray crystal structure of YtfE, in combination with EPR, magnetic circular dichroism (MCD), UV, and ¹⁷O‐labeling electron spin echo envelope modulation measurements, show that each iron of the oxo‐bridged Fe^II–Fe^III diiron core is coordinatively unsaturated with each iron bound to two bridging carboxylates and two terminal histidines in addition to an oxo‐bridge. Structural analysis reveals that there are two solvent‐accessible tunnels, both of which converge to the diiron center and are critical for capturing substrates. The reactivity of the reduced‐form Fe^II–Fe^II YtfE toward nitric oxide demonstrates that the prerequisite for N₂O production requires the two iron sites to be nitrosylated simultaneously. Specifically, the nitrosylation of the two iron sites prior to their reductive coupling to produce N₂O is cooperative. This result suggests that, in addition to any repair of iron centers (RIC) activity, YtfE acts as an NO‐trapping scavenger to promote the NO to N₂O transformation under low NO flux, which precedes nitrosative stress.     

Functional Characterization and Crystal Structure of the Type II Peptidyl Carrier Protein ColA1a in Collismycins Biosynthesis†

Collismycins (COLs) are antibiotics characterized by a 2,2′‐bipyridine (2,2′‐BP) core composed of a trisubstituted ring A and an unmodified ring B. The 2,2′‐BP core, which possesses metal‐chelating ability and plays key roles in various biological activities of COLs, is biosynthesized by a nonribosomal peptide synthetase (NRPS)‐polyketide synthase (PKS) hybrid machinery. The starter module of the NRPS‐PKS hybrid machinery consists of a type II peptidyl carrier protein (PCP) ColA1a and an adenylation protein ColA1b. We here report the functional characterization of ColA1a and ColA1b in vitro, confirming their functions in selection and loading of picolinic acid (PA), instead of normal amino acid substrates, as the origin of ring B in COLs. The 2.1 Å crystal structure of ColA1a was solved, revealing structural features including the additional helices α1a, α1b and missing helix α3, which may reflect unique interactions of ColA1a with other NRPS‐PKS proteins/domains or substrate. Primary and tertiary structural comparison of ColA1a with other PCPs revealed the structural basis for their typical α‐helical bundle, providing a better understanding of the structural flexibility of PCPs. These results facilitate the starter module engineering for the generation of COL derivatives with ring B modifications in the future.     

一种新的环状膦酸酐的合成与X射线晶体结构

2 ,4 二叔丁基 6 甲氧基苯膦二硫化物 [MoxP(S) 2 ,1]与H2 O/吡啶封管反应分别以 3 0 %和 2 2 %的收率得到一种新的环状膦酸酐的异构体 2a和 2b .通过3 1PNMR对反应过程的追踪探讨了 2的形成机制 .X射线晶体结构解析表明异构体 2b分子中有一个不规则的含磷六元杂环 .环内最大键角为 12 7.7° ,最小键角为 97.9° .六元杂环中 ,除CC具有双键性质外 ,其余的CP ,CO和PO键均具有典型的单键特征 .两个环外PS双键处于含磷杂环的同一侧 ,表明 2b分子是一个cis结构 .该晶体属单斜晶系 ,C2 /c空间群 ,a =2 .0 486( 3 )nm ,b =2 .2 3 66( 4 )nm ,c =1.63 3 4( 3 )nm ,β =112 .98( 1)° ,Z =8,V =6.890 ( 1)nm3 ,Dc=1.2 61g·cm-3 ,F( 0 0 0 ) =2 80 0 .0 0 ,μ(MoKα) =3 .40cm-1.最终偏差因子R =0 .0 63 ,Rw=0 .0 83 .最高和最低残余峰分别为 0 .82和 -0 .48× 10 3 e-/nm3 .     

A Designed Conformational Shift To Control Protein Binding Specificity

In a conformational selection scenario, manipulating the populations of binding‐competent states should be expected to affect protein binding. We demonstrate how in silico designed point mutations within the core of ubiquitin, remote from the binding interface, change the binding specificity by shifting the conformational equilibrium of the ground‐state ensemble between open and closed substates that have a similar population in the wild‐type protein. Binding affinities determined by NMR titration experiments agree with the predictions, thereby showing that, indeed, a shift in the conformational equilibrium enables us to alter ubiquitin’s binding specificity and hence its function. Thus, we present a novel route towards designing specific binding by a conformational shift through exploiting the fact that conformational selection depends on the concentration of binding‐competent substates.     

新型含硫半抗原的晶体结构、构象分析及其电性研究

分析了一种新设计的含硫半抗原N-苯甲酰牛磺酰基苯丙氨酸的晶体结构,确证了其中的S原子为四面体构型,可以用来模拟酰胺键水解的过渡态,并且发现了几个分子间氢键.运用分子力学程序MOLGEN对此化合物进行了优化,并与含P半抗原进行了比较.然后又比较了N_S_C键和N_P_C键的旋转构象分析图,发现N_S_C键只有一个低能构象.最后用MOPAC程序(AM1参数)计算了S原子周围的电荷分布.发现S原子周围的电荷分布与P原子周围的电荷分布相似.     

混合配体的铜(Ⅱ)配合［Cu(C2H4O2N)(C12H8N2)(H2O)］.ClO4.2.5H2O的合成、性质及晶体结构

本文对甘氨酸、1,10-邻菲咯啉铜(Ⅱ)固体配合物进行了合成和表征,并利用X射线分析研究了其晶体结构。     

Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily

Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63‐residue member of this family, snakin‐1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin‐1, determined by a novel combination of racemic protein crystallization and radiation‐damage‐induced phasing (RIP), is reported. Racemic crystals of snakin‐1 and quasi‐racemic crystals incorporating an unnatural 4‐iodophenylalanine residue were prepared from chemically synthesized d ‐ and l ‐proteins. Breakage of the C?I bonds in the quasi‐racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces.     

Catalysis and Electron Transfer in De Novo Designed Helical Scaffolds

The relationship between protein structure and function is one of the greatest puzzles within biochemistry. De novo metalloprotein design is a way to wipe the board clean and determine what is required to build in function from the ground up in an unrelated structure. This Review focuses on protein design efforts to create de novo metalloproteins within alpha‐helical scaffolds. Examples of successful designs include those with carbonic anhydrase or nitrite reductase activity by incorporating a ZnHis₃ or CuHis₃ site, or that recapitulate the spectroscopic properties of unique electron‐transfer sites in cupredoxins (CuHis₂Cys) or rubredoxins (FeCys₄). This work showcases the versatility of alpha helices as scaffolds for metalloprotein design and the progress that is possible through careful rational design. Our studies cover the invariance of carbonic anhydrase activity with different site positions and scaffolds, refinement of our cupredoxin models, and enhancement of nitrite reductase activity up to 1000‐fold.     

Hydrothermal Synthesis,Crystal Structure and Characterizations of Two New Manganese(II) Phosphonates with a 3D Framework Structure

Two new manganese(II) phosphonates, (NH₄)Mn_2.5[(O₃PCH(OH)CO₂)₂(H₂O)] ( 1 ) and [NH₃(CH₂)₄NH₃]_0.5Mn_2.5[(O₃PCH(OH)CO₂)₂(H₂O)] ( 2 ) have been synthesized under hydrothermal conditions and structurally characterized by single‐crystal X‐ray diffraction as well as with infrared spectroscopy, elemental analysis and thermogravimetric analysis. The two isomorphous compounds feature a 3D framework structure. The Mn(1)O₆ and Mn(3)O₅ polyhedra are bridged by the CPO₃ tetraheda into a Mn^II phosphonate layer in ac‐plane. Mn(2)O₆ polyhedra are linked to each other by CPO₃ tetraheda to form infinite chains, which are connected to layers by carboxylate groups to form a 3D framework structure with channels along the a‐ and c‐axis, respectively. The NH₄⁺ ions or protonated 1, 4‐butylenediamine cations are located inside the channels along the a axis.