酪氨酸酶催化氧化对丝素蛋白结构与性能的影响

采用酪氨酸酶对丝素蛋白催化氧化,考察了酶促氧化反应对丝素蛋白结构及丝素膜性能的影响。研究结果表明,酪氨酸酶可催化氧化丝素蛋白中酪氨酸残基生成多巴和多巴醌结构衍生物,并且两者含量随催化反应时间延长呈波动性变化;酶促反应后丝素蛋白中游离氨基含量下降,丝素风干膜断裂强度增加,表明酶促氧化中丝素大分子间发生自交联。XRD结果表明酪氨酸酶催化氧化对丝素蛋白二级结构有一定影响;SEM显示酶促改性可能影响丝素蛋白冷冻干燥膜多孔形态结构。     

Sulfated fibroin, a novel sulfated peptide derived from silk, inhibits human immunodeficiency virus replication in vitro

We prepared two kinds of sulfated silk fibroins, SclFib30 and SclFib31, which contain different amounts of sulfate. These sulfated silk fibroins have anti-HIV-1 activity in vitro, apparently due to interference with the adsorption of virus particles to CD4+ cells, and completely blocked virus binding to the cells at a concentration of 100 microg/ml. Sulfated fibroins also abolished cell-to-cell infection-induced syncytium formation upon cocultivation of MOLT-4 and MOLT-4/HIV-IIIB cells, suggesting that they would interfere with gp120 and prevent the formation of gp120/CD4 complex. Silk is used in biomaterials such as surgical sutures and is believed to be a safe material for humans. In accordance with low anticoagulant activity and high anti-HIV-1 activity against both X4 HIV-1 and R5 HIV-1 strains, sulfated silk fibroins have potential as antiviral material such for a vaginal anti-HIV formulation.     

Sulfated Fibroin,a Novel Sulfated Peptide Derived from Silk,Inhibits Human Immunodeficiency Virus Replication in Vitro

We prepared two kinds of sulfated silk fibroins, SclFib30 and SclFib31, which contain different amounts of sulfate. These sulfated silk fibroins have anti-HIV-1 activity in vitro, apparently due to interference with the adsorption of virus particles to CD4⁺ cells, and completely blocked virus binding to the cells at a concentration of 100 μg/ml. Sulfated fibroins also abolished cell-to-cell infection-induced syncytium formation upon cocultivation of MOLT-4 and MOLT-4/HIV-1_IIIB cells, suggesting that they would interfere with gp120 and prevent the formation of gp120/CD4 complex. Silk is used in biomaterials such as surgical sutures and is believed to be a safe material for humans. In accordance with low anticoagulant activity and high anti-HIV-1 activity against both X4 HIV-1 and R5 HIV-1 strains, sulfated silk fibroins have potential as antiviral material such for a vaginal anti-HIV formulation.     

Transitions in microtubule C-termini conformations as a possible dendritic signaling phenomenon

We model the dynamical states of the C-termini of tubulin dimers that comprise neuronal microtubules. We use molecular dynamics and other computational tools to explore the time-dependent behavior of conformational states of a C-terminus of tubulin within a microtubule and assume that each C-terminus interacts via screened Coulomb forces with the surface of a tubulin dimer, with neighboring C-termini and also with any adjacent microtubule-associated protein 2 (MAP2). Each C-terminus can either bind to the tubulin surface via one of the several positively charged regions or can be allowed to explore the space available in the solution surrounding the dimer. We find that the preferential orientation of each C-terminus is away from the tubulin surface but binding to the surface may also take place, albeit at a lower probability. The results of our model suggest that perturbations generated by the C-termini interactions with counterions surrounding a MAP2 may propagate over distances greater than those between adjacent microtubules. Thus, the MAP2 structure is able to act as a kind of biological wire (or a cable) transmitting local electrostatic perturbations resulting in ionic concentration gradients from one microtubule to another. We briefly discuss the implications the current dynamic modeling may have on synaptic activation and potentiation.     

蜘蛛丝的分子结构与力学性能研究

蜘蛛丝尤其是蜘蛛大囊状腺产生的拖丝,具有独特的机械性能,是自然界颇具应用潜力的生物材料。现代分子生物学技术使蜘蛛丝蛋白基因得以克隆,通过高分子物理化学手段方法的利用,有利于揭示蜘蛛丝蛋白质序列、分子结构、以及分子结构和力学性能之间的关系。对不同种类蜘蛛丝蛋白的深入研究,将为基因工程方法人工合成并改造蜘蛛丝成为可能。     

Structural analysis of silk with 13C NMR chemical shift contour plots.

The polymorphic structures of silk fibroins in the solid state were examined on the basis of a quantitative relationship between the 13C chemical shift and local structure in proteins. To determine this relationship, 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues, and the C alpha chemical shift plot for Gly residues were prepared using atomic co-ordinates from the Protein Data Bank and 13C NMR chemical shift data in aqueous solution reported for 40 proteins. The 13C CP/MAS NMR chemical shifts of Ala, Ser and Gly residues of Bombyx mori silk fibroin in silk I and silk II forms were used along with 13C CP/MAS NMR chemical shifts of Ala residues of Samia cynthia ricini silk fibroin in beta-sheet and alpha-helix forms for the structure analyses of silk fibroins. The allowed regions in the 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues for the structures in silk fibroins, i.e. Silk II, Silk I and alpha-helix, were determined using their 13C isotropic NMR chemical shifts in the solid state. There are two area of the phi,psi map which satisfy the observed Silk I chemical shift data for both the C alpha and C beta carbons of Ala and Ser residues in the 13C chemical shift contour plots.     

The carboxy-terminus,a key regulator of protein function

All proteins end with a carboxyl terminus that has unique biophysical properties and is often disordered. Although there are examples of important C-termini functions, a more global role for the C-terminus is not yet established. In this review, we summarize research on C-termini, a unique region in proteins that cells exploit. Alternative splicing and proteolysis increase the diversity of proteins and peptides in cells with unique C-termini. The C-termini of proteins contain minimotifs, short peptides with an encoded function generally characterized as binding, posttranslational modifications, and trafficking. Many of these activities are specific to minimotifs on the C-terminus. Approximately 13% of C-termini in the human proteome have a known minimotif, and the majority, if not all of the remaining termini have conserved motifs inferring a function that remains to be discovered. C-termini, their predictions, and their functions are collated in the C-terminome, Proteus, and Terminus Oriented Protein Function INferred Database (TopFIND) database/web systems. Many C-termini are well conserved, and some have a known role in health and disease. We envision that this summary of C-termini will guide future investigation of their biochemical and physiological significance.     

Spider minor ampullate silk proteins are constituents of prey wrapping silk in the cob weaver Latrodectus hesperus

Spiders spin high performance fibers with diverse biological functions and mechanical properties. Molecular and biochemical studies of spider prey wrapping silks have revealed the presence of the aciniform silk fibroin AcSp1-like. In our studies we demonstrate the presence of a second distinct polypeptide present within prey wrapping silk. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called MiSp1-like and demonstrate that its protein product is a constituent of prey wrap silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein database using the amino acid sequence of MiSp1-like revealed similarity to the conserved C-terminal domain of silk family members. In particular, MiSp1-like showed the highest degree of sequence similarity to the nonrepetitive C-termini of published orb-weaver minor ampullate fibroin molecules. Analysis of the internal amino acid sequence of the black widow MiSp1-like revealed polyalanine stretches interrupted by glycine residues and glycine-alanine couplets within MiSp1-like as well as repeats of the heptameric sequence AGGYGQG. Real-time quantitative PCR analysis demonstrates that the MiSp1-like gene displays a minor ampullate gland-restricted pattern of expression. Furthermore, amino acid composition analysis, coupled with scanning electron microscopy of raw wrapping silk, supports the assertion that minor ampullate silks are important constituents of black widow spider prey wrap silk. Collectively, our findings provide direct molecular evidence for the involvement of minor ampullate fibroins in swathing silks and suggest composite materials play an important role in the wrap attack process for cob-weavers.     

Mechanical features of various silkworm crystalline considering hydration effect via molecular dynamics simulations

Silk materials are receiving significant attention as base materials for various functional nanomaterials and nanodevices, due to its exceptionally high mechanical properties, biocompatibility, and degradable characteristics. Although crystalline silk regions are composed of various repetitive motifs with differing amino acid sequences, how the effect of humidity works differently on each of the motifs and their structural characteristics remains unclear. We report molecular dynamics (MD) simulations on various silkworm fibroins composed of major motifs (i.e. (GAGAGS)_n, (GAGAGA)_n, and (GAGAGY)_n) at varying degrees of hydration, and reveal how each major motifs of silk fibroins change at each degrees of hydration using MD simulations and their structural properties in mechanical perspective via steered molecular dynamics simulations. Our results explain what effects humidity can have on nanoscale materials and devices consisting of crystalline silk materials.     

Regulation of serotonin receptor function in the nervous system by lipid rafts and adaptor proteins

5-HT is a phylogenetically conserved monoaminergic neurotransmitter which is crucial for a number of physiological processes and is dysregulated in several disease states including depression, anxiety and schizophrenia. 5-HT neurons in the central nervous system are localized in the raphe nuclei and project to a wide range of target areas. 5-HT exerts its functions through 14 subtypes of 5-HT receptors. The tertiary structures of seven transmembrane 5-HT receptors contain several important features, including cholesterol consensus motifs, prominent intracellular loops and free C-termini. Alterations of cholesterol levels affect binding of ligands to 5-HT receptors and cholesterol-enriched microdomains in the cell membrane, termed lipid rafts, regulate 5-HT receptor internalization and signaling. The intracellular loops and the C-termini of 5-HT receptors provide binding sites for interacting adaptor proteins. Adaptor proteins affect internalization, desensitization as well as G-protein dependent and independent signaling via 5-HT receptors. We will here briefly review recent progress on the role of lipid rafts and adaptor proteins in the regulation of localization, trafficking, signaling and ligand bias of 5-HT receptors.     

Comparison of fibroin cDNAs from webspinning insects: insight into silk formation and function

Embiopterans (webspinning insects) are renowned for their prolific use of silk. These organisms spin silk to construct elaborate networks of tubes in which they live, forage, and reproduce. The silken galleries are essential for protecting these soft-bodied insects from predators and other environmental hazards. Despite the ecological importance of embiopteran silk, very little is known about its constituent proteins. Here, we characterize the silk protein cDNAs from four embiopteran species to better understand the function and evolution of these adaptive molecules. We show that webspinner fibroins (silk proteins) are highly repetitive in sequence and possess several conserved characteristics, despite differences in habitat preferences across species. The most striking similarities are in the codon usage biases of the fibroin genes, particularly in the repetitive regions, as well as sequence conservation of the carboxyl-terminal regions of the fibroins. Based on analyses of the silk genes, we propose hypotheses regarding codon bias and its effect on the translation and replication of these unusual genes. Furthermore, we discuss the significance of specific fibroin motifs to the mechanical and structural characteristics of silk fibers. Lastly, we report that the conservation of webspinner fibroin carboxyl-terminal regions suggests that fiber formation may occur through a mechanism analogous to that found in Lepidoptera. From these results, insight is gained into the tempo and mode of evolution that has shaped embiopteran fibroins.     

Egg case protein-1. A new class of silk proteins with fibroin-like properties from the spider Latrodectus hesperus

Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In our studies we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus. Analysis of the physical structure of egg case silk using scanning electron microscopy demonstrates the presence of small and large diameter fibers. By using the strong protein denaturant 8 M guanidine hydrochloride to solubilize the fibers, we demonstrated by SDS-PAGE and protein silver staining that an abundant component of egg case silk is a 100-kDa protein doublet. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called ecp-1, which encodes for one of the protein components of the 100-kDa species. BLAST searches of the NCBInr protein data base using the primary sequence of ECP-1 revealed similarity to fibroins from spiders and silkworms, which mapped to two distinct regions within the ECP-1. These regions contained the conserved repetitive fibroin motifs poly(Ala) and poly(Gly-Ala), but surprisingly, no larger ensemble repeats could be identified within the primary sequence of ECP-1. Consistent with silk gland-restricted patterns of expression for fibroins, ECP-1 was demonstrated to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands. ECP-1 monomeric units were also shown to assemble into higher aggregate structures through the formation of disulfide bonds via a unique cysteine-rich N-terminal region. Collectively, our findings provide new insight into the components of egg case silk and identify a new class of silk proteins with distinctive molecular features relative to traditional members of the spider silk gene family.     

Quaternary dynamics of αB-crystallin as a direct consequence of localised tertiary fluctuations in the C-terminus

The majority of proteins exist in vivo within macromolecular assemblies whose functions are dependent on dynamical processes spanning a wide range of time scales. One such assembly is formed by the molecular chaperone αB-crystallin that exists in a variety of exchanging oligomeric states, centred on a mass of approximately 560 kDa. For many macromolecular assemblies, including αB-crystallin, the inherent dynamics, heterogeneity and high mass contribute to difficulties in quantitative studies. Here, we demonstrate a strategy based on correlating solution-state nuclear magnetic resonance spectroscopy and mass spectrometry data to characterize simultaneously the organization and dynamics of the polydisperse αB-crystallin ensemble. We show that protomeric dimers assemble into oligomers via the binding of extended C-termini, with each monomer donating and receiving one terminus. Moreover, we establish that the C-termini undergo millisecond fluctuations that regulate the interconversion of oligomeric forms. The combined biophysical approach allows construction of an energy profile for a single monomer that completely describes the equilibrium dynamics of the ensemble. It also facilitates an analysis of dynamics spanning the millisecond to hour time scales and secondary to quaternary structural levels, and provides an approach for, obtaining simultaneously detailed structural, thermodynamic and kinetic information on a heterogeneous protein assembly.     

The C-termini of tubulin and the specific geometry of tubulin substrates influence the depolymerization activity of MCAK

MCAK is a Kinesin-13 that depolymerizes microtubules (MTs) and regulates MT dynamics. We used subtilisin-treated MTs (MTs lacking the C-termini of α- and β-tubulin) and alternative tubulin substrates to study which structural and geometrical features of the MT are critical for MCAK activity. We found that removal of the C-termini significantly decreased the efficiency of MCAK-induced depolymerization, which was not due to a reduction of end-specific binding. We also found that depolymerization of SMTs led to an increase in the stabilization of curved oligomeric tubulin products. Using alternative tubulin substrates with different geometries, we found that MCAK depolymerized parallel and anti-parallel tubulin sheets. However, MCAK did not depolymerize tubulin rings regardless of the presence or absence of the tubulin C-termini. We propose that localization of MCAK to the ends of MTs is independent of tubulin C-termini, that MCAK stabilizes a curved conformation at the end of the MT, and that efficient release of this complex is dependent on the presence of the C-termini of tubulin.αβ     

Multimerization of the mouse TATA-binding protein (TBP) driven by its C-terminal conserved domain.

The conformational states of the mouse TATA-binding protein (TBP) in solution were studied. A histidine tag and a factor Xa recognition site-carrying mouse TBP was expressed in E. coli, highly purified, and its fundamental functions as a TBP were demonstrated. We analyzed the molecular states of mouse TBP by gel filtration and glycerol gradient sedimentation, and found that TBP forms heterogeneous multimers in solution. Direct binding of TBP molecules to each other was proven by the far-Western procedure. Analyses using TBPs truncated at the N- and C-termini demonstrated that the functionally important C-terminal domain was responsible for homomultimer formation, and the N-terminal domain enhances multimerization. Furthermore, it was found that the TATA sequence dissociates homomultimers, and only monomeric TBP binds to the TATA-box. We suggest that TBP shares structural motifs in the C-terminal conserved domain for intermolecular interaction and TATA-binding.     

The N-terminus of KIR6.2 limits spontaneous bursting and modulates the ATP-inhibition of KATP channels

KATP channels are heteromultimers of a sulfonylurea receptor SUR and KIR6.2 with the inward rectifier forming the pore which is regulated by SUR. We have examined the contributions of the cytoplasmic domains of KIR6.2 to control of spontaneous bursting and ATP-inhibition in human SUR1/KIR6.2 KATP channels. Truncations of the N-terminus of KIR6.2 nearly eliminate transitions to interburst closed states without affecting the open or intraburst closed states, thus producing SUR1/DeltaNKIR6.2 channels with an extremely high open probability in the absence of nucleotides. These channels have a decrease apparent ATP-sensitivity which is consistent with the involvement of the N-terminus in a transition to an interburst closed state that preferentially binds inhibitory ATP. Mutations in both the N- and proximal C-termini of KIR6.2 can synergistically attenuate the ATP-inhibition. The results identify the N-terminus of KIR6.2 as a determinant of the interburst kinetics of KATP channels and suggest that the two cytoplasmic domains of KIR6.2 participate in ATP-inhibitory gating through distinct mechanisms.     

大腹园蛛拖丝蛋白一级结构初步研究

采用部分酸水解的方法对大腹园蛛（Ａｒａｎｅｕｓｖｅｎｔｒｏｃｏｓｕｓ）拖丝纤维蛋白进行水解,通过反相高液相色谱分离到一些重复小肽片段,对其序列分析表明,该蜘蛛拖丝蛋白与肖蛸科的棒络新妇蛛（Ｎｅｐｈｉｌａｃｌａｖｉｐｅｓ）丝蛋白的序列比较,有一个完全相同的肽段ＧＹＧＰＧ,其余所测片段测存在差异,显示不同种属的蜘蛛丝在一级结构上的同异,并探讨了结构和功能的关系。     

Scavenger receptor class B, type I (SR-BI) homo-dimerizes via its C-terminal region: fluorescence resonance energy transfer analysis

Expression of the scavenger receptor class B, type I (SR-BI) receptor facilitates high density lipoprotein cholesterol transport and correlates with protection against atherosclerosis. Studies have shown that SR-BI self-associates, but many of the techniques used to characterize SR-BI homo-oligomerization were wrought with the prospect of producing artifacts. Therefore, we employed fluorescence resonance energy transfer (FRET) to visualize SR-BI homo-oligomerization with the benefit of gaining information about its quaternary structure in the absence of typical membrane receptor artifacts. To this end, SR-BI was tagged at the N- or C-termini with either cyan (CFP) or yellow (YFP) fluorescent protein. To test whether SR-BI subunits oligomerize through N-N, N-C or C-C terminal interactions, we co-expressed the appropriate SR-BI fusion protein combinations in COS-7 cells and measured live-cell FRET following acceptor photobleaching. We did not observe FRET with co-transfection of SR-BI with CFP and YFP at the N-termini nor at the N- and C-termini, suggesting that the N-termini are not proximal to each other or to the C-termini. However, FRET was observed with co-transfection of SR-BI with CFP and YFP at the C-termini, suggesting that the C-terminal ends are within 10 nm of each other, consistent with SR-BI dimerization via its C-terminal region.     

Structural characterization of intracellular C-terminal domains of group III metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) are regulated by interacting proteins that mostly bind to their intracellular C-termini. Here, we investigated if mGluR6, mGluR7a and mGluR8a C-termini form predefined binding surfaces or if they were rather unstructured. Limited tryptic digest of purified peptides argued against the formation of stable globular folds. Circular dichroism, ¹H NMR and ¹H¹⁵N HSQC spectra indicated the absence of rigid secondary structure elements. Furthermore, we localized short linear binding motifs in the unstructured receptor domains. Our data provide evidence that protein interactions of the analyzed mGluR C-termini are mediated rather by short linear motifs than by preformed folds.