The rough energy landscape of superfolder GFP is linked to the chromophore

Many green fluorescent protein (GFP) variants have been developed for use as fluorescent tags, and recently a superfolder GFP (sfGFP) has been developed as a robust folding reporter. This new variant shows increased stability and improved folding kinetics, as well as 100% recovery of native protein after denaturation. Here, we characterize sfGFP, and find that this variant exhibits hysteresis as unfolding and refolding equilibrium titration curves are non-coincident even after equilibration for more than eight half-lives as estimated from kinetic unfolding and refolding studies. This hysteresis is attributed to trapping in a native-like intermediate state. Mutational studies directed towards inhibiting chromophore formation indicate that the novel backbone cyclization is responsible for the hysteresis observed in equilibrium titrations of sfGFP. Slow equilibration and the presence of intermediates imply a rough landscape. However, de novo folding in the absence of the chromophore is dominated by a smoother energy landscape than that sampled during unfolding and refolding of the post-translationally modified polypeptide.     

Molecular mechanisms of aquaporin biogenesis by the endoplasmic reticulum Sec61 translocon

The past decade has witnessed remarkable advances in our understanding of aquaporin (AQP) structure and function. Much, however, remains to be learned regarding how these unique and vitally important molecules are generated in living cells. A major obstacle in this respect is that AQP biogenesis takes place in a highly specialized and relatively inaccessible environment formed by the ribosome, the Sec61 translocon and the ER membrane. This review will contrast the folding pathways of two AQP family members, AQP1 and AQP4, and attempt to explain how six TM helices can be oriented across and integrated into the ER membrane in the context of current (and somewhat conflicting) translocon models. These studies indicate that AQP biogenesis is intimately linked to translocon function and that the ribosome and translocon form a highly dynamic molecular machine that both interprets and is controlled by specific information encoded within the nascent AQP polypeptide. AQP biogenesis thus has wide ranging implications for mechanisms of translocon function and general membrane protein folding pathways.     

GFP在绿色荧光裸鼠不同组织中的表达及分析

目的研究外源绿色荧光蛋白（green fluorescent protein,简称GFP）基因在BALB/c绿色荧光裸鼠主要器官组织中的表达及其差异。方法小动物成像系统和RT-PCR方法检测GFP的组织分布以及荧光表达水平情况。结果经活体荧光影像系统观察及PCR方法检测发现GFP可以在裸鼠多个器官组织中表达,其中在胰腺、心脏、全脑、皮肤、睾丸中表达量较高。结论外源绿色荧光蛋白可以在模型动物体内成功表达且稳定遗传,其中在胰腺组织中高表达。     

Role of the cytoplasmic segments of Sec61alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex

The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH(2) and COOH terminus of Sec61alpha was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61alpha. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.     

Green fluorescent protein (GFP) expression patterns in the olfactory epithelium of GFP transgenic cloned Jinhua pigs

Domestic pigs possess a well‐developed sense of smell. However, the morphology of the porcine olfactory epithelium (OE) is poorly understood. Recently, several strains of transgenic cloned pigs that are presumed to ubiquitously express green fluorescent protein (GFP) have been created. Thus, the purpose of this study was to elucidate the features of porcine OE using the tissues of GFP transgenic cloned pigs. Based on observations of Hematoxylin and Eosin staining and measurements of thickness, porcine OE tissue portions were classified into three categories (thick, standard, and thin). Cryosections revealed that the prominent GFP signals were expressed in olfactory sensory neurons (OSN), Bowman's glands, and olfactory nerve. A few GFP‐expressing sustentacular cells were seen; however, the intensity of GFP fluorescence was slight. In the thick portion, numerous GFP‐expressing polygonal OSN that did not possess dendrites were found. In the standard portions, GFP‐expressing cells had longitudinal dendrites. A few GFP‐expressing cells were found in the thin portion. In the thick and standard portions, most of the prominent GFP‐expressing cells were positive for olfactory marker protein. Moreover, double immunofluorescence staining with boiled GFP and Sox2 antibody revealed that GFP expression patterns in OSN are synchronized with Sox2 immunoreactive patterns.     

Analysis of allosteric signal transduction mechanisms in an engineered fluorescent maltose biosensor

We previously reported the construction of a family of reagentless fluorescent biosensor proteins by the structure-based design of conjugation sites for a single, environmentally sensitive small molecule dye, thus providing a mechanism for the transduction of ligand-induced conformational changes into a macroscopic fluorescence observable. Here we investigate the microscopic mechanisms that may be responsible for the macroscopic fluorescent changes in such Fluorescent Allosteric Signal Transduction (FAST) proteins. As case studies, we selected three individual cysteine mutations (F92C, D95C, and S233C) of Escherichia coli maltose binding protein (MBP) covalently labeled with a single small molecule fluorescent probe, N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), each giving rise to a robust FAST protein with a distinct maltose-dependent fluorescence response. The fluorescence emission intensity, anisotropy, lifetime, and iodide-dependent fluorescence quenching were determined for each conjugate in the presence and absence of maltose. Structure-derived solvent accessible surface areas of the three FAST proteins are consistent with experimentally observed quenching data. The D95C protein exhibits the largest fluorescence change upon maltose binding. This mutant was selected for further characterization, and residues surrounding the fluorophore coupling site were mutagenized. Analysis of the resulting mutant FAST proteins suggests that specific hydrogen-bonding interactions between the fluorophore molecule and two tyrosine side-chains, Tyr171 and Tyr176, in the open state but not the closed, are responsible for the dramatic fluorescence response of this construct. Taken together these results provide insights that can be used in future design cycles to construct fluorescent biosensors that optimize signaling by engineering specific hydrogen bonds between a fluorophore and protein.     

Biochemical evidence for the presence of two alpha-glucoside ABC-transport systems in the hyperthermophilic archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus can utilize different carbohydrates, such as starch, maltose and trehalose. Uptake of alpha-glucosides is mediated by two different, binding protein-dependent, ATP-binding cassette (ABC)-type transport systems. The maltose transporter also transports trehalose, whereas the maltodextrin transport system mediates the uptake of maltotriose and higher malto-oligosaccharides, but not maltose. Both transport systems are induced during growth on their respective substrates.     

Copper inhibits protein maturation in the secretory pathway by targeting the Sec61 translocon in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, proteins destined for secretion utilize the post-translational translocon machinery to gain entry into the endoplasmic reticulum. These proteins then mature by undergoing a number of post-translational modifications in different compartments of the secretory pathway. While these modifications have been well established for many proteins, to date only a few studies have been conducted regarding the conditions and factors affecting maturation of these proteins before entering into the endoplasmic reticulum. Here, using immunoblotting, microscopy, and spot test assays, we show that excess copper inhibits the Sec61 translocon function and causes accumulation of two well-known post-translationally translocated proteins, Gas1 (glycophospholipid-anchored surface protein) and CPY (carboxypeptidase Y), in the cytosol. We further show that the copper-sensitive phenotype of sec61-deficient yeast cells is ameliorated by restoring the levels of SEC61 through plasmid transformation. Furthermore, screening of translocation-defective Sec61 mutants revealed that sec61-22, bearing L80M, V134I, M248V, and L342S mutations, is resistant to copper, suggesting that copper might be inflicting toxicity through one of these residues. In conclusion, these findings imply that copper-mediated accumulation of post-translationally translocated proteins is due to the inhibition of Sec61.     

Chromophore Packing Leads to Hysteresis in GFP

Green fluorescent protein (GFP) possesses a unique folding landscape with a dual basin leading to the hysteretic folding behavior observed in experiment. While theoretical data do not have the resolution necessary to observe details of the chromophore during refolding, experimental results point to the chromophore as the cause of the observed hysteresis. With the use of NMR spectroscopy, which probes at the level of the individual residue, the hysteretic intermediate state is further characterized in the context of the loosely folded isomerized native-like state {N_iso} predicted in simulation. In the present study, several residues located in the lid of GFP indicate heterogeneity of the native states. Some of these residues show chemical shifts when the native-like intermediate {N_iso} responsible for GFP's hysteretic folding behavior is trapped. Observed changes in the chromophore are consistent with increased flexibility or isomerization in {N_iso} as predicted in recent theoretical work. Here, we observed that multiple chromophore environments within the native state are averaged in the trapped intermediate, linking chromophore flexibility to mispacking in the trapped intermediate. The present work is experimental evidence for the proposed final “locking” mechanism in GFP folding forming an incorrectly or loosely packed barrel under intermediate (hysteretic) folding conditions.     

Dual inducible expression of recombinant GFP and targeted antisense RNA in Lactococcus lactis

The food-grade status and probiotic activity of lactic acid bacteria (LAB) make them attractive hosts for production and oral delivery of therapeutic heterologous vaccines and other proteins, yet these bacteria currently do not achieve recombinant protein expression at levels comparable to those seen in Escherichia coli and Saccharomyces cerevisiae. Limited levels of expressed recombinant protein per cell most likely constrain the vaccine’s immunogenic potential with respect to the magnitude and specificity of the immune response. With the goal of increasing recombinant protein expression per cell in Lactococcus lactis IL1403, a model LAB, we have constructed and evaluated a new vector that permits simultaneously-induced expression of GFP, a model recombinant protein, and antisense RNA inhibition of the clpP-encoded intracellular protease. While silencing of the rational target clpP does not lead to increased GFP per cell, the new dual-expression system provides an efficient and potentially high-throughput metabolic engineering tool for strain improvement.     

绿色荧光蛋白标记下镰刀菌侵染地黄的组织学观察

为分析对地黄有较强致病性的尖孢镰刀菌的侵染机制,本研究采用携带有潮霉素抗性标记的强组成型表达载体pCT74,经PEG-CaCl₂介导的原生质体转化法导入镰刀菌,获得荧光信号强烈的sGFP标记菌株,并采用喷施接种和根部灌根接种。研究发现镰刀菌首先在地黄外部聚集繁殖,并通过伤口或气孔等通道侵入地黄维管组织,后逐渐导致周围海绵组织破裂,进而致使地下根茎逐渐变黑腐烂;由于地黄叶部气孔发达,使得镰刀菌由叶部侵入速度要快于根部灌根接种;不同孢子浓度接种实验表明镰刀菌对寄主的致害程度与其在叶部与根部的接种浓度并不呈相关性。进一步在接种处理后采集地黄叶部、茎部和根部组织提取真菌DNA后进行PCR扩增发现：在根部接种60h后即能检测到镰刀菌的侵入,经84h后侵入到地黄茎部组织,经96h后侵入到地黄叶部组织。该标记菌株可为今后探索地黄连作栽培中真菌病害大规模爆发的根际生物学过程提供研究材料。     

Cell-to-cell transport via the lumen of the endoplasmic reticulum

Plasmodesmata are plasma membrane‐lined channels through which cytoplasmic molecules move from cell‐to‐cell in plants. Most plasmodesmata contain a desmotubule, a central tube of endoplasmic reticulum (ER), that connects the ER of adjacent cells. Here we demonstrate that molecules of up to 10.4 kDa in size can move between the ER lumen of neighbouring leaf trichome or epidermal cells via the desmotubule lumen. Fluorescent molecules of up to 10 kDa, microinjected into the ER of Nicotiana trichome cells, consistently moved into the ER and nuclei of neighbouring trichome cells. This movement occurred more rapidly than movement via the cytoplasmic pathway. A fluorescent 3‐kDa dextran microinjected into the ER of a basal trichome cell moved into the ER and nuclei of epidermal cells across a barrier to cytoplasmic movement. We constructed a 10.4‐kDa recombinant ER‐lumenal reporter protein (LRP) from a fragment of the endogenous ER‐lumenal binding protein AtBIP1. Following transient expression of the LRP in the ER of Tradescantia leaf epidermal cells, it often moved into the nuclear envelopes of neighbouring cells. However, green fluorescent protein targeted to the ER lumen (ER‐GFP) did not move from cell to cell. We propose that the ER lumen of plant cells is continuous with that of their neighbours, and allows movement of small ER‐lumenal molecules between cells.     

Molecular strategies for protein stabilization: the case of a trehalose/maltose-binding protein from Thermus thermophilus

The trehalose/maltose-binding protein (MalE1) is one component of trehalose and maltose uptake system in the thermophilic organism Thermus thermophilus. MalE1 is a monomeric 48 kDa protein predominantly organized in alpha-helix conformation with a minor content of beta-structure. In this work, we used Fourier-infrared spectroscopy and in silico methodologies for investigating the structural stability properties of MalE1. The protein was studied in the absence and in the presence of maltose as well as in the absence and in the presence of SDS at different p(2)H values (neutral p(2)H and at p(2)H 9.8). In the absence of SDS, the results pointed out a high thermostability of the MalE1 alpha-helices, maintained also at basic p(2)H values. However, the obtained data also showed that at high temperatures the MalE1 beta-sheets underwent to structural rearrangements that were totally reversible when the temperature was lowered. At room temperature, the addition of SDS to the protein solution slightly modified the MalE1 secondary structure content by decreasing the protein thermostability. The infrared data, corroborated by molecular dynamics simulation experiments performed on the structure of MalE1, indicated that the protein hydrophobic interactions have an important role in the MalE1 high thermostability. Finally, the results obtained on MalE1 are also discussed in comparison with the data on similar thermostable proteins already studied in our laboratories.     

Exploring the folding pathway of green fluorescent protein through disulfide engineering

We have introduced two disulfide crosslinks into the loop regions on opposite ends of the beta barrel in superfolder green fluorescent protein (GFP) in order to better understand the nature of its folding pathway. When the disulfide on the side opposite the N/C‐termini is formed, folding is 2× faster, unfolding is 2000× slower, and the protein is stabilized by 16 kJ/mol. But when the disulfide bond on the side of the termini is formed we see little change in the kinetics and stability. The stabilization upon combining the two crosslinks is approximately additive. When the kinetic effects are broken down into multiple phases, we observe Hammond behavior in the upward shift of the kinetic m‐value of unfolding. We use these results in conjunction with structural analysis to assign folding intermediates to two parallel folding pathways. The data are consistent with a view that the two fastest transition states of folding are "barrel closing" steps. The slower of the two phases passes through an intermediate with the barrel opening occurring between strands 7 and 8, while the faster phase opens between 9 and 4. We conclude that disulfide crosslink‐induced perturbations in kinetics are useful for mapping the protein folding pathway.     

Construction of an Expression System for Aqualysin I in Escherichia coli That Gives a Markedly Improved Yield of the Enzyme Protein

An expression system for aqualysin I from Thermus aquaticus YT-1, a thermophilic serine protease belonging to the proteinase K family, in Escherichia coli is available, but the efficiency of production has been rather low for detailed analysis of the product. We developed a maltose biding protein (MBP)-fused proaqualysin I expression plasmid (pMAQ-c2Δ) in which MBP is attached to the N-terminus of proaqualysin I. MBP appeared effectively to suppress the folding-promoting activity of the N-terminal propeptide when the bacteria were grown at 30 °C, leading to a massive accumulation of fusion aqualysin I precursor. The precursor was converted efficiently to mature aqualysin I by heat treatment at 70 °C, enabling us to obtain 40 times more aqualysin I than is available using expression systems such as pAQNΔC105. By analyzing the product of the pMAQ-c2Δ-derived inactive mutant expression vector, pMAQ-S222A, it was confirmed that aqualysin I was initially expressed as a whole fusion protein and then processed autocatalytically.     

Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungusUstilago maydis

Pathogenic development ofUstilago maydis, the causative agent of corn smut disease, is a multistep process. Compatible yeast-like cells fuse and this generates the infectious dikaryon which grows filamentously. Having entered the plant the dikaryon induces tumors in its host in which massive proliferation of fungal material, karyogamy and spore formation occur. In order to follow fungal development from the initial steps to the final stage we have expressed the green fluorescent protein (GFP) fromAequorea victoria as a vital marker inU. maydis and demonstrate that GFP-tagged strains can be used to study host-pathogen interactions in vivo.     

Molecular adaptation strategies to high temperature and thermal denaturation mechanism of the D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose uptake system. In the hyperthermophilic archaeon T. litoralis this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds both trehalose and maltose with high affinity. In this work we used differential scanning calorimetry studies together with a detailed analysis, at the molecular level, of the three-dimensional protein structure to shed light on the basis of the high thermostability exhibited by the recombinant TMBP from the archaeon T. litoralis. The obtained data suggest that the presence of trehalose does not change the overall mechanism of the denaturation of this protein but it selectively modifies the stability of the TMBP structural domains.     

Green fluorescence induced by EF‐hand assembly in a split GFP system

The affinity between the 1–157 and 158–238 fragments of green fluorescent protein (GFP) is too low for spontaneous in vivo reassembly of the protein upon co-expression of the two fragments. This prevents chromophore maturation and the cells lack GFP fluorescence. We have utilized the very high affinity between the two EF-hands of calbindin D_9k to facilitate GFP assembly from its fragments and to introduce a calcium dependent molecular switch. In GFPN-EF1, residues 1–157 of GFP are fused to residues 1–43 of calbindin, and in EF2-GFPC, residues 44–75 of calbindin are fused to residues 158–238 of GFP. When co-expressed, GFPN-EF1 and EF2-GFPC associate spontaneously and rapidly resulting in a folded reconstituted protein with bright GFP fluorescence. The high affinity of GFPN-EF1 for EF2-GFPC leads to brighter fluorescence of the cells compared to cells with a control constructs carrying leucine zippers (Wilson et al., Nature Methods 2004;3:255). The complex of GFPN-EF1 and EF2-GFPC was purified from cells using metal-ion chelate chromatography and the temperature dependence of GFP fluorescence was found to be calcium dependent. The GFPN-EF1 and EF2-GFPC fragments were separated by ion exchange chromatography. The assembly of the fragments was found to be reversible and the complex was regained upon mixing, as evidenced by surface plasmon resonance (SPR) data. The affinity between GFPN-EF1 and EF2-GFPC as well as rates of association and dissociation were found to be Ca²⁺-dependent.     

PvdM of fluorescent pseudomonads is required for the oxidation of ferribactin by PvdP in periplasmic pyoverdine maturation

Fluorescent pseudomonads such as Pseudomonas aeruginosa or Pseudomonas fluorescens produce pyoverdine siderophores that ensure iron-supply in iron-limited environments. After its synthesis in the cytoplasm, the nonfluorescent pyoverdine precursor ferribactin is exported into the periplasm, where the enzymes PvdQ, PvdP, PvdO, PvdN, and PtaA are responsible for fluorophore maturation and tailoring steps. While the roles of all these enzymes are clear, little is known about the role of PvdM, a human renal dipeptidase–related protein that is predicted to be periplasmic and that is essential for pyoverdine biogenesis. Here, we reveal the subcellular localization and functional role of PvdM. Using the model organism P. fluorescens, we show that PvdM is anchored to the periplasmic side of the cytoplasmic membrane, where it is indispensable for the activity of the tyrosinase PvdP. While PvdM does not share the metallopeptidase function of renal dipeptidase, it still has the corresponding peptide-binding site. The substrate of PvdP, deacylated ferribactin, is secreted by a ΔpvdM mutant strain, indicating that PvdM prevents loss of this periplasmic biosynthesis intermediate into the medium by ensuring the efficient transfer of ferribactin to PvdP in vivo. We propose that PvdM belongs to a new dipeptidase-related protein subfamily with inactivated Zn²⁺ coordination sites, members of which are usually genetically linked to TonB-dependent uptake systems and often associated with periplasmic FAD-dependent oxidoreductases related to d-amino acid oxidases. We suggest that these proteins are necessary for selective binding, exposure, or transfer of specific d- and l-amino acid–containing peptides and other periplasmic biomolecules in manifold pathways.     

Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli

A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structure-function relationships. Although these maps can be predicted directly from amino acid sequence, the predictions are more accurate if combined with experimental data, which are usually obtained by fusing a reporter protein to the C-terminus of the protein. However, as reporter proteins are large, they cannot be used to report on the cytoplasmic/periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli.