Picosecond time-resolved fluorescence from blue-emitting chromophore variants Y66F and Y66H of the green fluorescent protein

The origin of the low steady-state fluorescence quantum yield of some blue-emitting variants of the green fluorescent protein (GFP) is investigated in single-site mutants in which the tyrosine residue at position 66 has been replaced by phenylalanine or by histidine. Time-resolved fluorescence measurements reveal excited-state lifetimes of 74 ps (Y66F) and 0.9 ns (Y66H) at room temperature that increase to values close to the radiative limit as the temperature is lowered. These short lifetimes are explained by temperature-dependent internal conversion. The pronounced difference between the room-temperature lifetimes of the two mutants suggests that hydrogen bonding of the distal aromatic ring plays a more important role than tight packing in the fixation of the chromophore.     

应用固定化金属亲和层析纯化His6融合甲状旁腺激素相关蛋白

应用原核细胞高效表达载体在大肠杆菌表达了N端带 6个组氨酸的重组人PTHrP。利用其对金属鳌合层析介质的亲合性 ,采用含盐咪唑梯度洗脱 ,一步纯化即可得到纯度大于 90 %且具有生物学活性的重组PTHrP ,并进一步制备了抗PTHrP单克隆抗体。应用His6纯化标签的固定化金属亲和层析 (IMAC) ,极大地方便了目的蛋白的纯化 ,为进一步研究PTHrP的生物学特性及其在临床诊断治疗中的应用奠定了基础。     

The effects of ligand exchange and mobility on the peroxidase activity of a bacterial cytochrome c upon unfolding

The effect on the heme environment upon unfolding Paracoccus versutus ferricytochrome c-550 and two site-directed variants, K99E and H118Q, has been assessed through a combination of peroxidase activity increase and one-dimensional NMR spectroscopy. At pH 4.5, the data are consistent with a low- to high-spin heme transition, with the K99E mutation resulting in a protein with increased peroxidase activity in the absence of or at low concentrations of denaturant. Furthermore, the mobility of the polypeptide chain at pH 4.5 for the wild-type protein has been monitored in the absence and presence of denaturant through heteronuclear NMR experiments. The results are discussed in terms of local stability differences between bacterial and mitochondrial cytochromes c that are inferred from peroxidase activity assays. At pH 7.0, a mixture of misligated heme states arising from protein-based ligands assigned to lysine and histidine is detected. At low denaturant concentrations, these partially unfolded misligated heme forms inhibit the peroxidase activity. Data from the K99E mutation at pH 7.0 indicate that K99 is not involved in heme misligation, whereas histidine coordination is proven by the data from the H118Q variant.     

Histone Tail Sequences Balance Their Role in Genetic Regulation and the Need To Protect DNA against Destruction in Nucleosome Core Particles Containing Abasic Sites

Abasic sites (AP) are produced 10 000 times per day in a single cell. Strand cleavage at AP is accelerated ≈100-fold within a nucleosome core particle (NCP) compared to free DNA. The lysine-rich N-terminal tails of histone proteins catalyze single-strand breaks through a mechanism used by base-excision-repair enzymes, despite the general dearth of glutamic acid, aspartic acid, and histidine—the amino acids that are typically responsible for deprotonation of Schiff base intermediates. Incorporating glutamic acid, aspartic acid, or histidine proximal to lysine residues in histone N-terminal tails increases AP reactivity as much as sixfold. The rate acceleration is due to more facile DNA cleavage of Schiff-base intermediates. These observations raise the possibility that histone proteins could have evolved to minimize the presence of histidine, glutamic acid, and aspartic acid in their lysine-rich N-terminal tails to guard against enhancing the toxic effects of DNA damage.     

Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position

In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.     

Functional tuning of a salvaged green fluorescent protein variant with a new sequence space by directed evolution

We previously reported a method, designated functional salvagescreen (FSS), to generate protein lineages with new sequencespaces through the functional or structural salvage of a defectiveprotein by employing green fluorescent protein (GFP) as a modelprotein. Here, in an attempt to mimic a step in the naturalevolution process of proteins, the functionally salvaged mutantGFP-I5 with new sequence space, but showing low fluorescenceintensity and stability, was selected and fine-tuned by directedevolution. During a course of functional tuning, GFP-I5 wasfound to evolve rapidly, recovering the spectral traits to thoseof the parent GFPuv. The mutant 3E4 from the third round ofdirected evolution possessed four substitutions; three (F64L,E111V and K166Q) were at the original GFP gene and the other(K8N) at the inserted segment. The fluorescence intensity of3E4 was 28-fold stronger than GFP-I5, and other spectral propertieswere retained. Biochemical and biophysical investigations suggestedthat the fine-tuning by directed evolution led the salvagedvariant GFP-I5 to a functionally favorable structure, resultingin recovery of stability and fluorescence. Site-directed mutagenesisof the mutated amino acid residues in both GFPuv and GFP-I5revealed that each amino acid residue has a different effecton the fluorescence intensity, which implies that 3E4 adopteda new evolutionary path with respect to fluorescence characteristicscompared with the parent GFPuv. Directed evolution in conjunctionwith FSS is expected to be used for generating protein lineageswith new fitness landscapes. Received March 31, 2003; revised October 30, 2003; accepted October 31, 2003     

流行性乙型脑炎病毒E蛋白上与病原性相关的氨基酸

目的 分析比较乙脑病毒弱毒株at222与其强毒株AT31全基因核苷酸序列，探索与乙脑病毒病原性相关的氨基酸。方法 由日本基因公司合成多核苷引物，以RT-PCR合成cDNA，筛选出阳性克隆。用Sanger法测定at222与AT31株的全基因序列。结果 全基因序列分析比较显示，非编码区3个不同核苷苷酸均出现在3’－端。编码蛋白氨基酸变化分别是E蛋白上3个，NSI蛋白上3个，NS蛋白上2个，NS4a蛋白上1个，NS4b蛋白上1个，NS5蛋白上2个。结论 强弱毒株间2个变异部位与以前报道一致，且其中AT31的E138位点上E氨基酸为强毒株JaGAr01、JaOArS982、Beijing－l和SA14所共有，而at222E138位点上K氨基酸为弱毒株SA14－14－2所共有。E蛋白上E138、E176位点上氨基酸决定了乙脑病毒病原性。     

Optimized fluorescent trimethoprim derivatives for in vivo protein labeling

The combined technologies of optical microscopy and selective probes allow for real-time analysis of protein function in living cells. Synthetic chemistry offers a means to develop specific, protein-targeted probes that exhibit greater optical and chemical functionality than the widely used fluorescent proteins. Here we describe pharmacokinetically optimized, fluorescent trimethoprim (TMP) analogues that can be used to specifically label recombinant proteins fused to E. coli dihydrofolate reductase (eDHFR) in living, wild-type mammalian cells. These improved fluorescent tags exhibited high specificity and fast labeling kinetics, and they could be detected at a high signal-to-noise ratio by using fluorescence microscopy and fluorescence-activated cell sorting (FACS). We also show that fluorescent TMP-eDHFR complexes are complements to green fluorescent protein (GFP) for two-color protein labeling experiments in cells.     

Mutagenic stabilization of the photocycle intermediate of green fluorescent protein (GFP)

The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited-state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild-type(wt)-GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.     

重组大肠杆菌可溶表达并快速定量GFP-hepA融合蛋白

To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C terminus of a green fluorescent protein mutant （GFPmutl）. As a result, not only was the functional recombinant expression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluorescence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.     

绿色荧光蛋白融合表达HBsAg基因载体的构建及其在COS-7细胞中的表达

目的　构建绿色荧光蛋白融合表达HBsAg基因的载体 ,并检测其在COS 7细胞中的表达。方法　采用PCR获得HBsAg基因 ;利用该基因片段和质粒pEGFP 3.1的HindⅢ /KpnⅠ限制性酶切位点构建融合表达载体pGHBs 3.1;通过脂质体介导的方法将该载体转染到COS 7细胞中 ,用PCR法检测基因转染 ,ELISA检测HBsAg的瞬时表达 ,荧光显微镜检测绿色荧光蛋白表达。结果　融合表达载体pGHBs 3.1转染COS 7细胞后 ,在细胞中检测到HBsAg基因片段 ,转染细胞培养上清中检测到HBsAg表达 ,用荧光显微镜观察到转染细胞中有绿色荧光蛋白表达。结论　表达载体pGHBs 3.1在COS 7细胞中获得了表达 ,表达的融合蛋白具有HBsAg和绿色荧光蛋白的双重活性 ,该载体可用绿色荧光蛋白作为报告基因观察HBsAg的表达及定位。     

Expression, immobilization and enzymatic properties of glutamate decarboxylase fused to a cellulose-binding domain

Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamic acid to gamma-aminobutyric acid (GABA), was fused to the cellulose-binding domain (CBD) and a linker of Trichoderma harzianum endoglucanase II. To prevent proteolysis of the fusion protein, the native linker was replaced with a S(3)N(10) peptide known to be completely resistant to E. coli endopeptidase. The CBD-GAD expressed in E. coli was successfully immobilized on Avicel, a crystalline cellulose, with binding capacity of 33 ± 2 nmol(CBD-GAD)/g(Avicel) and the immobilized enzymes retained 60% of their initial activities after 10 uses. The results of this report provide a feasible alternative to produce GABA using immobilized GAD through fusion to CBD.     

Some environmental factors affecting free amino acid composition in six varieties of peanuts

This study involved six Spanish type entries (five commercial varieties and a plant introduction) grown in the National Variety Tests in Oklahoma and Georgia under both irrigated and nonirrigated conditions. Significant effects (Georgia vs Oklahoma) were observed on aspartic acid, proline, glycine, valine, isoleucine, peptide, ammonia, and histidine. Significant differences for irrigated vs nonirrigated in the two states for aspartic acid, threonine, proline, glutamic acid, isoleucine, leucine, tyrosine, phenylalanine, peptide, ammonia, and histidine were observed. Significant differences among the six entries were observed for glutamic acid, leucine, tyrosine, phenylalanine, ammonia, histidine, arginine, tryptophan, and total amino acids. None of the treatments produced significant changes in measured amounts of serine, alanine, methionine, and lysine. Significant differences for Georgia vs Oklahoma and irrigated vs nonirrigated for Kjeldahl nitrogen of the whole peanut were noted. Presented at the AOCS Spring Meeting, New Orleans, May 1973. Journal paper 2636 of the Oklahoma Agricultural Experiment Station. ARS, USDA.     

Acid-Denatured Green Fluorescent Protein (GFP) as Model Substrate to Study the Chaperone Activity of Protein Disulfide Isomerase

Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI inhibitors.     

Use of additives to enhance the properties of cottonseed protein as wood adhesives

Soy protein is currently being used commercially as a “green” wood adhesive. Previous work in this laboratory has shown that cottonseed protein isolate, tested on maple wood veneer, produced higher adhesive strength and hot water resistance relative to soy protein. In the present study, cottonseed protein and soy protein isolates were tested on different wood types, and cottonseed protein again showed better performance relative to soy protein. Furthermore, the effects of several protein modifiers were evaluated, including amino acids, fatty acids, and other organic molecules with cationic or anionic charges. Aspartic acid, glutamic acid, acetic acid, butyric acid, and adipic acid gave improved performance when included with cottonseed protein isolate whereas no significant effect was observed on soy protein isolate. Both dry adhesive strength and hot water resistance were tested. The enhanced performance observed with these additives provides an additional incentive for the use of cottonseed protein in this application.     

Can Modern Molecular Modeling Methods Help Find the Area of Potential Vulnerability of Flaviviruses?

Flaviviruses are single-stranded RNA viruses that have emerged in recent decades and infect up to 400 million people annually, causing a variety of potentially severe pathophysiological processes including hepatitis, encephalitis, hemorrhagic fever, tissues and capillaries damage. The Flaviviridae family is represented by four genera comprising 89 known virus species. There are no effective therapies available against many pathogenic flaviviruses. One of the promising strategies for flavivirus infections prevention and therapy is the use of neutralizing antibodies (NAb) that can disable the virus particles from infecting the host cells. The envelope protein (E protein) of flaviviruses is a three-domain structure that mediates the fusion of viral and host membranes delivering the infectious material. We previously developed and characterized 10H10 mAb which interacts with the E protein of the tick-borne encephalitis virus (TBEV) and many other flaviviruses’ E proteins. The aim of this work was to analyze the structure of E protein binding sites recognized by the 10H10 antibody, which is reactive with different flavivirus species. Here, we present experimental data and 3D modeling indicating that the 10H10 antibody recognizes the amino acid sequence between the two cysteines C92-C116 of the fusion loop (FL) region of flaviviruses’ E proteins. Overall, our results indicate that the antibody-antigen complex can form a rigid or dynamic structure that provides antibody cross reactivity and efficient interaction with the fusion loop of E protein.     

Tuning HP1α chromodomain selectivity for di- and trimethyllysine

Histone lysine methylation is a critical marker for controlling gene expression. The position and extent of methylation (mono-, di-, or tri-) controls the binding of effector proteins that determine whether the associated DNA is expressed or not. Dysregulation of histone protein methylation has been associated with a number of types of cancer, and development of inhibitors for the effector proteins is becoming an active area of research. For this reason, understanding the mechanism by which effector proteins obtain selectivity for the different methylation states of lysine is of great interest. To this end, we have performed mutation studies on the Drosophila HP1α chromodomain, which binds H3K9Me(2) and H3K9Me(3) with approximately equal affinities. The selectivity of HP1α chromodomain for H3K9Me(3) over H3K9Me(2) was investigated by mutating E52 to remove or weaken the hydrogen bond to K9Me(2) while maintaining affinity for K9Me(3,) including E52F, E52I, E52V, E52D, an E52Q. The E52Q mutant exhibited the greatest degree of selectivity for KMe3, with 3.5-fold weaker binding to the dimethylated peptide (K(D) =52 μM) compared to the trimethylated peptide (K(D) =15 μM). These studies provide insight into the role of electrostatic interactions and hydrogen bonding in the differentiation of methylation states and have implications regarding the evolutionary pressure for selectivity in this protein-protein interaction. Moreover, the information from this study may help guide inhibitor development for this class of proteins.     

A Genetically Encodable System for Sequence‐Specific Detection of RNAs in Two Colors

Multicolor readout is an important feature of RNA detection techniques aiming at the investigation of RNA localization. Several detection methods have been developed, however they require either transfection of cells with the probe or prior tagging of the target RNA. We report a fully genetically encodable system for simultaneous detection of two RNAs by using green and yellow fluorescence based on tetramolecular fluorescence complementation (TetFC). To obtain yellow fluorescent protein (YFP), substitution T203Y was introduced into one of the three non‐fluorescent GFP fragments; this was fused to different variants of the Homo sapiens Pumilio homology domain. Using different sets of fusion proteins we were able to discriminate between two closely related target RNAs based on the fluorescence signals at the respective wavelengths.