Amino Acid Sequence of an Excitatory Insect-selective Toxin (BmK IT) From Venom of the Scorpion Buthus martensi Karsch

The insect-selective neurotoxin(BmK IT) of scorpion Buthus martensi Karsch was first reduced and S-alkylated, and then digested by TPCK-trypsin and Staphylococcus aureus V-8 Protease. The enzymatic peptides were purified on TLC-plastic sheet and submitted to determine their amino acid compositions and sequences. The sequence of the 70 amino acid residues of BmK IT was established with reference to the primary structure of AaH IT, another excitatory insect-selective toxin from the venom of North African scorpion Androctonus australis Hector. About 75% of the homologous sequence was found in the molecules of BmK IT and AaH IT. It is obvious that the results contribute toward better understanding of the molecular structure characteristics, structure/activity relationship of scorpion insect-selective toxins, and they can serve as the molecular basis for utilizing the toxins as a tool to clarify molecular mechanism involved in channel gating, and to infer the possibility of developing them as new selective b     

A Novel Peptide from Buthus martensii Karch

A novel peptide was purified and characterized from Buthus martensii Karch.The peptide,named BmK M6,is a single-chain polypeptide cross-linked by four intramolecular disulfide bridges.The molecular weight of the peptide was determined by MOLDI-TOF-MS as 7034 Da.The partial amino acid sequence of BmK M6 from N-terminal is VRDAYIAKPEN CVYECGITQDCNKLCTENG.     

A Novel β-1, 4-N, 6-O-Diacetylmuramidase from Streptomyces griseus and its Chemical Modification

A novel lysozyme namedβ-1, 4-N, 6-O-diacetylmuramidase R2 was purified and characterized from Streptomyces griseus. The molecular weight of the enzyme was determined by MALDI-TOF-MS as 23.5 kDa. The N-terminal amino acid sequence was DTSGVQGIDVS-HWQG.Chemical modification ofβ-1, 4-N, 6-O-diacetylmuramidase R2 indicated that sulfhydry1 group and carbamidine of arginine residues are not essential for the activity of the enzyme, but lysine residues and imidazole of histidine residues are essential for the activity. The number of essential tryptophan and carboxyl groups was found that only one tryptophan residue and three carboxyl groups in the active site.     

Structure determination of glycopeptide of β-momorcharin

The structure of the N-linked oligosaccharide chain of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae), was determined. A glycopeptide liberated by pronase digestion of the glycoprotein was subjected to amino acid and neutral carbohydrate analysis to establish the composition of amino acid and sugar residues. The sequences and glycosylation hnkages of the sugar and amino acid residues in the glycopeptide were determined as Manαl-6(XyIβ1-2)-Manβ1-4GlcNAcβ1-4(Fucαl-3)-GlcNAc-Asn-Leu by 2D-NMR spectroscopy and FAB-MS data.     

Primary structure of p-momorcharin, a ribosome-inactivating protein from the seeds of Momordica Charantia Linn (Cucurbitaceae)

The complete amino acid sequence of β-momorcharin, a ribosome-inactivating protein from the seeds of Momordica charantia Linn (Cucurbitaceae) has been determined. This has been done by the sequence analysis of peptides obtained by enzymatic digestion with trypsin, chymotrypsin and S. aureus V8 protease, as well as by chemical cleavage with BNPS-skatole. The protein consists of 249 amino acid residues containing one asparagine - linked sugar group attached to the site of Asn 5 1 and has a calculated relative molecular mass of 28,452 Da without addition of the carbohydrate. Comparison of this sequence with those of trichosanthin and other ribosome-inactivating proteins from different species of plants shows a significant homology with each other. Regarding the similarity of their biological properties, an active domain of these proteins has been predicted here.     

Structure Analysis of Streptococcal Protein G Fc Binding Domain

The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG when expressed in Escherichia coli. The complete nucleotide sequence of the gene fragment was determined. One base pair differs from previously reported protein Gnucleotide sequences, and resultsin an amino acid change (Ala-Thr), but this variation makes no difference in binding to the IgG Fc part by ELISA.The secondary structure of the protein G IgG Fc binding domain has been estimated by circular dichroism and assigned by computer algorithm.It shows a typical α-helix region in this domain.By breaking this α-helix region with recombinant DNA techniques, a 44 peptide, which contained the N-terminal 27 amino acid residues of this domain, was expressed in E. coli and showed no reactivity to IgG.The hydropathicity of this domain was also analyzed and compared with that of protein A relevant     

Synthesis and Expression in Escherichia coli of a Human Neutrophil Activating Protein-1/Interleukin-8 Gene

The complete gene coding for human neutrophilactivating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temperature-regulated control of the P_RP_L tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein comprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. The recombinant NAP-1/IL-8 was then purified to>95% homogeneity by gel filtration and cation exchange chromatography. The purified protein appeared as a single band on the SDS-PAGE gel and possessed potent chemotactic activity in the concentration of <10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP1/IL-8 was determined using the Edman method and was sho     

THE PRIMARY STRUCTURE OF THE LACTATE DEHYDROGENASE ISOZYME M. FROM GIANT PANDA

The amino acid sequences of tryptic, chymotryptic and cyanogen bromide cleavage peptides of the lac-tate dehydrogenase isozyme M4 from giant panda have been determined. Based on the overlapping peptidesand by a comparison with the known sequence of porcine lactate dehydrogenase isozyme M subunit, thecomplete primary structure of the giant panda lactate dehydrogenase isozyme M subunit has been estab-lished. The polypeptide chain of giant panda lactate dehydrogenase isozyme M subunit consists of 331amino acid residues. There is a variance of 17 amino acid residues between the porcine and the giantpanda lactate dehydrogenase isozyme M subunits. Most of the variable residues are substitutions bychemically similar amino acids and take place at residues not related to the active center of the enzyme.     

Study on Fmoc Solid-phase Synthesis and Activity of Thymosin α1

Thymosin α1 (Tα1), an immunologically polypeptide, [1] is highly acidic composed of 28 amino acid residues with acetylserine as the NH2 terminus. The MW of this peptide is 3108, with pI 4.2. There are many Asp and Glu in this molecule and the complete amino acid sequence of Tα1 is Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-IleThr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH. This peptide has potent biological activity and has been found to be 10～ 1000 times as active as thymosin F5. In this paper, a Tα1 has been synthe sized by a solid-phase method. Peptide synthesis was performed manually by the stepwise solid-phase method using the base-labile Fmoc group for protecting the α-amino acid. [2]     

马氏钳蝎蝎毒短肽BmK622的分离纯化和一级结构测定

应用凝胶过滤、离子交换和HPLC反相色谱法从马氏蝎粗毒中分离纯化得到蝎毒 多肽BmK622．联合运用串联质谱法和Edman降解法，鉴定了KmK622N端19个残基的序 列，经过数据库检索，发现数据库中用cDNA克隆方法鉴定了序列的马氏钳蝎蝎毒短 肽BmTX3一级序列N端19个残基与BmK622已测定的N端19个残基序列完全相同， BmK622的分子量测定和氨基酸组成分折的结果表明，BmK22与BmTX3分子量相同、氨 基酸组成一致，从而BmK22的一级结构为：GFLID VKCFA SSECW TACKK VTGSG QGKCQ NNQCR CY．     

Purification, characterization and functional expression of a new peptide with an analgesic effect from Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU1)

In this study, a new peptide named BmK AGP‐SYPU1 with an analgesic effect was purified from the venom of Chinese scorpion Buthus martensi Karsch (BmK) through a four‐step chromatographic process. The mouse twisting test was used to identify the target peptides in every separation step. The purified BmK AGP‐SYPU1 was further qualified by RP‐HPLC and HPCE. The molecular mass determined by the MALDI‐4800‐TOF/TOF MS for BmK AGP‐SYPU1 was 7544 Da. Its primary structure of the N‐terminal was obtained using Edman degradation. The gene sequence of BmK AGP‐SYPU1 was cloned from the cDNA pool and genomic of scorpion glands, respectively, and then expressed in Escherichia coli. The sequence determination showed that BmK AGP‐SYPU1 was composed of 66 amino acid residues with a new primary structure. The metal chelating affinity column and cation exchange chromatography were used to purify the recombinant BmK AGP‐SYPU1. Consequently, the native and recombinant BmK AGP‐SYPU1 showed similar analgesic effects on mice as assayed using a mouse twisting model. These results suggested that BmK AGP‐SYPU1 is a new analgesic component found in the Chinese scorpion Buthus martensi Karsch. Copyright © 2010 John Wiley & Sons, Ltd.     

Oligonucleotides Functionalized by Fluorescein and Rhodamine Dyes: Michael Addition of Methyl Acrylate to 2′‐Deoxypseudouridine

The 2′‐deoxypseudouridine ( 5 ) was functionalized at N(1) with methyl acrylate by Michael addition. The resulting methyl 2′‐deoxypseudouridine‐1‐propanoate ( 6 ) was converted to the phosphoramidite 8 and to the amino‐functionalized derivative 9 , which was transformed into the fluorescein‐labeled phosphoramidites 14 and 16 . Fluorescent oligonucleotides were synthesized either from these building blocks or by post‐synthetic modification of oligomers containing 2′‐deoxypseudouridine subunits. The stability of oligonucleotide duplexes was determined from the melting profiles, measured by UV‐ or VIS‐light absorbance, as well as from the fluorescence emission spectra. While small spacer residues did not affect the thermal stability of the 2′‐deoxypseudouridine‐containing duplexes, large dye residues led to destabilization.     

Spontaneous Aminoacylation of a RNA Sequence Containing a 3′‐Terminal 2′‐Thioadenosine

We report the synthesis of a modified 8mer RNA sequence, (C‐C‐C‐C‐A‐C‐C‐(2′‐thio)A)‐RNA 5′‐(dihydrogen phosphate) ( 9 ) containing a 3′‐terminal 2′‐thioadenosine (Schemes 2 and 3), and its spontaneous and site‐specific aminoacylation with the weakly activated amino acid thioester H Phe SPh ( 12 ). This reaction, designed in analogy to the ‘native chemical ligation’ of oligopeptides, occurs efficiently in buffered aqueous solutions and under a wide range of conditions (Table). At pH values between 5.0 and 7.4, two products, the 3′‐O‐monoacylated and the 3′‐O,2′‐S‐diacylated RNA sequences 10 and 11 are formed fast and quantitatively (Scheme 4). At pH 7.4 and 37°, the 3′‐O‐monoacylated product 10 is formed as major product in situ by selective hydrolysis of the O,S‐diacylated precursor 11 . Additionally, the preparation and isolation of the relevant 3′‐O‐monoacylated product 10 was optimized at pH 5. The here presented concept could be employed for a straightforward aminoacylation of analogously modified tRNAs.     

Fine Tuning of β‐Peptide Foldamers: a Single Atom Replacement Holds Back the Switch from an 8‐Helix to a 12‐Helix

Cyclic homologated amino acids are important building blocks for the construction of helical foldamers. N‐aminoazetidine‐2‐carboxylic acid (AAzC), an aza analogue of trans‐2‐aminocyclobutanecarboxylic acid (tACBC), displays a strong hydrazino turn conformational feature, which is proposed to act as an 8‐helix primer. tACBC oligomers bearing a single N‐terminal AAzC residue were studied to evaluate the ability of AAzC to induce and support an 8‐helix along the oligopeptide length. While tACBC homooligomers assume a dominant 12‐helix conformation, the aza‐primed oligomers preferentially adopt a stabilized 8‐helix conformation for an oligomer length up to 6 residues. The (formal) single‐atom exchange at the N terminus of a tACBC oligomer thus contributes to the sustainability of the 8‐helix, which resists the switch to a 12‐helix. This effect illustrates atomic‐level programmable design for fine tuning of peptide foldamer architectures.     

Geometry‐Retentive C‐Alkenylation of Lithiated α‐Aminonitriles: Quaternary α‐Alkenyl Amino Acids and Hydantoins

α‐Amino nitriles tethered to alkenes through a urea linkage undergo intramolecular C‐alkenylation on treatment with base by attack of the lithionitrile derivatives on the N′‐alkenyl group. A geometry‐retentive alkene shift affords stereospecifically the E or Z isomer of the 5‐alkenyl‐4‐iminohydantoin products from the corresponding starting E ‐ or Z ‐N ′‐alkenyl urea, each of which may be formed from the same N ‐allyl precursor by stereodivergent alkene isomerization. The reaction, formally a nucleophilic substitution at an sp² carbon atom, allows the direct regioselective incorporation of mono‐, di‐, tri‐, and tetrasubstituted olefins at the α‐carbon of amino acid derivatives. The initially formed 5‐alkenyl iminohydantoins may be hydrolyzed and oxidatively deprotected to yield hydantoins and unsaturated α‐quaternary amino acids.     

Controlling the Helix Handedness of ααβ‐Peptide Foldamers through Sequence Shifting

Peptide foldamers containing both cis ‐β‐aminocyclopentanecarboxylic acid and α‐amino acid residues combined in various sequence patterns (ααβ, αααβ, αβααβ, and ααβαααβ) were screened using CD and NMR spectroscopy for the tendency to form helices. ααβ‐Peptides were found to fold into an unprecedented and well‐defined 16/17/15/18/14/17‐helix. By extending the length of the sequence or shifting a fragment of the sequence from one terminus to another in ααβ‐peptides, the balance between left‐handed and right‐handed helix populations present in the solution can be controlled. Engineering of the peptide sequence could lead to compounds with either a strong propensity for the selected helix sense or a mixture of helical conformations of opposite senses.     

Design of two series of 1:1 cocrystals involving 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine and carboxylic acids

Two series of a total of ten cocrystals involving 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine with various carboxylic acids have been prepared and characterized by single‐crystal X‐ray diffraction. The pyrimidine unit used for the cocrystals offers two ring N atoms (positions N1 and N3) as proton‐accepting sites. Depending upon the site of protonation, two types of cations are possible [Rajam et al. (2017). Acta Cryst. C 73 , 862–868]. In a parallel arrangement, two series of cocrystals are possible depending upon the hydrogen bonding of the carboxyl group with position N1 or N3. In one series of cocrystals, i.e. 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine–3‐bromothiophene‐2‐carboxylic acid (1/1), 1 , 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine–5‐chlorothiophene‐2‐carboxylic acid (1/1), 2 , 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine–2,4‐dichlorobenzoic acid (1/1), 3 , and 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine–2‐aminobenzoic acid (1/1), 4 , the carboxyl hydroxy group (–OH) is hydrogen bonded to position N1 (O—H…N1) of the corresponding pyrimidine unit (single point supramolecular synthon). The inversion‐related stacked pyrimidines are doubly bridged by the carboxyl groups via N—H…O and O—H…N hydrogen bonds to form a large cage‐like tetrameric unit with an R₄²(20) graph‐set ring motif. These tetrameric units are further connected via base pairing through a pair of N—H…N hydrogen bonds, generating R₂²(8) motifs (supramolecular homosynthon). In the other series of cocrystals, i.e. 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine–5‐methylthiophene‐2‐carboxylic acid (1/1), 5 , 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine–benzoic acid (1/1), 6 , 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine–2‐methylbenzoic acid (1/1), 7 , 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine–3‐methylbenzoic acid (1/1), 8 , 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine–4‐methylbenzoic acid (1/1), 9 , and 4‐amino‐5‐chloro‐2,6‐dimethylpyrimidine–4‐aminobenzoic acid (1/1), 10 , the carboxyl group interacts with position N3 and the adjacent 4‐amino group of the corresponding pyrimidine ring via O—H…N and N—H…O hydrogen bonds to generate the robust R₂²(8) supramolecular heterosynthon. These heterosynthons are further connected by N—H…N hydrogen‐bond interactions in a linear fashion to form a chain‐like arrangement. In cocrystal 1 , a Br…Br halogen bond is present, in cocrystals 2 and 3 , Cl…Cl halogen bonds are present, and in cocrystals 5 , 6 and 7 , Cl…O halogen bonds are present. In all of the ten cocrystals, π–π stacking interactions are observed.     

One‐Pot Four‐Segment Ligation Using Seleno‐ and Thioesters: Synthesis of Superoxide Dismutase

The synthesis of a peptide selenoester was efficiently carried out by the 9‐fluorenylmethoxycarbonyl (Fmoc) method using N‐alkylcysteine, at the C‐terminus of the peptide, as the N‐to‐S acyl shift device. The selenoester selectively reacted with the terminal amino group of the peptide aryl thioester in the presence of N ,N ‐diisopropylethylamine and dipyridyldisulfide, thus leaving the aryl thioester intact. Combined with silver‐ion‐promoted and silver‐ion‐free thioester activation methods, a one‐pot four‐segment ligation was realized. The method was successfully used to assemble the entire sequence of superoxide dismutase (SOD), which is composed of 153 amino‐acid residues, in one pot. After the folding reaction, the fully active SOD was obtained.