DNA recognition of a 24-mer peptide derived from RecA protein

A novel 24-residue peptide (L2-G), Ile-Arg-Met-Lys-Ile-Gly-Val-Met-Phe-Gly-Asn-Pro-Glu-Thr-Thr-Thr-Gly-Gly-Asn-Ala-Leu-Lys-Phe-Tyr, derived from RecA can discriminate a single-stranded DNA (ssDNA) from a double-stranded DNA (dsDNA) and a new developed support with this peptide recognizes not dsDNA but ssDNA. The 24-mer peptide with L2 and helix G amino acids of Escherichia coli RecA protein showed the ssDNA binding property with more than 1000 times affinity difference for the dsDNA. However, truncated 15-mer peptide showed no ssDNA binding activity. In the ssDNA binding, L2-G changed its conformation with the perturbation of an alpha-helix structure. The ssDNA binding and the DNA discrimination property of this peptide were due to almost all L2 and helix G amino acids, respectively. This result is useful to design synthetic peptides as functional materials for DNA recognition.     

Structural studies with the uveopathogenic peptide M derived from retinal S-antigen

The 18-residue fragment of bovine S-antigen, corresponding to amino acid positions 303-320, is highly immunogenic and is known to induce experimental autoimmune uveitis. The solution conformation of this immunogenic peptide, known as peptide M, was studied by Fourier-transform infrared spectroscopy and by circular dichroism. In the pH range between approximately 4 and 9.5, peptide M has a strong tendency to form macromolecular assemblies in which it adopts an intermolecular beta-sheet structure. The intermolecular beta-sheets are stabilized by ionic interactions ("salt bridges") between the carboxylate groups and basic residues of the neighboring peptide molecules. These interactions can be disrupted by neutralization of either acidic (pH range below 4) or basic residues (pH range above 9.5) or by elevated hydrostatic pressure. The secondary structure of the peptide under conditions favoring the monomeric state appears to be a mixture of unordered structure and beta-sheets. The present data are consistent with a recently proposed model [Sette, A., Buns, S., Colon, S., Smith, J. A., Miles, C., & Grey, H. M. (1987) Nature 328, 395-399], which assumes that certain immunogenic peptides adopt an extended beta-type conformation in which they are "sandwiched" between the major histocompatibility complex and the T-cell receptor.     

Transfer-NMR and docking studies identify the binding of the peptide derived from activating transcription factor 4 to protein ubiquitin ligase beta-TrCP. Competition STD-NMR with beta-catenin

ATF4 plays a crucial role in the cellular response to stress. The E3 ubiquitin ligase, SCF beta-TrCP protein responsible for ATF4 degradation by the proteasome, binds to ATF4 through a DpSGXXXpS phosphorylation motif, which is similar but not identical to the DpSGXXpS motif found in most other substrates of beta-TrCP. NMR studies were performed on the free and bound forms of a peptide derived from this ATF4 motif that enabled the elucidation of the conformation of the ligand complexed to the beta-TrCP protein and its binding mode. Saturation transfer difference (STD) NMR allowed the study of competition for binding to beta-TrCP, between the phosphorylation motifs of ATF4 and beta-catenin, to characterize the ATF4 binding epitope. Docking protocols were performed using the crystal structure of the beta-catenin-beta-TrCP complex as a template and NMR results of the ATF4-beta-TrCP complex. In agreement with the STD results, in order to bind to beta-TrCP, the ATF4 DpSGIXXpSXE motif required the association of two negatively charged areas, in addition to the hydrophobic interaction in the beta-TrCP central channel. Docking studies showed that the ATF4 DpSGIXXpSXE motif fits the binding pocket of beta-TrCP through an S-turning conformation. The distance between the two phosphate groups is 17.8 A, which matched the corresponding distance 17.1 A for the other extended DpSGXXpS motif in the beta-TrCP receptor model. This study identifies the residues of the beta-TrCP receptor involved in ligand recognition. Using a new concept of STD competition experiment, we show that ATF4 competes and inhibits binding of beta-catenin to beta-TrCP.     

Species-specific inhibition of fertilization by a peptide derived from the sperm protein bindin.

The sperm protein bindin is responsible for the species-specific adhesion of the sperm to the egg. The regions of the bindin molecule responsible for forming the contact between the sperm and the egg were investigated by measuring the ability of peptides representing various regions of the bindin sequence to inhibit fertilization. Twenty-four peptides were studied: 7 based on the Strongylocentrotus purpuratus bindin sequence, 11 based on the S. franciscanus bindin sequence, and 6 control peptides. Values for the concentration of peptide required to inhibit 50% of the productive sperm contacts (IC50) were extracted from experimental measurements of the extent of fertilization in the presence of various concentrations. of these peptides. The IC50 value averaged 220 microM for the control peptides. Active peptides representing certain specific subregions of the bindin sequence displayed IC50 values < 10% of the average value for control peptides, and the IC50 for the most potent of the peptides tested was only approximately 1% of the control peptide value (IC50 = 2.2 microM). Furthermore, we found that a peptide representing a particular region of the S. franciscanus bindin sequence that differs from the S. purpuratus bindin sequence inhibits fertilization species specifically. For the reaction of S. purpuratus sperm and eggs, the IC50 of this peptide was approximately 120 microM, whereas for the reaction of S. franciscanus sperm and eggs it was only 8.6 microM. These results demonstrate that a few specific regions of the bindin molecule are involved in the sperm-egg contact and that certain of these regions mediate the species specificity of the interaction in a sequence-specific manner.     

NMR studies of the prionogenic peptide derived from Sup35 protein

The NMR studies of the prionogenic peptide derived from Sup35 are presented. The peptide molecules were dissolved in the half-aqueous solution to prevent severe aggregation, and were found to be in an extended conformation from the chemical shift and the coupling constant data. They could form higher order multimers by making intermolecular hydrogen bonds, judging from the observation that the NMR sample became a gel-like state at lower temperatures. This work reports the first structural information in the solution state about the prionogenic peptide mimicking the state of amyloid fibrils, and provides a solid foundation for further structural analysis of peptide molecules forming insoluble aggregates.     

Structural studies of a synthetic peptide derived from the carboxyl-terminal domain of RNA polymerase II

The conformation of the repeating heptapeptide unit of the carboxyl-terminal domain of RNA Polymerase II, Y¹S²P³T⁴S⁵P⁶S⁷ has been examined using nuclear magnetic resonance spectroscopy and circular dichroism. Nuclear Overhauser effects and CD spectra for the synthetic 56-residue peptide H₂N-(S²P³T⁴S⁵P⁶S⁷Y^l)₈-COOH in water indicate that the peptide is largely unordered. A small population of folded molecules is observed to contain β-turns located at Ser²-Pro³-Thr⁴-Ser⁵ (SPTS) and Ser⁵-Pro⁶-Ser⁷-Tyr¹ (SPSY). CD and NMR results in 90% TFE also indicate an equilibrium population of structures, but the fraction of turns is higher. Similarities of nuclear Overhauser effects in water and in 90% TFE suggest that the structures in TFE are biologically relevant. Based on these observations, the average structure of a single conformer of the heptapeptide repeat in 90% TFE was obtained by a distance geometry-simulated annealing method, using distance restraints extracted from nuclear Overhauser data. NMR spectra of the 56-mer show signals corresponding to only one repeat indicating that each repeat is in an identical environment. Thus it is possible to obtain an average structure of the heptapeptide repeat from NOE data on the 56-mer. Twenty-seven final structures were calculated and the root mean square deviations between the 27 structure and the mean coordinates was 1.52 Å for the backbone and 2.2 Å for all nonhydrogen atoms. The heptapeptide repeat consists of two overlapping β-turns which are potentially stabilized by hydrogen bonds. The hydroxyl side chains of Ser², Ser⁵, Thr⁴, and Ser⁷ all appear to be equally exposed for potential phosphorylation. The tyrosyl side chain of each repeat is folded inwards to the backbone and can potentially hydrogen bond to the carbonyl oxygen of the tyrosine in the preceding repeat. Iteration of the average structure of the heptapeptide repeat results in a model of the carboxyl-terminal domain with a regular but unusual secondary structure consisting of a series of staggered β-turns. © 1995 Wiley-Liss, Inc.     

Structural studies on 26RFa, a novel human RFamide-related peptide with orexigenic activity

A novel hypothalamic neuropeptide of the RFamide family, comprising 26 amino acids residues and thus termed 26RFa, has been recently characterized in human, and was found to be the endogenous ligand for the orphan G protein-coupled receptor GPR103. Intracerebroventricular injection of 26RFa provokes a robust increase in food intake in rodents. In the present study, we have investigated the solution conformation of 26RFa by using two-dimensional NMR spectroscopy in different media. In water, 26RFa exhibits mainly a random coil conformation although the presence of a nascent helix was detected between residues 6 and 15. In methanol, 26RFa adopts a well-defined conformation consisting of an amphipathic alpha-helical structure (Pro4-Arg17), flanked by two N- and C-terminal disordered regions. The strong conservation, from amphibians to mammals, of the amino acid sequence corresponding to the amphipathic helix and to the C-terminal flexible octapeptide of 26RFa, suggests that these two domains are crucial for the interaction of the peptide with its receptor.     

Structural characterization of a mutant peptide derived from ubiquitin: implications for protein folding

The formation of the N-terminal beta-hairpin of ubiquitin is thought to be an early event in the folding of this small protein. Previously, we have shown that a peptide corresponding to residues 1-17 of ubiquitin folds autonomously and is likely to have a native-like hairpin register. To investigate the causes of the stability of this fold, we have made mutations in the amino acids at the apex of the turn. We find that in a peptide where Thr9 is replaced by Asp, U(1-17)T9D, the native conformation is stabilized with respect to the wild-type sequence, so much so that we are able to characterize the structure of the mutant peptide fully by NMR spectroscopy. The data indicate that U(1-17)T9D peptide does indeed form a hairpin with a native-like register and a type I turn with a G1 beta-bulge, as in the full-length protein. The reason for the greater stability of the U(1-17)T9D mutant remains uncertain, but there are nuclear Overhauser effects between the side chains of Asp9 and Lys 11, which may indicate that a charge-charge interaction between these residues is responsible.     

On the non-existence of a tryptic peptide with biological activity derived from mouse nerve growth factor

Reverse-phase high-performance liquid chromatography was used to analyse the products of treatment of mouse nerve growth factor with cyanogen bromide followed by trypsin as described by Mercanti et al. All the biological activity was found to be due to incompletely cleaved starting material. Total digestion with trypsin led to complete loss of activity.     

来自穿膜肽的新肽的抗菌活性及抑菌机制

[目的]研究基于穿膜肽和抗菌肽构效关系改造获得的新肽P7的抗菌活性及其对大肠杆菌(E.coli)的抑菌机制.[方法]微量稀释法和溶血实验分析P7的抑菌活性及其对正常细胞的细胞毒性;采用膜荧光探针、流式细胞术和扫描电镜分析P7对E.coli膜通透性、膜完整性的影响和细胞超微结构变化;通过激光共聚焦分析P7在E.coli细胞中的定位;凝胶阻滞实验测定P7与E.coli基因组DNA结合能力.[结果]P7比母肽显示更强的抑菌活性,最低抑菌浓度范围为4-32 μmol/L,且在作用浓度范围内具有较弱的溶血活性.P7可以增加E.coli外膜和内膜的通透性,使E.coli细胞膜的完整性和细胞表面结构受损.同时P7可以穿过E.coli细胞膜在细胞质聚集并与基因组DNA结合.[结论]P7通过增加E.coli内外膜通透性,穿过细胞膜与胞内DNA结合发挥抑菌活性.     

TRUSS, a novel tumor necrosis factor receptor 1 scaffolding protein that mediates activation of the transcription factor NF-kappaB

We describe the cloning and characterization of tumor necrosis factor receptor (TNF-R)-associated ubiquitous scaffolding and signaling protein (TRUSS), a novel TNF-R1-interacting protein of 90.7 kDa. TRUSS mRNA was ubiquitously expressed in mouse tissues but was enriched in heart, liver, and testis. Co-immunoprecipitation experiments showed that TRUSS was constitutively associated with unligated TNF-R1 and that the complex was relatively insensitive to stimulation with TNF-alpha. Deletion mutagenesis of TNF-R1 indicated that TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. In addition, the N-terminal region of TRUSS (residues 1 to 440) contains sequences that permit association with the cytoplasmic domain of TNF-R1. Transient overexpression of TRUSS activated NF-kappaB and increased NF-kappaB activation in response to ligation of TNF-R1. In contrast, a COOH-terminal-deletion mutant of TRUSS (TRUSS(1-723)) was found to inhibit NF-kappaB activation by TNF-alpha. Co-precipitation and co-immunoprecipitation assays revealed that TRUSS can interact with TRADD, TRAF2, and components of the IKK complex. These findings suggest that TRUSS may serve as a scaffolding protein that interacts with TNF-R1 signaling proteins and may link TNF-R1 to the activation of IKK.     

Heterologous expression, characterization and structural studies of a hydrophobic peptide from the HIV-1 p24 protein

Proteins from the inner core of HIV-1, such as the capsid protein (p24), are involved in crucial processes during the virus life cycle. The p24 protein plays an active structural role in the Gag protein and in its mature form. This work describes the production of a peptide derived from the p24 C-terminal, TLRAEQASQEVKNWMTETLLVQNA, using recombinant technology. This region (p24-3) is involved in interfaces during the p24 dimerization, which occurs during capsid assembly. The p24-3 sequence was obtained by a synthetic gene strategy and inserted into the pET 32a expression vector to produce soluble fusion protein in Escherichia coli BL21(DE3). This strategy leads to an incorporation of three amino acid residues (AMA) in the N-terminal of the native sequence to form the recombinant p24-3 (rp24-3). The rp24-3 was purified by reverse phase chromatography to homogeneity, as inferred by mass spectrometry and protein sequence analysis. Structural studies using circular dichroism and steady-state fluorescence showed that the rp24-3 is structured by helical and beta elements. As a function of its hydrophobic character it can self-associate forming oligomers. We present in this paper the first development of a suitable expression system for rp24-3, which provides high amounts of the peptide. This strategy will allow the development of new antiviral (HIV) agents.