Protein and Peptide Letters - Volume 18, Issue 3, 2011

This is the third issue of Protein & Peptide Letters in which all the manuscripts have been contributed by the members of the Editorial Advisory Board of the journal. The papers included here demonstrate the wide variety of research areas covered by the Editorial Board. The field of proteins & peptides is very broad, encompassing wide areas of current chemistry, biology, biomaterials and related areas. This is true because proteins and peptides can be found in all living system and are responsible for both form and function, i.e., structural protein and enzymes, to name just two examples. The accelerating discoveries of the roles of these molecules in the pathology of disease and in new biomaterials with useful promise to expand the field even further. I am sure you will find papers in this issue quite interesting to read: The paper by Dr. John Wade et al. discusses the relaxin peptide family in humans consisting of seven members: relaxin-1, -2 and -3 and insulin-like (INSL) peptides 3, 4, 5 and 6. In the paper by Dr. Vladimir Uversky et al. it has been found that the soluble oligomers are important players in the multipathway aggregation of α-synuclein and should be taken into account in studies on the molecular mechanisms of this protein Dr. Robert Hodges and colleagues describe a series of 26-residue, amphipathic α-helical antimicrobial peptides consisting of all D-amino acid residues. A synthetic human L-LL37 (L-enantiomer) and D-LL37 (D-enantiomer) were investigated against M. tuberculosis susceptible strain (H37Rv) and a clinical multi-drug resistant strain (Vertulo). Structural stability of soybean (Glycine max) α-amylase by Dr. Arvind Kayastha et al. reports the stability of soybean α- amylase (GMA) against elevated temperatures and chemical denaturants (GndHCl) by employing circular dichroism and fluorescence spectroscopy. Dr. Hong-Bin Shen et al. developed a novel approach called PredCSF for predicting the conotoxin superfamily from the amino acid sequence directly based on fusing different kinds of sequential features by using modified one-versus-rest SVMs. In the paper by Dr. Saul Tendler and colleagues study of five analogues of a fragment from the shaft domain of the adenovirus fibre protein that readily form fibrils under a range of conditions is presented. Dr. Kuo-Chen Chou et al. describes that the region L5-7 (β5-β7, 68-82 residues) plays an important role for the oligomerization of SsHSP14.1 and its chaperone function. Here, to validate the findings, an in-depth investigation was conducted of both the wild type SsHSP14.1 and its deletion mutant DEL75-79. With E. coli proteins and bromelain as substrate, the deletion mutant DEL75-79 can protect them from thermo-aggregating as effectively as the wild protein. Dr. Sabato D'Auria et al. found that commercial and cheap Myoglobin (Mb) can be successfully used as a biological probe for a fluorescence biosensor for H2S detection. I appreciate efforts from the Editorial Office and wish them and journal all the best.

The relaxin peptide family in humans consists of seven members, relaxin-1, -2 and -3 and insulin- like (INSL) peptides 3, 4, 5 and 6. It is an offshoot of the large insulin superfamily. Each member consists of two chains, commonly referred to as A and B, which are held together by two inter-chain disulfide bonds and another intra-chain disulfide bond present within the A chain. The cysteine residues present in each chain, together with the distinctive disulfide bonding pattern, are conserved across all members of the superfamily. The chemical synthesis of these complex peptides poses a significant challenge. In the past, random combination of the two synthetic S-reduced chains under oxidizing conditions was utilized to form the three disulfide bonds. Nowadays, with the aid of highly efficient solid phase peptide synthesis methodologies, in conjunction with selective S- thiol-protecting groups, combination of individual A- and B- chains by sequential chemical formation of each of the three disulfide bonds is now possible resulting in good yields of these peptides. The relaxin peptide family members bind to G- protein coupled receptors (GPCRs) which have been classified as relaxin family peptide (RXFP) receptors. The various unique receptor-ligand interactions are outlined in this review, together with the physiological roles of the relaxin peptide family members and lastly their past and present clinical applications.

Under certain in vitro conditions, α-synuclein, an abundant 14kDa presynaptic intrinsically disordered protein involved in the pathogenesis of Parkinson's disease (PD), forms amyloid fibrils which resemble those found in Lewy bodies of PD patients. However, a substantial fraction of α-synuclein molecules (10-20%) does not form fibrils during fibrillation and exists in a form of soluble oligomers. In this study, we examined these soluble oligomers by a variety of biophysical techniques including atomic force microscopy (AFM), circular dichroism, Fourier-transform infrared spectroscopy and thioflavin T fluorescence. We observed that the fibrillation kinetics is affected by the variation in salt and protein concentrations. Although both high salt and high protein concentrations noticeably accelerated α-synuclein fibrillation, the amount of non-fibrillar oligomers is independent of the salt content. The oligomers formed at low salt concentration adopt more β-sheet structure and are smaller in size than those formed at high salt concentration. AFM analysis shows that the low salt oligomers represent a mixture of small oligomers and some amorphous aggregates, whereas oligomers formed at high salt concentrations are noticeably larger, more homogenous, and are mostly spherical in shape. All the late stage non-fibrillar oligomers do not form fibrils even when seeded with pre-formed fibrils, are characterized by negligible rates of dissociation, likely due to their intertwined structure, and are able to disrupt the integrity of the biological membrane. These findings suggest that these soluble oligomers are important players in the multi-pathway aggregation of α-synuclein and should be taken into account in studies on the molecular mechanisms of this protein fibrillation.

With the emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (Mtb), new classes of anti-mycobacterial agents with very different modes of action compared to classical antibiotics, are urgently needed. In this study, a series of 26-residue, amphipathic α-helical antimicrobial peptides consisting of all D-amino acid residues and synthetic human L-LL37 (L-enantiomer) and D-LL37 (D-enantiomer) were investigated against M. tuberculosis susceptible strain (H37Rv) and a clinical multi-drug resistant strain (Vertulo). Minimal inhibitory concentrations (MICs) were determined through a peptide killing assay. D5, the most active analog against M. tuberculosis had a MIC value of 11.2 μM (35.2 μg/ml) against H37Rv strain and 15.6 μM (49 μg/ml) against the MDR strain. Peptide D1 had similar activity as D5 against the MDR strain (57 μg/mL), a 9-fold improvement in hemolytic activity and a 7.4-fold better therapeutic index compared to D5. Surprisingly, LL37 enantiomers showed little to no activity compared to the de-novo designed α-helical antimicrobial peptides.

Stability and unfolding of mammalian and microbial α-amylases have been intensively investigated. However, there is only limited information available on the structural stability of plant α-amylases, namely of the two isoenzymes from barley AMY1 and AMY2, of the α-amylase from mung bean (Vigna radiata), and of the α-amylase from malted sorghum (Sorghum bicolor). We report here the stability of soybean α-amylase (GMA), against elevated temperatures and chemical denaturants (GndHCl) by employing circular dichroism and fluorescence spectroscopy. Since it is well-known that calcium ions play a crucial role for enzymatic activity and stability of α-amylases, we performed our studies with calcium bound and calcium free GMA. The thermal unfolding transition temperature decreased from 72 °C for calcium saturated samples to 57 °C for the case of calcium depleted GMA. Similarly, the GndHCl transition concentration was lowered from 0.70 M for calcium bound GMA to 0.41 M in the absence of calcium. Thermal unfolding of GMA is irreversible due to aggregation of the unfolded state. GMA unfolded in 6 M GndHCl shows high degree of reversibility after diluting the unfolded enzyme in native buffer containing 7 M glycerol. Furthermore, the refolded enzyme showed 93% of activity.

Conotoxins are small disulfide-rich peptides that are invaluable channel-targeted peptides and target neuronal receptors. They show prospects for being potent pharmaceuticals in the treatment of Alzheimer's disease, Parkinson's disease, and epilepsy. Accurate and fast prediction of conotoxin superfamily is very helpful towards the understanding of its biological and pharmacological functions especially in the post-genomic era. In the present study, we have developed a novel approach called PredCSF for predicting the conotoxin superfamily from the amino acid sequence directly based on fusing different kinds of sequential features by using modified one-versus-rest SVMs. The input features to the PredCSF classifiers are composed of physicochemical properties, evolutionary information, predicted secondary structure and amino acid composition, where the most important features are further screened by random forest feature selection to improve the prediction performance. The results show that PredCSF can obtain an overall accuracy of 90.65% based on a benchmark dataset constructed from the most recent database, which consists of 4 main conotoxin superfamilies and 1 class of non-conotoxin class. Systematic experiments also show that combing different features is helpful for enhancing the prediction power when dealing with complex biological problems. PredCSF is expected to be a powerful tool for in silico identification of novel conotonxins and is freely available for academic use at http://www.csbio.sjtu.edu.cn/bioinf/PredCSF. #Author's Profile: Dr. Hong-Bin Shen is a professor of Institute of Image Processing and Pattern Recognition, Shanghai Jiao Tong University. His research interests include data mining, protein structure and function prediction, drug discovery and biological network. Dr. Shen has published more than 60 papers and constructed 20 bioinformatics servers in these areas.

Here we present a study of five analogues of a fragment from the shaft domain of the adenovirus fibre protein that readily form fibrils under a range of conditions. Using atomic force microscopy the fibrillisation of these peptides at the liquid/solid interface utilizing ordered crystalline substrates has been investigated. Our results demonstrate that the assembly pathway at the liquid/solid interface enables only the formation of truncated fibrillar structures, which align along the substrate's underlying atomic lattice during growth. Furthermore, that the concentration and volume of solution applied can be used to directly control the density of fibrillar coverage at the surface.

The small heat shock protein SsHSP14.1 from the hyper-thermophilic archeaon, Sulfolobus solfataricus (S. solfataricus) was able to protect proteins from thermal aggregation and prevent enzymes from heat induced inactivation. According to the 3D (dimensional) structural model of SsHSP14.1 developed by us before, the region L5-7 (β5-β7, 68-82 residues) plays an important role for the oligomerization of SsHSP14.1 and its chaperone function. Here, to validate the findings, an in-depth investigation was conducted of both the wild type SsHSP14.1 and its deletion mutant DEL75-79. With E. coli proteins and bromelain as substrate, the deletion mutant DEL75-79 can protect them from thermo-aggregating as effective as the wild protein. Interestingly, unlike the wild protein, DEL75-79 was unable to prevent bromelain and EcoRI from thermo-inactivating. Results of size exclusion HPLC showed that the oligomerization state was changed in mutant protein. This was in accordance with the changed structure and lower hydrophobicity of DEL75-79. These outcomes proved that the L5-7 loop did play a role for the oligomerizing SsHSP14.1, and that the residues 75-79 were indispensable for its function of prevent enzymes from thermo-inactivating.

A new, fast, simple and cost-effective sensing device for monitoring H2S has been developed. Proof-of-principle results showing that a commercial and cheap Myoglobin (Mb) can be successfully used as a biological probe for a fluorescence biosensor for H2S detection are reported. Two different commercial labels Cy3 and Atto620 were selected for this study. A high selectivity for detecting H2S against other thiols was found. The applicability of the proposed sensing system was successfully explored not only in solution but also when applied in the form of a solid state device.

Vibriolysin is among several zinc metalloproteases produced by Vibrio cholerae. It is involved in the molecular pathogenicity of cholera. Here, we cloned and expressed full-length vibriolysin gene from V. cholerae. Electrophoretic and mass spectrometric data showed that the N-terminal pro-peptide was removed from pro-vibriolysin generating a 45- kDa segment containing the metalloprotease plus the C-terminal domains, and the 35 kDa metalloprotease. The 35 kDa metalloprotease segment of vibriolysin was purified to homogeneity using ion-exchange and gel filtration chromatography. Circular dichroism (CD) analysis of vibriolysin indicated α+β secondary structure, similar to other closely related metalloproteases of known structure. Positive dichroic absorption maxima in near-UV CD spectrum provided evidence for bound metal atom(s). Dynamic Light Scattering (DLS) measurements at different pHs were also performed to establish the aggregational properties of purified vibriolysin in solution. The results of DLS studies revealed that vibriolysin exists as a homomer with a hydrodynamic radius of 56.7 nm ± 2% under physiological conditions and remains catalytic when BSA was used as a protein substrate. While, extreme acidic (pH 3.5-5.4; RH = 65-239 nm) and alkaline (pH 8.5-9.4; RH = 57-72 nm) buffering conditions induced further aggregation of vibriolysin, without any trace of the monomeric state in solution.

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The gene for encoding the full length light chain with HCC (binding) domain of Clostridium botulinum neurotoxin A was synthesized and cloned into a bacterial expression vector pQE30-UA and produced as an Nterminally six-histidine-tagged fusion protein (rBoNT/A LC-HCC). This protein was expressed in two different strains of Escherichia coli namely BL21(DE3) and SG13009. Expression at 37 °C revealed localization of rBoNT/A LC- HCC in inclusion body whereas it was expressed in soluble form at 21°C. The recombinant fusion protein was purified by nickel affinity gel column chromatography and identified by monoclonal antibody and peptide mass fingerprinting. The recombinant protein was shown to bind with synaptic vesicles and gangliosides (GT1b) using enzyme-linked immunosorbent assay. The rBoNT/A LC-HCC was also found to be highly active on its substrate (SNAP-25) from rat brain, indicating that the expressed and purified rBoNT/A LC-HCC protein retains a functionally active conformation. Biologically active recombinant fusion protein was also evaluated for its immunological potential.

The combination of several drugs is often necessary, especially during long-term therapy. A competitive binding of the drugs can cause a decrease of the amount of drugs actually bound to the protein and increase the biologically active fraction of the drug. The aim of this study has been to analyze the interactions of tamoxifen (TMX) and aspirin (ASA) with human serum albumin (HSA) and to evaluate the mechanism of a simultaneous binding of TMX and ASA to the protein. Fluorescence analysis was used to estimate the effect of the drugs on the protein fluorescence and to define the binding and quenching properties of drug-HSA complexes. The binding sites for TMX and ASA were identified in ternary structures of HSA by means of spectrofluroscence. The analysis of the fluorescence quenching of HSA in binary and ternary systems pointed at TMX and ASA having an effect on the HSA-ASA and HSA-TMX complexes. Furthermore, the results of synchronous fluorescence, resonance light scattering and circular dichroism of the binary and ternary systems showed that the binding of TMX and ASA to HSA could induce conformational changes in HSA. Moreover, the simultaneous presence of TMX and ASA during binding to HSA should be taken into account in multi-drug therapy, as it induces the necessity of a monitoring therapy owing to the possible increase of uncontrolled toxic effects. Competitive site marker experiments demonstrated that the binding site of ASA and TMX to HSA differed in the binary system as opposed to in its ternary counterpart. Finally, molecular modeling of the possible binding sites of TMX and ASA in binary and ternary systems to HSA confirmed the experimental results.

Temperature dependence of the α-helix conformation of bee venom melittin in methanol-water mixed solvents has been examined by NMR, in order to elucidate conformation stability and a phase diagram. At high methanol concentration of 100 - ca. 80 wt.%, melittin forms a full α-helix conformation in the temperature range from 25 °C to 60 °C. At intermediate methanol concentration of ca. 80 - ca. 25 wt.%, it undergoes a thermal transformation from a full α-helix to a partial α-helix. In solutions of low methanol concentrations of ca. 25 - 0 wt.%, partial α-helix monomers and their selfaggregated conformers coexist at low temperatures, and the relative number of the monomers increases with increase in temperature. The monomers turn to a random coil state at high temperatures only below ca. 10 wt. % methanol concentrations. The thermal transitions are discussed from the viewpoint of stability of intra-molecular hydrogen bonds and intermolecular hydrophobic interactions.