Protein and Peptide Letters - Volume 15, Issue 9, 2008

First a few notes on the operation of the journal: We have just made the decision to change to 12 issues per year starting in 2009, up from 10 issues this year. This is due to the very heavy manuscript flow that we have been experiencing in 2008. In addition, the larger size and two column format, initiated in 2005 with volume 12, have been well received. Improvements have also been made with respect to reproduction of figures, although this will always be dependent upon getting high quality files from the authors. We have seen a growth in the Impact Factor and expect this to continue. I want to express my appreciation to Miss Sarwat Aziz Abbasi of the publisher's office for all her help with all matters involving the journal. We are very grateful to the members of the Editorial Advisory Board (EAB) for Protein and Peptide Letters for their efforts over the years to review manuscripts, to submit their own work, to recommend referees, and to critique the operation of all aspects of the journal. Over the past 15 years we have added new members to the EAB, thanked retiring members, and added new Regional Editors to help with the increasing manuscript flow we have experienced. While we are actively soliciting new individuals at this time, we are always open to self-nomination from interested readers of the journal. In this issue, we inaugurate a special feature that we hope will prove to be valuable for all readers. This issue features contributions from members of the EAB. This should help other scientists to identify with some EAB member when submitting their work to PPL. By identifying an EAB member who works in the area that you do to oversee the peer review process, you can insure that your manuscript will get a fair analysis by an expert in the field. For the same reason, to insure fair peer review, we ask that all submissions be accompanied by a list of four or more potential referees. The contributions in this issue were subjected to the same peer review process as all manuscripts and all went through a round of revision. The contributions cover a wide range of protein and peptide science. The first five papers loosely deal with studies of binding interactions between proteins and other molecules. Dunn and colleagues report on of a series of statine-based inhibitors binding to enzymes of the plasmepsin family as part of a study on potential antimalarial drugs. Ajoy Basak and coworkers report on the properties of heptad repeats from a fragment of the spike surface glycoprotein of the SARS virus. Monique Cosman, Daniel Barsky and their colleagues discuss the function of a specific Yersinia pestis gene product and conclude that it is most likely a glutamine binding protein. Odorant binding proteins are β-barrel proteins with the cavity inside the barrel serving as the binding site. Mamone and D'Auria report on the effects of deamination of a porcine odorant binding protein on its function. Claudia Regina Batista de Souza and her colleagues examine the possible cross reaction between sensitivity to latex and to proteins from the cassava plant. The next ten papers deal with structural analysis and prediction. David Craik's lab has contributed two papers to this issue: in the first, Schirra, Anderson, and Craik report on the structural refinement of NMR analysis of protease inhibitors from the tobacco plant; in the second paper, Westermann, Clark, and Craik use NMR to study the binding mode of α-conotoxins to an acetylcholine binding protein. Predicting membrane protein types is a new process of great importance in this post-genomic world. Kuo-Chen Chou and his colleagues apply a new program, called Local Linear Discriminant Analysis, to this job. The matrix protein M1 plays an important role in influenza virus growth and Kordyukova and her colleagues have used bromelain digestion to obtain information on the properties of M1. Identifying peptide segments hidden within protein sequences, which are termed cryptides, is described by Mukai and his colleagues in their discussion of the effects of several peptides on histamine release from mast cells. Kazuyasu Sakaguchi and co-authors discuss a subfamily of the protein phosphatases, the magnesium dependent phosphatases and use bioinformatic methods to identify active site residues and to analyze substrate specificity. The prion protein has an N-terminus that includes four repeats of the peptide sequence Gly-Gln- Phe-His-Gly-Gly-Gly-Trp. Shimohigashi and his colleagues analyze the amino acid composition of different parts of the prion protein using the Radar Chart method. An exciting new area of protein research involves intrinsically disordered proteins. Vladimir Uversky, Keith Dunker and their associates discuss the use of the TOP-IDP-Scale, a new prediction tool for measuring the propensity for intrinsic disorder. Willy Taylor and his colleagues Hollup and Jonassen describe the use of protein fragments in protein modeling and structure prediction. You-Ming Feng and his co-workers report on studies of the equilibrium folding of the proinsulin precursor protein. B. Moon Kim and his colleagues describe the synthesis of a conformationally constrained γ-turn mimic.......

Plasmepsin 4 (PM4) is a digestive vacuole enzyme found in all Plasmodium species examined to date. While P. falciparum has three additional aspartic proteinases in its digestive vacuole in addition to plasmepsin 4, other Plasmodium species have only PM4 in their digestive vacuole. Therefore, PM4 may be a good target for the development of an antimalarial drug. This study presents data obtained with PM4s from several Plasmodium species. Low nanomolar Ki values have been observed for all PM4s studied.

hSARS-CoV is the causative agent for SARS infection. Its spike glycoprotein (S) is processed by host furin enzyme to produce S1 and S2 fragments, the latter being crucial for fusion with the host membrane. This takes place via formation of a coiled coil 6-helix bundle involving N and Cterminal heptad repeat domains (HR-N and HR-C) of S2. Several fluorescent and non-fluorescent peptides from these domains were synthesized to examine their interactions by circular dichroism, thermal denaturation, native-page, mass spectrometry and fluorescence spectroscopy studies. Data revealed that HR-C domains (1153-1189), (1153-1172) and (1164-1184) all exhibit potent binding interactions with HR-N892-931 domain. These peptides may find useful therapeutic applications in SARS intervention.

Here we present modeling and NMR spectroscopic evidence that the function of a Yersinia pestis pMT1 plasmid protein, designated as orf38, is most likely a glutamine binding protein. The modeling was homology-based at a very low level of sequence identity (∼ 16%) and involved structural comparison of multiple templates, as well as template-substrate interaction analyses. Transferred nuclear Overhauser and saturation transfer difference experiments were used to characterize the binding of sugars and amino acids to orf38. The identification and characterization of an unknown protein function using the strategy presented here has applicability to a variety of research areas, including functional genomics and proteomics efforts.

Odorant-binding proteins are biomolecules belonging to the lipocalin family. Among all the odorant-binding proteins, the porcine odorant-binding protein has been well characterized. This protein is a monomer that is characterized by the presence of the β-barrel structure and of the disulphide bridge. The internal cavity of the β-barrel is the binding site. In this study we have investigated the structural properties of the porcine odorant-binding protein by mass spectrometry experiments. Our data allow us to hypothesize that specific deamidation mechanisms of specific amino acid residues can be responsible for the binding properties of this class of proteins

Pt2L4 is a protein from cassava homologue to Hevb5, a principal allergen from latex. Here we aimed to elucidate immunological relationships between these proteins. Our results revealed that epitopes found in Hev b 5 are not entirely conserved in Pt2L4 which is not recognized by IgE from patients allergic to Hev b 5.

Ornamental tobacco (Nicotiana alata) produces a series of 6 kDa proteinase inhibitors belonging to the potato type II inhibitor family. These proteins inhibit trypsin and chymotrypsin, the main digestive enzymes of predatory insects, thus leading to starvation, impaired larval development or death. In this context, the three-dimensional structures of these inhibitors are important for developing novel strategies for pest control. The solution structures of C1 and T1, the two main prototypes of the N. alata inhibitors, were originally determined more than a decade ago (J. Mol. Biol. 242, 231-243 (1994) and Biochemistry 34, 14304-14311 (1995)). Since then methods for NMR structure calculations have evolved considerably. Here we report the refinement of the structures of C1 and T1 with state-of-the-art protocols for NMR structure calculations. This refinement leads to an improved quality of the structures, making them a more reliable basis for the development of novel pesticides and modeling applications.

The saturation transfer difference (STD) NMR technique was employed to study the complex of the α-conotoxins Vc1.1 and MII bound to the acetylcholine binding protein (AChBP) from Lymnea stagnalis, a model system of the α7 subunit of the nicotinic acetylcholine receptor. MII was found to be the more potent ligand for AChBP, consistent with data from electrophysiology measurements for the nicotinic acetylcholine receptor. Both peptides displayed strong interactions on aromatic residues in the α-helical part of their sequences, i.e., Tyr10 in Vc1.1 and His9 in MII respectively. From the STD NMR spectra it was determined that the peptides are buried in the nicotinic binding site of ACBP as has been previously shown for the conotoxins PnIA[A10L, D14K], ImI and TxIA[A10L] by X-ray crystallography. This study demonstrates the value of STD NMR in the study of conotoxin binding to receptor proteins.

Membrane proteins are generally classified into the following eight types: (1) type I transmembrane, (2) type II, (3) type III, (4) type IV, (5) multipass transmembrane, (6) lipid-chain-anchored membrane, (7) GPI-anchored membrane, and (8) peripheral membrane (K.C. Chou and H.B. Shen: BBRC, 2007, 360: 339-345). Knowing the type of an uncharacterized membrane protein often provides useful clues for finding its biological function and interaction process with other molecules in a biological system. With the explosion of protein sequences generated in the Post-Genomic Age, it is urgent to develop an automated method to deal with such a challenge. Recently, the PsePSSM (Pseudo Position-Specific Score Matrix) descriptor is proposed by Chou and Shen (Biochem. Biophys. Res. Comm. 2007, 360, 339-345) to represent a protein sample. The advantage of the PsePSSM descriptor is that it can combine the evolution information and sequencecorrelated information. However, incorporating all these effects into a descriptor may cause the “high dimension disaster”. To overcome such a problem, the fusion approach was adopted by Chou and Shen. Here, a completely different approach, the so-called LLDA (Local Linear Discriminant Analysis) is introduced to extract the key features from the highdimensional PsePSSM space. The dimension-reduced descriptor vector thus obtained is a compact representation of the original high dimensional vector. Our jackknife and independent dataset test results indicate that it is very promising to use the LLDA approach to cope with complicated problems in biological systems, such as predicting the membrane protein type.

Influenza A virus matrix M1 protein is membrane associated and plays a crucial role in virus assembly and budding. The N-terminal two thirds of M1 protein was resolved by X-ray crystallography. The overall 3D structure as well as arrangement of the molecule in relation to the viral membrane remains obscure. Now a proteolytic digestion of virions with bromelain was used as an instrument for the in situ assessment of the M1 protein structure. The lipid bilayer around the subviral particles lacking glycoprotein spikes was partially disrupted as was shown by transmission electron microscopy. A phenomenon of M1 protein fragmentation inside the subviral particles was revealed by SDS-PAGE analysis followed by in-gel trypsin hydrolysis and MALDI-TOF mass spectrometry analysis of the additional bands. Putative bromelain-digestion sites appeared to be located at the surface of the M1 protein globule and could be used as landmarks for 3D molecular modeling.

Mastoparan, a toxic peptide from wasp venom, induces various biological functions including histamine release from rat peritoneal mast cells. Here we report that, for the activation of mast cells by mastoparan, at least two positively charged side chains are required on the hydrophilic side of the amphiphilic structure of the peptide. The present results are expected to be utilized for the bioinformatic and comprehensive identification of endogenous mast cell-stimulating cryptides.

Protein phosphatase magnesium-dependent 1, delta (PPM1D) is a member of the PPM1 (formerly PP2C) protein phosphatase family, and is induced in response to DNA damage. The overexpression of PPM1D is thought to exert oncogenic effects through the inhibition of tumor suppressor proteins. PPM1D shows high selectivity for the primary sequence in its substrates when compared with other phosphatases, but the mechanisms underlying substrate recognition by this enzyme is not clearly known. In our present study we wished to identify the active center and further elucidate the substrate preference of PPM1D, and to this end performed sequence alignments among the human PPM1 type phosphatases. The results of this analysis clearly showed that the putative active site residues of PPM1D are highly conserved among the PPM1 family members. Phosphatase analyses using PPM1D mutants further identified the metal-chelating residues and a phosphate binding residue. In kinetic analyses using a series of phosphorylated p53 peptide analogs, the introduction of acidic residues into the region flanking the sites of dephosphorylation enhanced their affinity with PPM1D. Homology modeling of PPM1D also revealed that PPM1D contains two characteristic loops, a Pro-residue rich loop on the opposite side of the active site and a basic-residue rich loop in the vicinity of the active site in the catalytic domain. Interestingly, nonhydrolyzable AP4-3E peptides derived from the acidic p53 peptide analogs very effectively blocked PPM1D activity in an uncompetitive manner, suggesting that AP4-3E peptides may be useful lead compounds in the development of novel inhibitors of PPM1D.

Analysis of the amino acid composition of prion protein using a newly developed program for radar-chart deviation analysis has identified an abnormality or irregularity of the N-terminal flexible domain. Aromatic amino acids Trp and His together with Gly are abnormally abounding in this Nterminal domain, in which octapeptide GQPHGGGW is connected four times in tandem. This tetrarepeat structure has been suggested to be essential for the prion protein not only to play an intrinsic functional role in the physiological condition, but also to bring on structural abnormalities in prion disease.

Intrinsically disordered proteins carry out various biological functions while lacking ordered secondary and/or tertiary structure. In order to find general intrinsic properties of amino acid residues that are responsible for the absence of ordered structure in intrinsically disordered proteins we surveyed 517 amino acid scales. Each of these scales was taken as an independent attribute for the subsequent analysis. For a given attribute value X, which is averaged over a consecutive string of amino acids, and for a given data set having both ordered and disordered segments, the conditional probabilities P(so|x)and P(sd|x) for order and disorder, respectively, can be determined for all possible values of X. Plots of the conditional probabilities P(so|x) and P(sd|x) versus X give a pair of curves. The area between these two curves divided by the total area of the graph gives the area ratio value (ARV), which is proportional to the degree of separation of the two probability curves and, therefore, provides a measure of the given attribute's power to discriminate between order and disorder. As ARV falls between zero and one, larger ARV corresponds to the better discrimination between order and disorder. Starting from the scale with the highest ARV, we applied a simulated annealing procedure to search for alternative scale values and have managed to increase the ARV by more than 10%. The ranking of the amino acids in this new TOP-IDP scale is as follows (from order promoting to disorder promoting): W, F, Y, I, M, L, V, N, C, T, A, G, R, D, H, Q, K, S, E, P. A web-based server has been created to apply the TOP-IDP scale to predict intrinsically disordered proteins (http://www.disprot.org/dev/disindex.php).

We survey a method that uses patterns of residue packing in known protein structures to refine structural models. The method can be used to refine models that include only one coordinate point per residue (Cα) and is not dependent on homology. We demonstrate that the method improves both decoy and CASP7 target models.

We use the procedure established for ‘disulfide stability analysis in redox system’ to investigate the unfolding process of porcine insulin precursor (PIP). Six major unfolding intermediates have been captured, in which four contain two disulfides, two contain one disulfide. Based on the characterization and analysis of the intermediates an unfolding pathway has been proposed, by which the native PIP unfolded through in turn 2SS and 1SS intermediates into fully reduced form. Besides, the comparison of the intermediates captured in PIP unfolding process with those intermediates captured in its refolding process revealed that some intermediates captured during both unfolding/refolding processes of PIP have identical disulfide pairing pattern, from which we suggest that the unfolding/refolding processes of PIP share some common intermediates but flow in the opposite direction.

An efficient asymmetric synthesis of 6-aminobicyclo[2.2.1]heptane-2-carboxylic acid as a novel γ-turn mimic has been achieved. Structural analysis of the γ-amino acid derivative was carried out using 1H NMR spectroscopy and intramolecular hydrogen bonding between side chain amides confirmed the turn structure, which had been predicted by Ab initio computational study.

Expression of two recombinant hepcidin homologues from Atlantic salmon, Salmo salar, characterization of their antimicrobial activity, and partial structural determination of the peptides is described. Expression was attempted in baculovirus and bacterial expression systems and the various purification and refolding methods used to determine the optimal strategy for production of active, correctly refolded hepcidin are reviewed.

The present review brings a timeline of some of the major steps given throughout the years towards the development of our knowledge regarding the biology of the neutrophil. The contribution of early articles and their elementary biochemical approach is highlighted. The importance of the development of proteomic techniques is paralleled to the shift in neutrophil research towards high throughput molecular methods. As a last change of standpoint, the study of the neutrophil is presented integrated with other lifesciences technologies such as lipidomics, genomics and systems biology. The paper also brings a perspective/ tendency overview at the same time that it discusses some of the difficulties encountered in the research of the neutrophil.

Bothropstoxin-II a calcium-dependent enzyme from Bothrops jararacussu venom causes tissue damage and several haemostatic disorders including platelet aggregation. In order to elucidate the structural determinants of its multiple pharmacological activities, we have studied the effects of suramin on Bothropstoxin-II and present details concerning the mode of binding.