Protein and Peptide Letters - Volume 17, Issue 4, 2010

We used a de novo designed, β-hairpin forming T1 peptide as a model to investigate the kinetics of peptide fibrogenesis by a combination of light scattering (LS), circular dichroism (CD), fluorescence, and atomic force microscopy (AFM). The results demonstrate that the T1 fibrogenesis undergoes a consecutive stepwise process, with a high degree of cooperation, presenting sigmoidal time-courses of the peptide aggregation, the subsequent conformational conversion of the backbone, and the peptide sidechains' rearrangement. We suggest that the conformational conversion was initiated after the peptide aggregates reach a dimensional size threshold, which could be a key step in the formation of β-structural nuclei that catalyze the subsequent reactions. Furthermore, besides triggering the peptide aggregation, the interactions between the peptide sidechains predominately facilitate the regular alignment of the peptide molecules and the formation of a well-defined suprastructure. This work provides an insight of the hierarchical self-assembly of β-hairpin forming peptides. It is helpful for designing β-structural peptides for self-assembly into nanowires, which would have potential applications in the construction of nano-materials.

To investigate the processing of transforming growth factor β1 (TGFβ1) pro-protein by furin protease we expressed a GST-pro-TGFβ1 fusion protein in bacteria. Analysis of the furin digestion pattern revealed the liberation of 12.5 kDa TGFβ1 monomers. There was no evidence for cleavage of an alternative furin site within the pro-protein.

Dynamic kinetic resolution (DKR) was demonstrated in the carbodiimide-mediated couplings of N-benzoyl-DLamino acids with L-amino acid esters: the yields of the D-L-peptides significantly exceeded 50% in some cases. NBenzoyl- DL-t-leucine afforded the D-L-peptide almost exclusively (up to 96% yield) in the reaction with methyl Lprolinate, which is the most efficient DKR obtained in the field of amino acids and derivatives.

X-ray crystallography is the most widely used method for protein 3-dimensional structure determination. Selection of target protein that can yield high quality crystal for X-ray crystallography is a challenging task. Prediction of protein crystallization propensity from sequence information is useful for the selection of target protein for crystallization. Recently, support vector machines have been widely used to solve various biological problems. In this work, we present a SVMCRYS method which use support vector machine to classify protein sequence into ‘amenable to crystallization’ and ‘resistant to crystallization’. SVMCRYS was trained on a dataset containing 728 sequences that gave diffraction quality crystal and 728 sequences where work had been stopped before obtaining crystal. The performance of SVMCRYS method was compared with other sequence-based crystallization prediction methods such as SECRET, CRYSTALP, OB-Score, ParCrys and XtalPred using three different datasets. SVMCRYS achieved better prediction rate with higher sensitivity and specificity. Our analysis suggests that SVMCRYS can be used to predict proteins which are amenable to crystallization and proteins which are difficult for crystallization. The SVMCRYS software, dataset and feature set can be obtained from http://www3.ntu.edu.sg/home/EPNSugan/index_files/svmcrys.htm.

In insects numerous physiological processes are regulated by neuropeptides. Two fluorescent analogues of the amino acids tryptophan and tyrosine were synthesized and incorporated in the diuretic neuropeptide helicokinin I from the moth Heliothis zea. By fluorescence emission measurements it was shown that both fluorescent helicokinin I analogues react sensitive on the dielectricity of their microenvironment. A helicokinin I analogue containing the fluorescent tryptophan mimic β-[6'-(N,N-dimethyl)-amino-2'-naphthoyl]alanine (Ald) was shown to bind to dodecylphosphocholine (DPC) micelles by the Ald residue. A membrane binding model for helicokinin I is proposed based on data from related mammalian and insect-neuropeptides.

The stereochemistry of the hydride transfer in reactions catalyzed by NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus HB27 was determined by means of 1H-NMR spectroscopy. The enzyme transfers the pro-S hydrogen of [4R-2H]NADH and exhibits Prelog specificity. Enzyme-substrate docking calculations provided structural details about the enantioselectivity of this thermophilic enzyme. These results give additional insights into the diverse active site architectures of the largely versatile short-chain dehydrogenase superfamily enzymes. A feasible protocol for the synthesis of [4R-2H]NADH with high yield was also set up by enzymatic oxidation of 2-propanol-d8 catalyzed by Bacillus stearothermophilus alcohol dehydrogenase.

Most common methods for isolation of the light-harvesting 2 (LH2) complex are time-consuming. In our present work, functional LH2 complex was one-step purified from Rhodobacter sphaeroides by immobilized metal-ion affinity chromatography. The purified LH2 complex exhibited high activity in light absorption and energy transfer. Our present work provides an efficient approach for preparation of functional LH2 complex.

Purpose: A number of proteome studies have been performed recently to identify pheromone-related protein expression and their molecular function using genetically modified rodents' urine. However, no such studies have used Indian commensal rodents; interestingly, in a previous investigation, we confirmed the presence of volatile molecules in commensal rodents urine and these molecules seem to be actively involved in pheromonal communication. Therefore, the present study aims to identify the major urinary protein [MUP] present in commensal rat urine, which will help us to understand the protein's expression pattern and intrinsic properties among the rodents globally. Experimental Design: Initially, the total urinary proteins were separated by 1-D and 2-D electrophoresis and then subsequently analyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight and Mass Spectrometer (MALDITOF/ MS). Furthermore, they were then fragmented with the aid of a Tandem Mass Spectrometer (TOF/TOF) and the identified sequences aligned and confirmed using similarity with the deduced primary structures of members of the lipocalin superfamily. Results: The SDS-PAGE protein profiles showed distinct proteins with molecular masses of 15, 22.4, 25, 28, 42, 50, 55, 68, and 91 kDa. Of these proteins, the 22.4 kDa protein was considered to be target candidate. When 2D electrophoresis was carried out, about ~50 spots were detected with different masses and various pI ranges. The 22.4 kDa protein was found to have a pI of about 4.9. This 22.4 kDa protein spot was digested and subjected to mass spectrometry; it was identified as rat MUP. The fragmented peptides from the rat MUP at 935, 1026, 1192, and 1303 m/z were further fragmented with the aid of MS/MS and generated de novo sequence and this confirmed this protein to be the MUP present in the urine of commensal rats. Conclusion: The present investigation confirms the presence of MUP with a molecular mass of 22.4 kDa in the urine of commensal rats. This protein may be involved in the binding of volatile molecules and opens up a discussion about how volatile and non-volatile molecules in the commensal rats' urine may contribute chemo-communic

We report that the addition of amino acids to the amyloid peptide dramatically affected the structure and the rate of formation of amyloid fibrils. The attachment of three lysines to Aβ(10-35) gave the formation of remarkably long fibrils, while three glutamates resulted in a faster formation rate of the fibrils.

In this paper, we propose a strategy to predict the subcellular locations of proteins by combining various feature selection methods. Firstly, proteins are coded by amino-acid composition and physicochemical properties, then these features are arranged by Minimum Redundancy Maximum Relevance method and further filtered by feature selection procedure. Nearest Neighbor Algorithm is used as a prediction model to predict the protein subcellular locations, and gains a correct prediction rate of 70.63%, evaluated by Jackknife cross-validation. Results of feature selection also enable us to identify the most important protein properties. The prediction software is available for public access on the website http://chemdata.shu.edu.cn/sub22/, which may play a important complementary role to a series of web-server predictors summarized recently in a review by Chou and Shen (Chou, K.C., Shen, H.B. Natural Science, 2009, 2, 63-92, http://www.scirp.org/journal/NS/).

Band assignment for oxy, deoxy and methemoglobin using orbital promotion is crucial to understanding inter-relation of electronic transitions. Spectral changes may be correlated with conformational alterations. Conformational changes of hemoglobin were interpreted using four-orbital model of Gouterman. Our results indicated that Goutherman model can predict the predominant conformations of hemoglobin.

Lipoxygenases (LOXs, EC 1.13.11.12) are a class of non-heme iron containing dioxygenases which catalyze the regiospecific and stereospecific hydroperoxidation of polyunsaturated fatty acids with 1,4-pentadiene system such as linoleic acid and linolenic acid in plants. In this work we studied the LOX activity in damaged as well as in distal leaves in response to specialist (Agraulis vanillae vanillae) or generalist (Spodoptera frugiperda) insect attack. Enzymatic assays showed that induction of LOX activity occurred locally and systemically in response to both insects' attacks. Northern blot analysis revealed that LOX expression is also insect-inducible in agreement with enzymatic assay results. In addition, northern analysis corroborated previous reports that LOX activity is wound- and methyl jasmonate-inducible. These results suggest that the herbivore-response in passion fruit is mediated by jasmonates, since a key enzyme of the biosynthetic pathway of jasmonic acid is induced upon lepidopteran insects' attacks.

Rho GTPase controls multiple signal-transduction pathways involving the actin cytoskeleton and the microtubule cytoskeleton in processes such as the cell cycle, morphogenesis, and cell migration. The activity of Rho GTPases, such as Rac1, Cdc42, and RhoA, is regulated by GTPase-activating proteins (GAPs), guanine nucleotide exchange factors (GEFs), and guanine nucleotide dissociation inhibitor (GDI). The GEFs activate Rho GTPases through exchange of GDP for GTP. A group of the GEF family, CDM-GEF, containing the Dock180 Homology Region 2 (DHR2) domain is subdivided into four groups based on amino acid sequences. One of these subgroups, DOCK-A, includes DOCK1, DOCK2, and DOCK5, and activates Rac1 Rho GTPase. Mouse Dock5 (mDock5) is structurally similar to DOCK1 and composed of 1868 amino acids containing Src homology 3 (SH3), DHR1, DHR2, and a proline-rich (PR) motif. We generated an mDock5-specific antibody, which detected denatured and nascent forms of mDock5. We determined tissue-specific expression patterns and subcellular localization of mDock5 using this antibody. This antibody provides a way to delineate the biological functions of mDock5 in vivo.

Hop is a tetratricopeptide repeat domain (TPR)-containing co-chaperone that is able to directly associate with both Hsp70 and Hsp90. Previous data showed that the TPR2A-domain is the primary site for dimerization and that the TPR2B-domain may also play a role in dimerization. We present Hop-D456G, a mutant within the TPR2B-domain, that is a mixture of monomeric and dimeric species.

A new class of 1,4-substituted 1,2,3-triazole-based unnatural amino acids is demonstrated by employing click reaction between N-Fmoc amino alkyl azides and propiolic acid. The resulting unnatural amino acids were isolated and then subjected to Fmoc deprotection to isolate 1,2,3-triazole based amino acids as stable solids. These new class of molecules were also used for chain extension from both N- and C-terminals to synthesize dipeptidomimetics bearing 1,2,3- triazole moiety in the backbone.

The Hierarchical Domain-Wise Alignment (HDWA) technique of domain identification in proteins is presented. HDWA is designed to identify hierarchically organized dynamic domains in proteins using the MD trajectories by eliminating systematic motions from MD trajectories recursively in a model-free manner. The method is tested on the proteins from different structural classes.

Granulysin is a human polypeptide produced by cytolytic cells active against a broad range of microbes. Three peptides covering the regions 25-50 (Gr-1 and Gr-2) and 39-62 (Gr-3) of granulysin were synthesized, and their in vitro activity against Mycobacterium tuberculosis was evaluated. The most active peptide was Gr-1C, containing a disulphide bridge, with Minimal Inhibitory Concentration value of 10.1 μM. In concentrations of up to 50 μM, Gr-1 and Gr2 didn't exceed 30% of hemolysis.

In this work, we isolated and characterized novel antifungal proteins from seeds of dandelion (Taraxacum officinale Wigg.). We showed that they are represented by five isoforms, each consisting of two disulphide-bonded large and small subunits. One of them, To-A1 was studied in detail, including N-terminal amino acid sequencing of both subunits, and shown to display sequence homology with the sunflower 2S albumin. Using different assays we demonstrated that dandelion 2S albumins possess inhibitory activity against phytopathogenic fungi and the oomycete Phytophtora infestans at micromolar concentrations with various isoforms differing in their antifungal activity. Thus, 2S albumins of dandelion seeds represent a novel example of storage proteins with defense functions.

The understanding of evolutionary mechanism is important, and equally important is to describe the evolutionary process. If so, we would know where the biological evolution will go. At species level, we would know whether and when a species will extinct or be prosperous. At protein level, we would know when a protein family will mutate more. In our previous study, we explored the possibility of using the differential equation to describe the evolution of protein family from influenza A virus based on the assumption that the mutation process is the exchange of entropy between protein family and its environment. In this study, we use the analytical solution of system of differential equations to fit the evolution of matrix protein 1 family from influenza A virus. Because the evolutionary process goes along the time course, it can be described by differential equation. The results show that the evolution of a protein family can be fitted by the analytical solution. With the obtained fitted parameters, we may predict the evolution of matrix protein 1 family from influenza A virus. Our model would be the first step towards the systematical modeling of biological evolution and paves the way for further modeling.

A novel small antifungal peptide-producing strain D4 was isolated from the dung of wild plateau yak and identified as Bacillus megaterium. The purification procedure of peptide consisted of acid precipitation, methanol extract and C18 reverse-phase chromatography. The amino acid composition of peptide YP differed from those of antifungal peptides which have been reported to date. Peptide YP was proved to be a novel small antifungal cyclic peptide. It exhibited strong inhibitory activity against many phytopathogenic fungi and had a wild range of thermo stability and pH stability and was not susceptible to all proteases tested.