Protein and Peptide Letters - Volume 17, Issue 12, 2010

The analysis of conformations corresponding to continuous amino acid repeat peptides (CARPs) comprising six or more residues in proteins of known three-dimensional structure revealed that alanine, glycine, glutamic acid, proline, valine, histidine, aspartic acid, glutamine and lysine were associated as repeating amino acid residues. Alanine, glycine and histidine CARPs were most common, although the histidine hexapeptide and large CARPs mainly correspond to affinity tags and are not part of the native protein sequence. The Ala and Glu CARPs were observed either as part of helix, or coil or a combination of these conformations. The octapeptide Ala CARP in six-hairpin glycosidases was observed as part of strand and coil conformation. The Gly and Pro CARPs were mainly associated with coil conformation. Majority of the coil regions in CARPs contained beta and gamma-turn structural motifs. The conformations of the Asp, Glu and Lys hexapeptide or larger CARPs were not defined in the corresponding protein three-dimensional structures analyzed. The longest CARP of known conformation was observed for alanine as a decapeptide in a lysozyme-like protein that corresponds to helix. A feature of CARPs is that a majority are exposed to solvent with accessible surface area greater than 200 Ų units in the protein three-dimensional structure.

Protein solubility plays a major role for understanding the crystal growth and crystallization process of protein. How to predict the propensity of a protein to be soluble or to form inclusion body is a long but not fairly resolved problem. After choosing almost 10,000 protein sequences from NCBI database and eliminating the sequences with 90% homologous similarity by CD-HIT, 5692 sequences remained. By using Chou's pseudo amino acid composition features, we predict the soluble protein with the three methods: support vector machine (SVM), back propagation neural network (BP Neural Network) and hybrid method based on SVM and BP Neural Network, respectively. Each method is evaluated by the re-substitution test and 10-fold cross-validation test. In the re-substitution test, the BP Neural Network performs with the best results, in which the accuracy achieves 92.88% and Matthews Correlation Coefficient (MCC) achieves 0.8513. Meanwhile, the other two methods are better than BP Neural Network in 10-fold cross-validation test. The hybrid method based on SVM and BP Neural Network is the best. The average accuracy is 86.78% and average MCC is 0.7233. Although all of the three methods achieve considerable evaluations, the hybrid method is deemed to be the best, according to the performance comparison.

Apoptosis is an essential process for controlling tissue homeostasis by regulating a physiological balance between cell proliferation and cell death. The subcellular locations of proteins performing the cell death are determined by mostly independent cellular mechanisms. The regular bioinformatics tools to predict the subcellular locations of such apoptotic proteins do often fail. This work proposes a model for the sorting of proteins that are involved in apoptosis, allowing us to both the prediction of their subcellular locations as well as the molecular properties that contributed to it. We report a novel hybrid Genetic Algorithm (GA)/Support Vector Machine (SVM) approach to predict apoptotic protein sequences using 119 sequence derived properties like frequency of amino acid groups, secondary structure, and physicochemical properties. GA is used for selecting a near-optimal subset of informative features that is most relevant for the classification. Jackknife cross-validation is applied to test the predictive capability of the proposed method on 317 apoptosis proteins. Our method achieved 85.80% accuracy using all 119 features and 89.91% accuracy for 25 features selected by GA. Our models were examined by a test dataset of 98 apoptosis proteins and obtained an overall accuracy of 90.34%. The results show that the proposed approach is promising; it is able to select small subsets of features and still improves the classification accuracy. Our model can contribute to the understanding of programmed cell death and drug discovery. The software and dataset are available at http://www.inb.uni-luebeck.de/tools-demos/apoptosis/GASVM

LIM Kinase 2 (LIMK2) is a LIM domain-containing protein kinase which regulates actin polymerization thorough phosphorylation of the actin depolymerizing factor cofilin. It is also known to function as a shuttle between the cytoplasm and nucleus in endothelial cells. A basic amino acid-rich motif in LIMK2 was previously identified to be responsible for this shuttling function, as a nucleolar localization signal (NoLS). Here it is shown that this nucleolar localization signal sequence also has the characteristic function of a cell-penetrating peptide (CPP). We synthesized LIMK2 NoLSconjugated peptides and a protein and analyzed their cell-penetrating abilities in various types of cells. The BC-box motif of the Von Hippel-Lindau (VHL) protein was used for the peptide. This motif previously has been reported to be involved in the neural differentiation of bone marrow stromal cells and skin-derived precursor cells. Green fluorescence protein (GFP) was used as a large biologically active biomolecule for the protein. The LIMK2 NoLS-conjugated peptides and protein translocated across the cell membranes of fibroblast cells, neural stem cells, and even iPS cells. These results suggest that LIMK2 NoLS acts as a cell-penetrating peptide and its cell-penetrating ability is not restricted by cell type. Moreover, from an in vivo assay using a mouse brain, it was confirmed that LIMK2 NoLS has potential for transporting biomolecules across the blood-brain barrier.

[PSI+] phenotype can be transiently induced when Magnesium chloride (MgCl2) was the selective pressure in SUP35 repeat-expansion mutant [psi-] yeast strains. We further investigated [PSI+] phenotype change under different MgCl2 conditions with native Sup35p and quantified the Sup35p status changes with fluorescence recovery after photobleaching (FRAP) and semi-denaturing detergent-agarose gel electrophoresis (SDD-AGE) analysis. It was found that the [PSI+] phenotype presented a dose-dependent relationship with the concentrations of MgCl2. Furthermore, Sup35p aggregated in MgCl2 treated cells but did not form large aggregates as it does in [PSI+] cells, and the size of Sup35p aggregates showed a time-dependent relationship with the MgCl2 application. The aggregation of Sup35p strictly depended on the presence of MgCl2 stress in our strains.

Rational drug design is an important step towards effective patient care enabling lead molecule discovery in a relatively faster and inexpensive way. This article describes a variable string length genetic algorithm with domain specific operators for de novo ligand design. The algorithm first mines the active site of the given protein receptor whose geometry and chemical composition guides the ligand building. Active site mined by the algorithm is compared with two more established active sites detecting schemes to evaluate its efficiency. Various combinations from a suite of forty one fragments are mined to design the ligands. Bond stretching, angle bending, torsional terms, van der Waals and electrostatic interaction energy with distance dependent dielectric constant contribute are used to compute the internal energy of the ligand and the interaction energy of the ligand receptor complex. Forty one fragments are used to design the ligands. Experimental results are provided for HIV-1 Protease, HIV-1 Nef and Thrombin demonstrating the superiority of the proposed scheme vis-a-vis three other approaches. Comparison with known inhibitors also demonstrates the effectiveness of the proposed approach.

Fowlicidin-3 and fowlicidin-1 are cathelicidin-type antimicrobial peptides found in chicken. They effectively inhibit the proliferation of many gram-positive and gram-negative bacteria. To obtain sufficient amounts of these peptides for possible use in therapeutic applications, DNA encoding each full-length gene, including all exons and introns, was fused to the β-casein promoter in a pBC1 vector that was then introduced into C127 cells. The full-length precursor proteins were expressed in response to a mixture of insulin, hydrocortisone, and prolactin. Processed fowlicidin-1 and fowlicidin- 3, as well as their precursors, were found in the cell culture media, which suggested that they could be processed and secreted. These transgenic peptides had antibacterial activity. Thus, transfected C127 cells may serve as an in vitro transgenic cell system that can be used to evaluate if specific gene constructs can be efficiently expressed in the mammary glands of transgenic mice.

The interaction between cyclophosphamide monohydrate with human serum albumin (HSA) and human serum transferrin (hTf) was studied with UV absorption, fluorescence and circular dichroism (CD) spectroscopies as well as molecular modeling. Based on the fluorescence quenching results, it was determined that HSA and hTf had two classes of apparent binding constants and binding sites at physiological conditions. The KSV1, KSV2, n1 and n2 values for HSA were found to be 8.6×108 Lmol-1, 6.34×108 Lmol-1, 0.7 and 0.8, respectively, and the corresponding results for hTf were 6.08×107 Lmol-1, 4.65×107 Lmol-1, 1.3 and 2.6, respectively. However, the binding affinity of cyclophosphamide monohydrate to HSA was more significant than to hTf. Circular dichroism results demonstrated that the binding of cyclophosphamide to HSA and hTf induced secondary changes in the structure and that the α-helix content became altered into β- sheet, turn and random coil forms. The participation of tyrosyl and tryptophan residues of proteins was also estimated in the drug-HSA and hTf complexes by synchronous fluorescence. The micro-environment of the HSA and hTf fluorophores was transferred to hydrophobic and hydrophilic conditions, respectively. The distance r between donor and acceptor was obtained by the Forster energy according to fluorescence resonance energy transfer (FRET) and found to be 1.84 nm and 1.73 nm for HSA and hTf, respectively. This confirmed the existence of static quenching for both proteins in the presence of cyclophosphamide monohydrate. Site marker competitive displacement experiments demonstrated that cyclophosphamide bound with high affinity to Site II, sub-domain IIIA of HSA, and for hTf, the C-lobe constituted the binding site. Furthermore, a study of molecular modeling showed that cyclophosphamide situated in domain II in HSA was bound through hydrogen bonding with Arg 257 and Ser 287, and that cyclophosphamide was situated in the C-lobe in hTf, presenting hydrogen bonding with Asp 625 and Arg 453. The modeling data thus confirmed the experimental results.

Information about interactions between enzymes and small molecules is important for understanding various metabolic bioprocesses. In this article we applied a majority voting system to predict the interactions between enzymes and small molecules in the metabolic pathways, by combining several classifiers including AdaBoost, Bagging and KNN together. The advantage of such a strategy is based on the principle that a predictor based majority voting systems usually provide more reliable results than any single classifier. The prediction accuracies thus obtained on a training dataset and an independent testing dataset were 82.8% and 84.8%, respectively. The prediction accuracy for the networking couples in the independent testing dataset was 75.5%, which is about 4% higher than that reported in a previous study [1]. The webserver for the prediction method presented in this paper is available at http://chemdata.shu.edu.cn/small-enz.

The present paper is an attempt to study the mechanism of ethanol induced aggregation of chicken egg albumin and to stabilize the protein against ethanol induced aggregation. The protein aggregation was determined by monitoring the light scattering of protein aggregates spectrophotometrically. The protein undergoes certain structural changes in water- ethanol solution and the degree of aggregation was found to be linearly depending upon the concentration of alcohol used. The intrinsic fluorescence study showed a large blue shift in the λmax (16 nm) in the presence of 50% ethanol. The ANS fluorescence intensity was found to be gradually increasing with increasing concentration of ethanol. This indicates an increase in the hydrophobic cluster on the protein surface and as a result the hydrophobic interaction is appeared to be the major driving force for the aggregate formation. Addition of sucrose significantly reduced the ethanol-induced aggregation of chicken egg albumin. In presence of 50% sucrose the ethanol induced aggregation was reduced to 5%. The study reveals that addition of sucrose brings out changes in the solvent distribution and prevents the structural changes in protein which lead the aggregation.

We propose a novel concept associated with the relationship between structure and function in biomolecular systems. We performed a 75 nanoseconds molecular dynamics (MD) simulation for an RNA-binding protein, neurooncological ventral antigen (NOVA), and examined its physico-chemical properties. NOVA dissociated from the NOVARNA complex showed a large conformational change: formation of intra-molecular hydrogen bonds between the Cterminal region and the loop structure located at the middle of amino acid sequence. The free energy analysis suggests that the deformed structure is more stabilized in macromolecular crowding environment where the dielectric constant is smaller than 5. The solvent accessible surface area (SASA) analysis indicates that NOVA enhances the efficiency of association with RNA by changing the relative SASA for the target sequence in RNA molecules. Based on the obtained results, we propose a novel concept of spontaneous adjustment mechanism to explain the structural and energetic changes observed for NOVA in the free state.

Multidrug-resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC transporter family. Proteins in this family share many characteristic structural features, including two membrane-spanning domains and two nucleotide-binding domains (NBD1 and NBD2), that function cooperatively but not equally bind and hydrolyze ATP. The MRP6 protein is structurally and functionally poorly characterized. Previously, we showed, by NMR spectroscopy, that a fragment of MRP6-NBD1 presents helical structure and fluorescence experiments demonstrated that peptide binds ATP. These data suggested that the study on selected regions could be a valid approach to define the structure of MRP6. In the present study, to better characterize MRP6-NBD1, we report data of CD spectroscopy, nucleotide binding and ATP hydrolysis on two different polypeptides, one corresponding to the full-length NBD1 domain (residues from Asp-627 to Leu-851) and the oth We report that both polypeptides are highly structured in aqueous buffer and in 20% trifluoroethanol showing considerable helical and β-structure content. The ATP hydrolysis activity is exhibited only by the full-length NBD1 domain. Comparison between our

Asparagine can be non-enzymatically deamidated and isomerized via succinimide to isoaspartate. This posttranslational modification can potentially alter the physical properties or the function of the parent protein. Asn32 of ribonuclease U2A from Ustilago sphaerogena is known to rapidly deamidate and isomerize in alkaline conditions. The crystal structure of ribonuclease U2A complexed with 2´-adenylic acid and calcium ions was determined at 1.03 Å resolution. In this structure, the region from Asp29 to Asp37 winds around a calcium ion, and the main-chain of Asn32-Gly33 adopts an extended conformation. Rotation of the side-chain of Asn32 could bring Asn32Cγ into close proximity to Gly33N, in a conformation suitable for succinimide formation. The structure suggests that in solution the region around Asn32-Gly33 is likely to be in equilibrium between multiple conformers, with the deamidation of Asn32 proceeding when the region adopts an extended conformation.

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents at high concentration to tumor tissues while minimizing systemic drug exposure. β-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity, which allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. This study used integrin αv β3 as a target for tumor-specific delivery of β- Lactamase. β-Lactamase was fused with ACDCRGDCFCG peptide (RGD4C) by recombinant DNA technology. Likewise, this study cloned a fused cDNA and successfully expressed active recombinant protein in E. coli purified with Ni- NTA resin. After purification, the protein showed the expected size of 42 kDa on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein was found to be active for specificity in breast cancer cell line, MCF-7, which supports the utility of the protein as an agent for ADEPT.

Caspases play an important role in many critical non-apoptosis processes by cleaving relevant substrates at cleavage sites. Identification of caspase substrate cleavage sites is the key to understand these processes. This paper proposes a hybrid method using support vector machine (SVM) in conjunction with position specific scoring matrices (PSSM) for caspase substrate cleavage sites prediction. Three encoding schemes including orthonormal binary encoding, BLOSUM62 matrix profile and PSSM profile of neighborhood surrounding the substrate cleavage sites were regarded as the input of SVM. The 10-fold cross validation results demonstrate that the SVM-PSSM method performs well with an overall accuracy of 97.619% on a larger dataset.