Protein and Peptide Letters - Volume 17, Issue 6, 2010

The sizes of the protein databases are growing rapidly nowadays thus clustering protein sequences based only on sequence information becomes increasingly important. In this paper, we analyze the limitation of Affinity propagation (AP) algorithm when clustering a dataset generated randomly. Then we propose a post-processing method to improve the AP algorithm. This method uses the median of the input similarities as the shared preference value, and then employs post-processing phase combined mergence and reassignment strategy on the results of the AP algorithm. We have tested our method extensively and compared its performance with other five methods on several datasets of COG (Clusters of Orthologous Groups of proteins) database, SCOP and G-protein family. The number of clusters obtained for a given set of proteins approximate to the correct number of clusters in that set. Moreover, in our experiments, the quality of the clusters as quantified by F-measure was better than that of others (on average, 9% better than BlastClust, 33% better than TribeMCL, 34% better than CLUSS, 59% better than Spectral clustering and 41% better than AP).

Lectins, a class of proteins that reversibly and non-enzymatically bind specific sugars, have been purified from different kinds of legumes. In this study, a 48-kDa lectin (KBL) was purified from Korean large black soybeans using liquid chromatography. The specific hemagglutinating activity of the KBL was 4096 titer/mg. EDTA-induced loss of hemagglutinating activity of KBL could be recovered by addition of Fe3+ ions and some divalent cations as Ca2+, Mn2+, Fe2+, Cu2+, Zn2+, and Pb2+. Sugars such as D-(+)-galactose, D-(+)-raffinose, L-(+)-arabinose, α-D-(+)-melibiose, and α-lactose could inhibit the hemagglutinating activity of the lectin. Furthermore, the protein showed high thermal stability as well as stability over a wide range of pH values. KBL inhibited HIV-1 reverse transcriptase activity with an IC50 of 1.38 μM. However, it was destitute of cytokine releasing, mitogenic, ribonuclease and antifungal activities. In addition, inhibitory activity toward nasopharyngeal cell lines was undetectable in KBL at concentrations up to 20 μM.

An all-atom level MD simulation in explicit solvent at high temperature is a powerful technique to increase our knowledge about the structurally important regions modulating thermal stability in thermoenzymes. In this respect, two large-sized thermoalkalophilic enzymes from Bacillus stearothermophilus L1 (L1 lipase) and Geobacillus zalihae strain T1 (T1 lipase) are well-established representatives. In this paper, comparative results from temperature-induced MD simulations of both model systems at 300 K, 400 K and 500 K are presented and discussed with respect to identification of highly flexible regions critical to thermostability. From our MD simulation results, specific regions along the L1 lipase and T1 lipase polypeptide chain including the small domain and the main catalytic domain or core domain of both enzymes show a marked increase in fluctuations and dynamics followed by clear structural changes. Overall, the N-terminal moiety of both enzymes and their small domains exhibit hyper-sensitivity to thermal stress. The results appear to propose that these regions are critical in determining of the overall thermal stability of both organisms.

Four AS-48 mutants (Trp24Ala, Gly13Lys, Leu40Lys and Ala53Ser) were obtained by site-directed mutagenesis. The minimal inhibitory concentration of each peptide showed that only residue Trp24 was unquestionably involved in the biological activity. Guanidine hydrochloride-induced unfolding assays showed a three-state transition denaturation process, suggesting a molten-globule-like conformation after the first transition.

The early determination of family for a newly found enzyme molecule becomes important because it is directly related to the detail information about which specific target it acts on, as well as to its catalytic process and biological function. Unfortunately, it is still a hard work to distinguish enzyme classes by experiments. With an enormous amount of protein sequences uncovered in the genome research, it is both challenging and indispensable to develop an automatic method for fast and reliably classifying the enzyme family. Using the concept of Chou's pseudo amino acid composition, we developed a new method that coupled discrete wavelet transform with support vector machine based on the amino acid hydrophobicity to predict enzyme family. The overall success rate obtained by the 10-cross-validation for the identification of the six enzyme families was 91.9%, indicating the current method could be an effective and promising highthroughput method in the enzyme research.

State-of-the-art biochemistry methods in combination with an automated phenotyping method demonstrate the high potential of transgenic tobacco plants in producing properly-folded therapeutic proteins for the treatment of protein-misfolding diseases (e.g., Alzheimer's disease). This molecular farming approach led to highest protein production of hydroponically- grown tobacco compared to other growth substrates generally used in plant cultivation.

Mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction. It is the objective of this communication to demonstrate that insight into protein-protein interactions in the Common Docking motif of yeast mitogen-activated protein kinases can be obtained based on homology models. Homology models for four yeast MAPKs, FUS3, KSS1, HOG1 and MPK1 were built based on the X-ray structures of active and inactive rat ERK2. The structural motifs required for the basis of specificity were rationalized based on these structures.

In this paper, we report our study of thermodynamic parameters of the interactions of antifreeze proteins (AFP) type I and it short segments with DMPC unilamellar vesicles as model for cell membrane. The heat of interactions between AFP's and the model cell membrane were studied by Isothermal Titration Calorimetry (ITC) at temperatures above and below phase transition temperatures of the membrane. It is shown that heat of interactions is linearly dependent on the temperatures below the phase transition of the membrane and constant at temperatures above phase. The heat of interaction above phase transition is assigned to the interaction of the AFP with the membrane, while below phase transition the ordering effect of the AFP influence the heat of interaction.

MSH proteins are capable of recognizing damage in DNA due to a common chemotheraputic, cisplatin, and consequently participate in the initiation of cell death pathways. While previous studies have used computational modeling and cell biology to demonstrate that there are specific structural responses to cisplatin damage that are critical to the initiation of apoptosis, this study demonstrates that there are also specific dynamical changes that are also associated with cisplatin binding. These changes further distinguish the undamaged MutS/DNA complex from the damaged MutS/DNA complex and suggest that there are dynamical aspects to the response of MSH proteins to the binding of DNA damaged by therapeutics; consideration of these responses may influence further drug design and development.

SsHSP14.1, a novel sHSP from the hyper-thermophilic archaeon Sulfolobus solfataricus (S. solfataricus), is reported herein to function to protect EcoR I from heat-induced inactivation. A predicted salt bridge and hydrophobic interactions were found to be important for this function.

The coupling of an aspartate residue with an active site histidine plays a pivotal role in enzyme catalysis. The His-Asp pair in glutamate mutase and other B12-dependent mutases is not only responsible for coenzyme-binding, but is also involved in fine-tuning the enzymatic activities. Our modeling results show that the His-Asp pair is arranged in a highly organized manner. Except for carboxymethylated Cys or Glu, a less hindered or non-charged amino acid residue is preferred between the conserved histidine and aspartate residue.

In this study, we have developed a standalone tool called as ANAMBS (Analysis of Metal Binding Site) to derive metal neighbourhood information using PERL as the programming language. The tool accepts the structures in the pdb format. The cut off distance to define the metal binding region can be specified. The metal binding site composition, orientation of various amino acids and atoms along with the Hydropathy index within the metal binding site region can be measured. Its speed and efficiency makes it a beneficial tool for various structural biology projects, especially when the characterization of the metal binding domain is needed. Additionally, the database MEDB (Metal Environment Database) was developed which presents quantitative information on metal-binding sites in protein structures. It can be used for identification of trends or patterns in the metal-binding sites. The information obtained can be used to generate structural templates from metal binding sites of known enzymes and to develop constraints for computational modeling of metalloproteins. The tool and database are available at http://www.uohyd.ernet.in/anambs/

Shigella dysenteriae type 1 and Escherichia coli O157:H7 produce Shiga toxin (Stx) and Shiga toxin (Stx1), respectively and these two toxins are almost identical. E. coli O157:H7 is the major cause of diarrhea-associated hemolytic uremic syndrome. Stx and Stx1 are AB5 type of toxin with a molecular weight of 70 kDa, comprising an enzymaticalyactive A subunit (32 kDa) and five receptor-binding B subunits (7.7 kDa). In this study DNA fragment (289 bp, Gene Bank Accn No. EF685161) coding for B chain of Stx was amplified from S. dysenteriae type1 and cloned. Shiga toxin-binding subunit was expressed and purified in native conditions by affinity and gel permeation chromatography with the yield of 5.1 mg/L in shake flask culture. For the purpose of immunization, the polypeptide was polymerized with glutaraldehyde. Hyper immune serum produced in mice reacted with the purified polypeptide and intact Shiga toxin. The anti- StxB antiserum effectively neutralized the cytotoxicity of Shiga toxin towards HeLa cells.

A 26-kDa trypsin inhibitor with an N-terminal sequence resembling storage proteins was purified from moth beans (Phaseolus acutifolius). It dose-dependently inhibited trypsin with an IC50 value of about 0.38 μM. It inhibited [methyl-3H] thymidine incorporation by lymphoma MBL2 cells with an IC50 value of 20 μM.

The prediction of protein function is a difficult and important problem in computational biology. In this study, an efficient method is presented to predict protein function with sequence composition information. Four kinds of basic building blocks of protein sequences are investigated, including N-grams, binary profiles, PFAM domains and InterPro domains. The protein sequences are mapped into high-dimensional vectors by using the occurrence frequencies of each kind of building blocks. The resulting vectors are then taken as input to support vector machine to predict their function based on gene ontology. Experiments are conducted over the subset of GOA database. The experimental results show that the protein function can be predicted from primary sequence information. The method based on InterPro domains outperforms the other building blocks, and gets an overall accuracy of 0.87 and ROC score is 0.93. We also demonstrate that the use of feature extraction algorithms such as latent semantic analysis and nonnegative matrix factorization, can efficiently remove noise and improve the prediction efficiency without significantly degrading the performance. The results obtained here are helpful for the prediction of protein function by using only sequence information.

Enzyme-substrate interaction under the presence of high concentration of salts is of great interest for biotechnology applications and basic enzymology. In our previous work, the salt effect on halophilic subtilase SR5-3 was evaluated with Suc-AAPF-MCA and with the FRET peptide Abz-AAPFSSKQ-EDDnp. It was demonstrated that the magnitude of catalytic activity enhancement was affected by the presence of the prime site residues. In this work, a detailed analysis of the salt effect on SR5-3 protease substrate specificity was performed using chromogenic and coumarin substrates as well as FRET peptides derived from Abz-KLRSSKQ-EDDnp. The followings were demonstrated: 1) Preference of amino acid of SR5-3 protease at the P3, P2, P1, P1' or P2' position of FRET substrates was almost similar with that of subtilisin. 2) Under the presence of the salts (3M NaCl or 1M Na2SO4), SR5-3 protease showed higher kcat values, lower Km values and totally 2-6 times higher kcat/Km values compared with those of control for FRET substrates, and salts did not significantly affect the preference of amino acid residues at the primary positions (P1 and P1'), but it affected the preference at the P2 and P2' position. In contrast, for smaller substrates with only non-prime sites, SR5-3 protease showed 20-75 times higher kcat/Km values compared with those of control. These findings are in agreement with the notion that increases in enzyme-substrate interactions in subtilases alter the rate-determining step in peptide hydrolysis.

A novel exo-glucanase gene (xeg5B) was isolated from a rumenal microbial metagenome, cloned, and expressed in E. coli. The 1548 bp gene coded for a protein of 516 amino acids, which assumed an (α/β)8 fold typical of glycoside hydrolase (GH) family 5. The protein molecule consisted of a loop segment blocking one end of the active site, which potentially provided the enzyme with exo-acting property. The recombinant enzyme showed exclusive specificity towards xyloglucan and oligoxyloglucan substrates with no detectable activity on unsubstituted linear glucans, CMC, laminarin, and lichenan. The major end products of exhaustive hydrolysis were XX (tetrasaccharide) and XG (trisaccharide). The hydrolysis of tamarind xyloglucan followed the Michaelis-Menten kinetics, yielding Km and Vmax of 2.12±0.13 mg/ml and 0.17±0.01 mg/ml/min (37°C, pH 6.0), respectively.