Loop Fragment Research Articles

Predicting the Most Stable Aptamer/Target Molecule Complex Configuration Using a Stochastic-Tunnelling Basin-Hopping Discrete Molecular Dynamics Method: A Novel Global Minimum Search Method for a Biomolecule Complex

This study proposed a novel global minimum search method for predicting the most stable biomolecule complex, which combines the strengths of three global minimum search methods (stochastic tunnelling, basin hopping, and discrete molecular dynamics) to efficiently improve the spatial domain search ability of the stochastic tunnelling–basin hopping (STUN–BH) method from our previous study. The epithelial cell adhesion molecule (EpCAM, PDB code: 4MZV) was used as a benchmark target molecule for the EpCAM aptamer EpA (AptEpA). For the most stable AptEpA/EpCAM complex predicted by our new method, the AptEpA was attached to the entangling loop fragments of the two EpCAM molecules with the most AptEpA residues. After the AptEpA/EpCAM complex had equilibrated with the water environment through a molecular dynamics simulation at 300 K for 10 ns, stable hydrogen bonds formed between the bases of AptEpA and EpCAM residues of the secondary structures, which included the alpha helix and beta sheet becoming less stable in the water environment. Those hydrogen bonds formed between the bases of AptEpA and EpCAM loop fragment residues remained stable in the water environment.

Aptasensor designed via the stochastic tunneling-basin hopping method for biosensing of vascular endothelial growth factor

The Systematic Evolution Ligands by Exponential Enrichment (SELEX) is common used for selection of high affinity single-stranded DNA (ssDNA) aptamer with target protein. However, we do not know what the most stable configuration of the selected aptamer bound with target protein is. Therefore, a systematic search process using the stochastic tunneling-basin hopping (STUN-BH) method is proposed to find the most stable configuration of the ssDNA aptamer specific for vascular endothelial growth factor (VEGF) capture (AptVEGF; 5′-TGTGGGGGTGGACGGGCCGGGTAGA-3′). After the most stable configuration was obtained by the STUN-BH method, molecular dynamics (MD) simulation was carried out to investigate the thermal stability of AptVEGF/VEGF at 300 K in both vacuum and water. All molecular simulations were conducted with the large-scale atomic/molecular massively parallel simulator (LAMMPS), and the AMBER99SB force field was used to describe the atomic interactions for the current AptVEGF/VEGF system. The three most stable AptVEGF/VEGF configurations obtained by the STUN-BH method indicated that AptVEGF residues exhibit greater affinity for VEGF surface loop fragments as compared with surface alpha helix and beta sheet fragments. Results indicated that after the first AptVEGF (AptVEGF I) occupies most of the VEGF loop fragment, the second AptVEGF (AptVEGF II) is adsorbed by the rest of the VEGF loop fragment and the VEGF Chain B beta sheet fragment, resulting in a 24.8% reduction in binding strength as compared to that of AptVEGF I. Furthermore, when AptVEGF I and AptVEGF II chains were stably adsorbed by VEGF, the third AptVEGF (AptVEGF III) chain can only partially attach to VEGF, as confirmed by real AptVEGF-VEGF binding experiments. Lastly, we demonstrated that the aptasensor constructed according to MD simulation is highly sensitive for VEGF with a linear detection range of 10 pg/mL–10 ng/mL.

Investigation of high pressure effect on the structure and adsorption of β-lactoglobulin

β-Lactoglobulin, being one of the principal whey protein, is of huge importance to the food industry. Temperature/pressure effects on this small protein has been extensively studied by industry. To characterize biochemical properties of β‐lactoglobulin after or during pressurization, a wide range of methods have been used thus far. In this study, for the first time, the pressure‐induced conformation of β‐lactoglobulin in the crystal state was determined, at pressure 430 MPa. Changes observed in the high pressure structure correlate with the physico‐chemical properties of pressure-treated β‐lactoglobulin obtained from dynamic light scattering, electrophoretic mobility and quartz crystal microbalance with dissipation monitoring measurements. A comparison between the β‐lactoglobulin structures determined at both high and ambient pressure contrasts the stable nature of the protein core and adjacent loop fragments. At high pressure the β‐lactoglobulin structure presents early signs of dimer dissociation, charge and conformational changes characteristic for initial unfolded intermediate as well as a significant modification of the binding pocket volume. Those observations are supported by changes in zeta potential values and results in increase affinity of the β‐lactoglobulin adsorption onto gold surface. Observed pressure-induced structural modifications were previously suggested as an important factor contributing to β‐lactoglobulin denaturation process. Presented studies provide detailed analysis of pressure‐associated structural changes influencing β‐lactoglobulin conformation and consequently its adsorption.

New insight into the mechanism of mitochondrial cytochrome c function.

We investigate functional role of the P76GTKMIFA83 fragment of the primary structure of cytochrome c. Based on the data obtained by the analysis of informational structure (ANIS), we propose a model of functioning of cytochrome c. According to this model, conformational rearrangements of the P76GTKMIFA83 loop fragment have a significant effect on conformational mobility of the heme. It is suggested that the conformational mobility of cytochrome c heme is responsible for its optimal orientation with respect to electron donor and acceptor within ubiquinol–cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV), respectively, thus, ensuring electron transfer from complex III to complex IV. To validate the model, we design several mutant variants of horse cytochrome c with multiple substitutions of amino acid residues in the P76GTKMIFA83 sequence that reduce its ability to undergo conformational rearrangements. With this, we study the succinate–cytochrome c reductase and cytochrome c oxidase activities of rat liver mitoplasts in the presence of mutant variants of cytochrome c. The electron transport activity of the mutant variants decreases to different extent. Resonance Raman spectroscopy (RRS) and surface-enhanced Raman spectroscopy (SERS) data demonstrate, that all mutant cytochromes possess heme with the higher degree of ruffling deformation, than that of the wild-type (WT) cytochrome c. The increase in the ruffled deformation of the heme of oxidized cytochromes correlated with the decrease in the electron transport rate of ubiquinol–cytochrome c reductase (complex III). Besides, all mutant cytochromes have lower mobility of the pyrrol rings and methine bridges, than WT cytochrome c. We show that a decrease in electron transport activity in the mutant variants correlates with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c.

Efficient conversion of N6-threonylcarbamoyladenosine (t6A) into a tRNA native hydantoin cyclic form (ct6A) performed at nucleoside and oligoribonucleotide levels.

A t6A nucleoside was efficiently and stereospecifically transformed into a hydantoin cyclic form of N6-l-threonylcarbamoyladenosine (ct6A) by the use of polymer bounded carbodiimide (EDC-P) and HOBt. The procedure was successfully applied for a post-synthetic conversion of t6A-containing RNA 17-mers (of the sequences of anticodon stem and loop (ASL) fragments of S. pombe tRNAi and E. coli tRNALys) into the products bearing the ct6A unit.

Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing.

The binding of sequence-specific RNA-interacting proteins, such as the bacteriophage MS2 or PP7 coat proteins, to their corresponding target sequences has been extremely useful and widely used to visualize single mRNAs in vivo. However, introduction of MS2 stem–loops into yeast mRNAs has recently been shown to lead to the accumulation of RNA fragments, suggesting that the loops impair mRNA decay. This result was questioned, because fragment occurrence was mainly assessed using ensemble methods, and their cellular localization and its implications had not been addressed on a single transcript level. Here, we demonstrate that the introduction of either MS2 stem–loops (MS2SL) or PP7 stem–loops (PP7SL) can affect the processing and subcellular localization of mRNA. We use single-molecule fluorescence in situ hybridization (smFISH) to determine the localization of three independent mRNAs tagged with the stem–loop labeling systems in glucose-rich and glucose starvation conditions. Transcripts containing MS2SL or PP7SL display aberrant localization in both the nucleus and the cytoplasm. These defects are most prominent in glucose starvation conditions, with nuclear mRNA processing being altered and stem–loop fragments abnormally enriching in processing bodies (PBs). The mislocalization of SL-containing RNAs is independent of the presence of the MS2 or PP7 coat protein (MCP or PCP).

Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase

Inulinases are classified as hydrolases and widely used in the food and medical industries. Here, we report the deletion of a six-membered adjacent active site loop fragment (74YGSDVT79 sequence) from third Ω-loop of the exo-inulinase containing aspartate residue from Aspergillus niger to study its structural and functional importance. Site-directed mutagenesis was used to create the mutant of the exo-inulinase (ο6SL). To investigate the stability of the region spanning this loop, MD simulations were performed 80ns for 20-85 residues. Molecular docking was performed to compare the interactions in the active sites of enzymes with fructose as a ligand. Accordingly, the functional thermostability of the exo-inulinase was significantly decreased upon loop fragment deletion. Evaluation of the kinetics parameters (Vmax, Km, kcat and, kcat/Km) and activation energy (Ea) of the catalysis of enzymes indicated the importance of the deleted sequence on the catalytic performance of the enzyme. In conclusion, six-membered adjacent active site loop fragment not only plays a crucial role in the stability of the enzyme, but also it involves in the enzyme catalysis through lowering the activation energy of the catalysis and effective improving the catalytic performance.

A Comprehensive Proteomic Study of the Skin Secretions of the Frog Lithobates spectabilis.

Disulfide C-terminal loop fragments derived from AMPs and the presence of peptidases have been previously reported in the skin secretions of different amphibians. However, there are only a few studies on the identification of enzymes in frog skin secretion based on the primary structure of these proteins. Similarly, little data exist regarding the identification of disulfide C-terminal loops at large scale. Therefore, a comprehensive study on this issue certainly could bring in much more information for understanding this molecular process and its biochemical consequences. Thus, the aim of this work was to characterize the presence of disulfide C-terminal loop fragments of AMPs and identify the proteins and probable enzymes present in the completely unknown secretion contents of the frog Lithobates spectabilis. For this purpose, high-resolution mass spectrometry was applied to analyze the skin secretions processed by two different protocols: (1) using a cocktail of enzymatic inhibitors and 2) without any protease inhibitors, maintaining the solution for 2 hours at 10°C. Results from procedure-1, revealed 122 molecular masses, whereas procedure-2 permitted 253 different molecular masses to be identified. Fifty-nine peptides including 22 disulfide C-terminal loop-containing peptides were obtained following procedure-2. Polyacrylamide gel electrophoresis separation, tryptic digestion and LCMS/ MS were used for "de novo" sequencing of 111 different peptides and the unequivocal identification of fifteen proteins including at least three different peptidases. Additionally, it was possible to fully sequence eight peptides, including a ranatuerin-related peptide identified here as Spectabilin, that was subsequently chemically synthesized and showed high antibacterial, antiparasitic and cytotoxic activities.

Development of methods for separation of binarized fragments of etching pits of semiconductor wafer

The article is devoted to the search of successful methods for separation of fragments belonging to the supposed etching pits of dislocation loops. The developed methods are revealed binarized fragments of etching pits among of the many other elements of surface image of a semiconductor wafer. The filtration method of binarized fragments of etching pits of wafer dislocation uses a roundness index of the specified range, received on the base of reference line width of dislocation loop at a ratio of 1:4. This optional feature allows separating fragments similar to lines of loops of etching pits on the basis of their size and shape. The method of removing the micro-defects loops reduces the number of fragments by eliminating of loops without signs of loops of etching pits. It is based on the use of the XOR subtraction operation between the binarized image of dislocation areas and the image with accentuated loops of the fragments. The criteria for allocation of the main significant loop fragments allow form the selection rules for the further processing of binarized image. The criteria for allocation of the main significant loop fragments, method of binarized fragments filtering, method of removing the loops of micro-defects of the semiconductor wafer are the part of a package of measures to carry out tasks on production management organization and creation of technical diagnostic system of output production quality.

Clustering and percolation in protein loop structures.

BackgroundHigh precision protein loop modelling remains a challenge, both in template based and template independent approaches to protein structure prediction.MethodWe introduce the concepts of protein loop clustering and percolation, to develop a quantitative approach to systematically classify the modular building blocks of loops in crystallographic folded proteins. These fragments are all different parameterisations of a unique kink solution to a generalised discrete nonlinear Schrödinger (DNLS) equation. Accordingly, the fragments are also local energy minima of the ensuing energy function.ResultsWe show how the loop fragments cover practically all ultrahigh resolution crystallographic protein structures in Protein Data Bank (PDB), with a 0.2 Ångström root-mean-square (RMS) precision. We find that no more than 12 different loop fragments are needed, to describe around 38 % of ultrahigh resolution loops in PDB. But there is also a large number of loop fragments that are either unique, or very rare, and examples of unique fragments are found even in the structure of a myoglobin.ConclusionsProtein loops are built in a modular fashion. The loops are composed of fragments that can be modelled by the kink of the DNLS equation. The majority of loop fragments are also common, which are shared by many proteins. These common fragments are probably important for supporting the overall protein conformation. But there are also several fragments that are either unique to a given protein, or very rare. Such fragments are probably related to the function of the protein. Furthermore, we have found that the amino acid sequence does not determine the structure in a unique fashion. There are many examples of loop fragments with an identical amino acid sequence, but with a very different structure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12900-015-0049-x) contains supplementary material, which is available to authorized users.

Selection of peptide mimics of HIV-1 epitope recognized by neutralizing antibody VRC01.

The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies.

Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

Three dimensional structure of the dimeric gene-engineered variant of green fluorescent protein EGFP-K162Q in P6(1) crystal space group

The crystal structure of the dimeric green fluorescent protein EGFP-K162Q with C-terminal deletion MDELYK (EGFPv) has been determined in space group P6 at resolution 1.34 A. The obtained structure has been compared with that of the monomeric form of EGFP (green biomarker with enhanced photophysical properties) determined in other crystal space group P2(1)2(1)2(1) at resolution 1.50 and 1.35 A [1, 2]. Two subunits in the EGFPv structure are packed at 75 degrees with the contact surface approximately 800 A2. The dimeric structure is stabilized by six hydrogen bonds and the central hydrophobic core built of six residues. The RMSD value for Calpha atoms of 3-230 residues in the superimposed P61 and P2(1)2(1)2(1) structures is 0.55 A. The distinguishing feature of EGFPv- P6(1) structure, compared with that of EGFP-P2(1)2(1)2(1), is the noticeable difference in orientation of the Glu222 side chain and also new conformation of the loop fragment 155-159 with deviations among the Calpha atoms of superimposed structures reaching for Lys156 - 4.6 A and Lys158 - 5.5 A

Molecular recognition of CCR5 by an HIV-1 gp120 V3 loop.

The binding of protein HIV-1 gp120 to coreceptors CCR5 or CXCR4 is a key step of the HIV-1 entry to the host cell, and is predominantly mediated through the V3 loop fragment of HIV-1 gp120. In the present work, we delineate the molecular recognition of chemokine receptor CCR5 by a dual tropic HIV-1 gp120 V3 loop, using a comprehensive set of computational tools predominantly based on molecular dynamics simulations and free energy calculations. We report, what is to our knowledge, the first complete HIV-1 gp120 V3 loop : CCR5 complex structure, which includes the whole V3 loop and the N-terminus of CCR5, and exhibits exceptional agreement with previous experimental findings. The computationally derived structure sheds light into the functional role of HIV-1 gp120 V3 loop and CCR5 residues associated with the HIV-1 coreceptor activity, and provides insights into the HIV-1 coreceptor selectivity and the blocking mechanism of HIV-1 gp120 by maraviroc. By comparing the binding of the specific dual tropic HIV-1 gp120 V3 loop with CCR5 and CXCR4, we observe that the HIV-1 gp120 V3 loop residues 13–21, which include the tip, share nearly identical structural and energetic properties in complex with both coreceptors. This result paves the way for the design of dual CCR5/CXCR4 targeted peptides as novel potential anti-AIDS therapeutics.

Construction of functional fragments of the cytoplasmic loop with the C-terminal region of PomA, a stator component of the Vibrio Na+ driven flagellar motor

The membrane motor proteins, PomA (polar flagellar motility protein A) and PomB (polar flagellar motility protein B), of Vibrio alginolyticus form a stator complex that converts energy from the ion flow to mechanical work in bacterial flagellar motors. The cytoplasmic domain of PomA is believed to interact with the rotor protein FliG to make a torque. In this study, to investigate the function of the cytoplasmic domain of PomA, we constructed a series of fragments that flank the cytoplasmic loop of PomA between the second and third transmembrane (TM) domains (A-loop) and the C-terminal region, and expressed them in Escherichia coli together with PomA and PotB (a chimeric protein of PomB and MotB). We observed a dominant-negative effect of one PomA fragment on motility. We confirmed that these PomA fragments localized both in the membrane fraction and in the cytoplasmic fraction, and induced bacterial growth delay. Effect of additional point and deletion mutations into this fragment implies that the C-terminal region and TM domains used as a linker play a significant part in these observations. From these results, we conclude that the PomA fragments retain the structure important for functions. We expect that further constructions will provide a variety of experimental approaches to characterize the interaction between PomA and FliG.

Mechanistic insights into CED-4-mediated activation of CED-3

Programmed cell death in Caenorhabditis elegans requires activation of the caspase CED-3, which strictly depends on CED-4. CED-4 forms an octameric apoptosome, which binds the CED-3 zymogen and facilitates its autocatalytic maturation. Despite recent advances, major questions remain unanswered. Importantly, how CED-4 recognizes CED-3 and how such binding facilitates CED-3 activation remain completely unknown. Here we demonstrate that the L2' loop of CED-3 directly binds CED-4 and plays a major role in the formation of an active CED-4-CED-3 holoenzyme. The crystal structure of the CED-4 apoptosome bound to the L2' loop fragment of CED-3, determined at 3.2 Å resolution, reveals specific interactions between a stretch of five hydrophobic amino acids from CED-3 and a shallow surface pocket within the hutch of the funnel-shaped CED-4 apoptosome. Structure-guided biochemical analysis confirms the functional importance of the observed CED-4-CED-3 interface. Structural analysis together with published evidence strongly suggest a working model in which two molecules of CED-3 zymogen, through specific recognition, are forced into the hutch of the CED-4 apoptosome, consequently undergoing dimerization and autocatalytic maturation. The mechanism of CED-3 activation represents a major revision of the prevailing model for initiator caspase activation.

Genotypic HIV‐coreceptor tropism prediction with geno2pheno [coreceptor]: differences depending on HIV‐1‐subtype

Purpose of the studyDetermination of HIV‐1 coreceptor tropism is a major prerequisite before starting treatment with a CCR5‐antagonist. While most of the patients currently under treatment with maraviroc are probably infected with HIV‐1 subtype B viruses, recently published data show differences in the distribution of coreceptor tropism in different HIV‐1 subtypes.MethodsIn a Germany‐wide project within the HIV‐GRADE society, V3‐loop sequences of 2466 isolates were analysed with geno2pheno for coreceptor tropism using a FPR cut‐off of 10%. HIV‐1 subtype was determined by using the COMET HIV subtyping tool. Sequences consisted of at least the V3 loop fragment. The ratio of CCR5 vs CXCR4 tropic viruses was calculated for each subtype. A normalized mean for all analyzed subtypes was calculated to extrapolate the overall ratio of coreceptor usage distribution. From this the expected distribution in the particular subtype was calculated and compared to the observed one. Statistical analysis was performed using the chi2 test.Summary of ResultsMost samples were classified as HIV‐1 subtype B (79%, n=1952). Other subtypes present in at least 23 samples were A1 (9.5%, n=234), C (4.8%, n=118), CRF01_AE (2.2%, n=55), G (1.6%, n=39), D (1.1%, n=27), F (0.9%, n=23). The calculated normalized mean distribution over all subtypes was 71% CCR5‐ vs. 29% CXCR4‐tropic viruses. No significant difference compared to the mean distribution could be observed for HIV‐1 subtypes B (71/29%), C (76/24%) and F (70/30%). Higher rates of CXCR4 tropic virus were detected in subtypes D (52/48%, p=0.01) and CRF01_AE (49/51%, p=0.001), while in HIV‐1 subtypes A1 (22/78%, p=0.02) and G (13/87%, p=0.02), a higher rate of CCR5‐tropic virus was observed.ConclusionsOur analysis shows a different distribution of CCR5 and CXCR4 tropic virus in some subtypes. In contrast to other publications, we could not observe a statistically significant difference in subtype C compared to the overall mean distribution, while we could confirm a higher rate of CXCR4‐tropic virus in subtype D, as previously described. Without further data on treatment success of patients with non‐B subtypes under treatment with maraviroc, it remains unclear if subtype‐specific differences in the distribution of tropism are biased by differences in clinical variables before test or if there is a bias in the tropism interpretation system. In the latter case, individual interpretation cut‐offs for different subtypes may be necessary.

Specific gene silencing of <I>At1g13770</I> and <I>At2g23470</I> by artificial microRNAs in <I>Arabidopsis</I>

DUF647 (domain of unknown function 647) protein family is found in diverse eukaryotic organisms and highly conserved in eukaryotes. It has 6 members in Arabidopsis genome. So far, the function of 4 members of Arabidopsis DUF647 family is unknown. In this report, using an endogenous Arabidopsis MIR319a precursor as the backbone, we constructed two artificial microRNAs (amiRNAs) to knock down the expression of two DUF647 family genes At1g13770 and At2g23470. Using the WMD (Web microRNA Designer) platform, we designed two amiRNAs targeting At1g13770 and At2g23470 genes, respectively. Both amiRNAs sequences were engineered into the MIR319a precursor using overlapping PCR and the amiRNAs backbones were transferred into the binary vector pCHF3. The resulting plasmids that harbor amiRNAs stem loop fragments were transformed into Arabidopsis by Agrobacterium-mediated floral diping. Upon constitutive expression of these two amiRNAs, the target genes were efficiently down-regulated in transgenic line. The decreased level of At2g23470 transcript in At2g23470-amiRNA transgenic plants resulted in severe sterility. This work will facilitate the functional analysis of At1g13770 and At2g23470 genes in Arabidopsis growth and development.

Modeling Structures and Motions of Loops in Protein Molecules

Unlike the secondary structure elements that connect in protein structures, loop fragments in protein chains are often highly mobile even in generally stable proteins. The structural variability of loops is often at the center of a protein’s stability, folding, and even biological function. Loops are found to mediate important biological processes, such as signaling, protein-ligand binding, and protein-protein interactions. Modeling conformations of a loop under physiological conditions remains an open problem in computational biology. This article reviews computational research in loop modeling, highlighting progress and challenges. Important insight is obtained on potential directions for future research.

Configurational Entropy Reallocation and Complex Loop Dynamics of the Mosquito-Stage Pvs25 Protein Complexed with the Fab Fragment of the Malaria Transmission Blocking Antibody 2A8.

Pvs25 is a protein of unique 3D structure, and it is characterized by the presence of repeated EGF-like domains and 11 disulfide bonds. It is a very important candidate for the transmission-blocking malaria vaccine, as it plays an important role in mosquito infection by Plasmodium parasites. Recently, the X-ray structure of the protein complexed with the transmission blocking antibody 2A8 has been reported. In this study, we report the loop reorganization of the Pvs25 protein based on configurational entropy calculations and dihedral principal component analysis as revealed from the protein complex and free molecular dynamics simulations. While the total entropy of the protein was estimated to be almost the same in the free and complex trajectories, the partition of the entropy contribution in the loop fragments of the protein revealed interesting entropy reallocation after the 2A8 antibody binding. Interestingly, the 51-71 protein loop experienced a significant reduction in its configurational entropy, while other parts of the protein did not show any difference in it, or even showed an entropy increase. This trend in entropy redistribution was found to be in direct relationship with specific interactions with the antibody's binding site. Results from root-mean-square fluctuations/deviations and dihedral angle principal component analysis further support this finding.