Α-helix Segments Research Articles

Characterization of the interaction of metal-protoporphyrins photosensitizers with β- lactoglobulin

We investigated the interaction of a series of metal-protoporphyrins (PPIXs) with bovine β- lactoglobulin (BLG) using a combination of optical spectroscopy and computational simulations. Unlike other studies, the simulations were not merely used to rationalize the experimental data but were employed to refine the experimental data itself. The study was carried out at two pH values, 5 and 9, where BLG is known to have different conformation dictated by the so-called Tanford transition which occurs near pH 7.5. The transition is postulated to regulate access to the interior binding cavity of the protein, thus the pH variation was used as a parameter to investigate whether PPIXs access the central cavity of BLG. The results of our study show that indeed binding increases significantly at alkaline pH, however, the increased affinity is not due to the accessibility of the central cavity. Instead, binding appears to be determined by the tendency of PPIXs to form large inhomogeneous aggregates at acidic pH which hinders interactions with proteins.The binding site determined through a combination of experimental and computational methods is located at the interface between two BLG monomers where the long α-helix segment of the protein face each other. This region is rich in positively charged Lys residues that interact with the propionic acid chains of the protoporphyrins.Establishing the modality of binding between protoporphyrins and BLG would have important consequences for the use of BLG:PPIX complexes in applications such as artificial photoreceptors, artificial metallo-enzymes, delivery of photosensitizers for phototherapy and even solar energy conversion.

A novel gyrase inhibitor from toxin-antitoxin system expressed by Staphylococcus aureus.

Toxin-antitoxin (TA) systems consist of a toxin inhibiting essential cellular functions (such as DNA, RNA and protein synthesis), and its cognate antitoxin neutralizing the toxicity. Recently, we identified a TA system termed TsbA/TsbT in the Staphylococcus aureus genome. The induction of the tsbT gene in Escherichia coli halted both DNA and RNA synthesis, reduced supercoiled plasmid and resulted in increasingly relaxed DNA. These results suggested that DNA gyrase was the target of TsbT. In addition, TsbT inhibited both E. coli and S. aureus DNA gyrase activity and induced linearization of plasmid DNA in vitro. Taken together, these results demonstrate that the TsbT toxin targets DNA gyrase in vivo. Site-directed mutagenesis experiments showed that the E27 and D37 residues in TsbT are critical for toxicity. Secondary structure prediction combining the analysis of vacuum-ultraviolet circular-dichroism spectroscopy and neural network method demonstrated that the 22nd-32nd residues of TsbT form an α-helix structure, and that the E27 residue is located around the centre of the α-helix segment. These findings give new insights not only into S. aureus TA systems, but also into bacterial toxins targeting DNA topoisomerases.

Computational MitoTarget Scanning Based on Topological Vacancies of Single-Walled Carbon Nanotubes with the Human Mitochondrial Voltage-Dependent Anion Channel (hVDAC1).

We present an in silico approach for modeling the noncovalent interactions between the human mitochondrial voltage-dependent anion channel (hVDAC1) and a family of single-walled carbon nanotubes (SWCNTs) with a defined pattern of topological vacancies ( v = 1-16), obtained by removing atoms from the SWCNT surface. The general results showed more stable docking interaction complexes (SWCNT-hVDAC1), with more negative Gibbs free energy of binding affinity values, and a strong dependence on the vacancy number ( R2 = 0.93) and vacancy formation energy ( R2 = 0.96). In addition, for most of the SWCNT vacancies that were analyzed, the interatomic distances for the interactions of the SWCNT-hVDAC1 complex with the functional catalytic residues (i.e., Pro7, Gln199, Gln182, Phe181, Val20, Asp19, Lys15, Gly14, Asp12, Ala11, and Arg18) that form the hVDAC1 active site (i.e., the voltage-sensing N-terminal α-helix segment) were very similar to or shorter than the interatomic distances of these residues for ATP-hVDAC1 interactions. In particular, the hVDAC1 residues that can be phosphorylated like Tyr10, Tyr198, and Se16 were significantly perturbed by the interactions with SWCNT with at least nine vacancies. In addition, the SWCNT vacancy family members can affect the flexibility properties of the hVDAC1 N-terminal α-helix segment inducing different patterns of local perturbations in inter-residue communication. Finally, vacancy quantitative structure-binding relationships (V-QSBRs) were unveiled for setting up a robust model that can predict the strength of docking interactions between SWCNTs with a specific topological vacancy and hVDAC1. The developed V-QSBR model classified properly all of the SWCNTs with a different number of SWCNT vacancies with exceptional sensitivity and specificity (both equal to 100%), indicating a strong potential to unequivocally predict the influence of SWCNT vacancies on the mitochondrial channel interactions.

Conformational changes, from β-strand to α-helix, of the fatty acid-binding protein ReP1-NCXSQ in anionic lipid membranes: dependence with the vesicle curvature.

We studied the conformational changes of the fatty acid-binding protein ReP1-NCXSQ in the interface of anionic lipid membranes. ReP1-NCXSQ is an acidic protein that regulates the activity of the Na+/Ca2+ exchanger in squid axon. The structure is a flattened barrel composed of two orthogonal β-sheets delimiting an inner cavity and a domain of two α-helix segments arranged as a hairpin. FTIR and CD spectroscopy showed that the interactions with several anionic lipids in the form of small unilamellar vesicles (SUVs) induced an increase in the proportion of helix secondary structure. Lower amount or no increase in α-helix was observed upon the interaction with anionic lipids in the form of large unilamellar vesicles (LUVs). The exception was 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) that was equally efficien to to induce the conformational change both in SUVs and in LUVs. In solution, the infrared spectra of ReP1-NCXSQ at temperatures above the unfolding displayed a band at 1617cm-1 characteristic of aggregated strands. This band was not observed when the protein interacted with DMPG, indicating inhibition of aggregation in the interface. Similarly to the observed in L-BABP, another member of the fatty acid binding proteins, a conformational change in ReP1-NCXSQ was coupled to the gel to liquid-crystalline lipid phase transition.

Metamorphic Proteins: Emergence of Dual Protein Folds from One Primary Sequence.

Every amino acid exhibits a different propensity for distinct structural conformations. Hence, decoding how the primary amino acid sequence undergoes the transition to a defined secondary structure and its final three-dimensional fold is presently considered predictable with reasonable certainty. However, protein sequences that defy the first principles of secondary structure prediction (they attain two different folds) have recently been discovered. Such proteins, aptly named metamorphic proteins, decrease the conformational constraint by increasing flexibility in the secondary structure and thereby result in efficient functionality. In this review, we discuss the major factors driving the conformational switch related both to protein sequence and to structure using illustrative examples. We discuss the concept of an evolutionary transition in sequence and structure, the functional impact of the tertiary fold, and the pressure of intrinsic and external factors that give rise to metamorphic proteins. We mainly focus on the major components of protein architecture, namely, the α-helix and β-sheet segments, which are involved in conformational switching within the same or highly similar sequences. These chameleonic sequences are widespread in both cytosolic and membrane proteins, and these folds are equally important for protein structure and function. We discuss the implications of metamorphic proteins and chameleonic peptide sequences in de novo peptide design.

Mutational analysis of hepatitis E virus ORF1 "Y-domain": Effects on RNA replication and virion infectivity.

AIMTo investigate the role of non-structural open reading frame 1 “Y-domain” sequences in the hepatitis E virus (HEV) life cycle.METHODSSequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry. Mutant virions’ infectivity was assayed on naïve HepG2/C3A cells.RESULTSIn silico analysis identified a potential palmitoylation-site (C336C337) and an α-helix segment (L410Y411S412W413L414F415E416) in the HEV Y-domain. Molecular characterization of C336A, C337A and W413A mutants of the three universally conserved residues showed non-viability. Further, of the 10 consecutive saturation mutants covering the entire Y-domain nucleotide sequences (nts 650-1339), three constructs (nts 788-994) severely affected virus replication. This revealed the indispensability of the internal sequences but not of the up- or downstream sequences at the transcriptional level. Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation, indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed formation of stable hairpins (nts 788-994) where saturation mutation drastically inhibited virion infectivity.CONCLUSIONThis is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, which may involve gene regulation and/or membrane binding in intracellular replication complexes.

Sarkosyl-Induced Helical Structure of an Antimicrobial Peptide GW-Q6 Plays an Essential Role in the Binding of Surface Receptor OprI in Pseudomonas aeruginosa.

The emergence of antibiotic-resistant microbial strains has become a public health issue and there is an urgent need to develop new anti-infective molecules. Although natural antimicrobial peptides (AMPs) can exert bactericidal activities, they have not shown clinical efficacy. The limitations of native peptides may be overcome with rational design and synthesis. Here, we provide evidence that the bactericidal activity of a synthetic peptide, GW-Q6, against Pseudomonas aeruginosa is mediated through outer membrane protein OprI. Hyperpolarization/depolarization of membrane potential and increase of membrane permeability were observed after GW-Q6 treatment. Helical structure as well as hydrophobicity was induced by an amphipathic surfactant, sarkosyl, for binding to OprI and possible to membrane. NMR studies demonstrated GW-Q6 is an amphipathic α-helical structure in DPC micelles. The paramagnetic relaxation enhancement (PRE) approach revealed that GW-Q6 orients its α-helix segment (K7-K17) into DPC micelles. Additionally, this α-helix segment is critical for membrane permeabilization and antimicrobial activity. Moreover, residues K3, K7, and K14 could be critical for helical formation and membrane binding while residues Y19 and W20 for directing the C-terminus of the peptide to the surface of micelle. Taken together, our study provides mechanistic insights into the mode of action of the GW-Q6 peptide and suggests its applicability in modifying and developing potent AMPs as therapeutic agents.

Assembly of the secretion pores GspD, Wza and CsgG into bacterial outer membranes does not require the Omp85 proteins BamA or TamA.

In Gram-negative bacteria, β-barrel proteins are integrated into the outer membrane by the β-barrel assembly machinery, with key components of the machinery being the Omp85 family members BamA and TamA. Recent crystal structures and cryo-electron microscopy show a diverse set of secretion pores in Gram-negative bacteria, with α-helix (Wza and GspD) or β-strand (CsgG) transmembrane segments in the outer membrane. We developed assays to measure the assembly of three distinct secretion pores that mediate protein (GspD), curli fibre (CsgG) and capsular polysaccharide (Wza) secretion by bacteria and show that depletion of BamA and TamA does not diminish the assembly of Wza, GspD or CsgG. Like the well characterised pilotins for GspD and other secretins, small periplasmic proteins enhance the assembly of the CsgG β-barrel. We discuss a model for integral protein assembly into the bacterial outer membrane, focusing on the commonalities and differences in the assembly of Wza, GspD and CsgG.

Cosolvent, ions, and temperature effects on the structural properties of Cecropin A-Magainin 2 hybrid peptide in solutions.

Antimicrobial peptides are promising alternative to traditional antibiotics and antitumor drugs for the battle against new antibiotic resistant bacteria strains and cancer maladies. The study of their structural and dynamics properties at physiological conditions can help to understand their stability, delivery mechanisms, and activity in the human body. In this article, we have used molecular dynamics simulations to study the effects of solvent environment, temperature, ions concentration, and peptide concentration on the structural properties of the antimicrobial hybrid peptide Cecropin A-Magainin 2. In TFE/water mixtures, the structure of the peptide retained α-helix contents and an average hinge angle in close agreement with the experimental NMR and CD measurements reported in literature. Compared to the TFE/water mixture, the peptide simulated at the same ionic concentration lost most of its α-helix structure. The increase of peptide concentration at both 300 and 310 K resulted in the peptide aggregation. The peptides in the complex retained the initial N-ter α-helix segment during all the simulation. The α-helix stabilization is further enhanced in the high salt concentration simulations. The peptide aggregation was not observed in TFE/water mixture simulations and, the peptide aggregate, obtained from the water simulation, simulated in the same conditions did dissolve within few tens of nanoseconds. The results of this study provide insights at molecular level on the structural and dynamics properties of the CA-MA peptide at physiological and membrane mimic conditions that can help to better understand its delivery and interaction with biological interfaces.

Probing Tertiary Structure of Proteins Using Single Trp Mutations with Circular Dichroism at Low Temperature

Trp is the most spectroscopically informative aromatic amino acid of proteins. However, the near-UV circular dichroism (CD) spectrum of Trp is complicated because the intensity and sign of 1La and 1Lb bands vary independently. To resolve vibronic structure and gain site-specific information from complex spectra, deconvolution was combined with cooling and site-directed tryptophan substitution. Low temperature near-UV CD was used to probe the local tertiary structure of a loop and α-helix in tear lipocalin. Upon cooling, the enhancement of the intensities of the near-UV CD was not uniform, but depends on the position of Trp in the protein structure. The most enhanced 1Lb band was observed for Trp at position 124 in the α-helix segment matching the known increased conformational mobility during ligand binding. Some aspects of the CD spectra of W28 and W130 were successfully linked to specific rotamers of Trp previously obtained from fluorescence lifetime measurements. The discussion was based on a framework that the magnitude of the energy differences in local conformations governs the changes in the CD intensities at low temperature. The Trp CD spectral classification of Strickland was modified to facilitate the recognition of pseudo peaks. Near-UV CD spectra harbor abundant information about the conformation of proteins that site directed Trp CD can report.

Dependence of Alamethicin Membrane Orientation on the Solution Concentration

Alamethicin has been extensively studied as an antimicrobial peptide and is widely used as a simple model for ion channel proteins. It has been shown that the antimicrobial activity of peptides is related to their membrane orientation. In this study, we determined the relationship between the solution concentration of alamethicin and its membrane orientation in lipid bilayers using sum frequency generation (SFG) vibrational spectroscopy. Our SFG results indicated that the alamethicin molecules more or less lay down on the surface of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers at a low peptide concentration of 0.84 μM; the α-helix segment tilts at about 88°, and 310-helix segment tilts at about 58° versus the surface normal. However, when the peptide concentration was increased to 15.6 μM, we observed that alamethicin molecules further inserted into the lipid bilayers: the α-helical component changes its orientation to make a 37° tilt from the lipid bilayer normal, and the 310-helical component tilts at about 50° versus the surface normal. This is in agreement with the barrel-stave mode for the alamethicin-cell membrane interaction as reported previously. Additionally, we have also studied membrane orientation of alamethicin as a function of peptide concentration with SFG. Our results showed that the membrane orientation of the alamethicin α-helical component changed substantially with the increase of the alamethicin concentration, while the membrane orientation of the 310-helical component remained more or less the same.

Designing of nitrile hydratase from alkaline protease using quanidine hydrochloride and cobalt metal ion

Alkaline protease (22.5kDa) from Bacillus licheniformis was denatured using 3–6M Guanidine hydrochloride followed by renaturation with cobalt metal ion in buffer solution (pH 7.5) in order to design cobalt dependent nitrile hydratase AP-xMGdn-Co, where x=3, 4, 5 and 6. The native alkaline protease showed a 20% residual molar ellipticity at 212nm while on treatment with Gdn-HCl, it got reduced to 12% due to nearly complete collapse of the globular structure of the protein. The observed circular dichroism spectra of the renatured proteases with Co metal ion are different from the native enzyme, because those molecules contain many metal chelating sites through its coordinating amino acid residues. The introduction of cobalt centered new active site was evidenced by the fluorescence spectroscopy (appearance of a new peak at 360nm) and ICP analysis (0.0476–0.0553mg/L). The specific activities of the modified enzymes were measured using the selective catalytic hydrolysis of 3-cyanopyridine to nicotinamide. The highest specific activity of renatured enzymes (AP-xMGdn-Co) were obtained at optimum pH of 7.5 and temperature of 50°C, due to the proper co-ordination of amino acid residue with cobalt metal ion and the appropriate arrangement of α-helix, β-strand, turn and unordered segment in the globular structure.

Structural Conversion Rate Changes of Recombinant Bovine Prion by Designed Synthetic Peptides

An understanding of structural changes and self-assembly of proteins, which are thought to involve specific peptide–peptide interactions, will contribute to the development of therapeutic agents and diagnosis for the detection of conformational diseases. We hypothesize that certain peptides may contribute to the conformational change of prion proteins. The present paper describes the discovery of prion-related synthetic peptides which influence structural conversion of recombinant bovine prion protein. The peptides designed are prion-protein fragments containing core domains consisting of α-helical (human prion protein fragment 180–195) and known β-sheet (human prion protein fragment 169–175) structures. Additionally several reported known β-sheet breaker peptides and a conjugate consisting of β-sheet and α-helix segments based on the secondary structures of human prion protein, designated HPPSH, have been chemically synthesized by the conventional Fmoc solid-phase method and characterized by circular dichroism and the Thioflavin T fluorescence method. Our data indicated that the co-existence of peptides, HPPSH or other prion fragment peptides involving toxic core sequence (the fragment 106–126), influenced the kinetic rate of aggregation and the lag-time of fibril formation of recombinant bovine prion protein except the core sequence itself. The method will be used for discovery of responsible material from natural resources. And designed peptides can be also used for bio-detection.

Secondary‐structure analysis of alcohol‐denatured proteins by vacuum‐ultraviolet circular dichroism spectroscopy

To elucidate the structural characteristics of alcohol-denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra of six proteins-myoglobin, human serum albumin, α-lactalbumin, thioredoxin, β-lactoglobulin, and α-chymotrypsinogen A-down to 170 nm in trifluoroethanol solutions (TFE: 0-50%) and down to 175 nm in methanol solutions (MeOH: 0-70%) at pH 2.0 and 25°C, using a synchrotron-radiation VUVCD spectrophotometer. The contents of α-helices, β-strands, turns, poly-L-proline type II helices (PPIIs), and unordered structures of these proteins were estimated using the SELCON3 program, including the numbers of α-helix and β-strand segments. Furthermore, the positions of α-helices and β-strands on amino acid sequences were predicted by combining these secondary-structure data with a neural-network method. All alcohol-denatured proteins showed higher α-helix contents (up to ~ 90%) compared with the native states, and they consisted of several long helical segments. The helix-forming ability was higher in TFE than in MeOH, whereas small amounts of β-strands without sheets were formed in the MeOH solution. The produced α-helices were transformed dominantly from the β-strands and unordered structures, and slightly from the turns. The content and mean length of α-helix segments decreased as the number of disulfide bonds in the proteins increased, suggesting that disulfide bonds suppress helix formation by alcohols. These results demonstrate that alcohol-denatured proteins constitute an ensemble of many long α-helices, a few β-strands and PPIIs, turns, and unordered structures, depending on the types of proteins and alcohols involved.

Studies on the rules of β-strand alignment in a protein β-sheet structure

To further disclose the underlying mechanisms of protein β-sheet formation, studies were made on the rules of β-strands alignment forming β-sheet structure using statistical and machine learning approaches. Firstly, statistical analysis was performed on the sum of β-strands between each β-strand pairs in protein sequences. The results showed a propensity of near-neighbor pairing (or called “first come first pair”) in the β-strand pairs. Secondly, based on the same dataset, the pairwise cross-combinations of real β-strand pairs and four pseudo-β-strand contained pairs were classified by support vector machine (SVM). A novel feature extracting approach was designed for classification using the average amino acid pairing encoding matrix (APEM). Analytical results of the classification indicated that a segment of β-strand had the ability to distinguish β-strands from segments of α-helix and coil. However, the result also showed that a β-strand was not strongly conserved to choose its real partner from all the alternative β-strand partners, which was corresponding with the ordination results of the statistical analysis each other. Thus, the rules of “first come first pair” propensity and the non-conservative ability to choose real partner, were possible important factors affecting the β-strands alignment forming β-sheet structures.

The Crystal Structure of the Murine Voltage Dependent Anion Channel 1 (mVDAC1) (Likely) Represents a Native Conformation

The Voltage Dependent Anion Channel (VDAC) constitutes the major pathway for the entry and exit of metabolites across the outer membrane of the mitochondria and can serve as a scaffold for molecules that modulate the organelle. After decades of trials, three groups independently reported the 3D structure of this eukaryotic membrane protein consisting of a 19-stranded β-barrel. Our group resolved the mVDAC1 structure at 2.3Å resolution revealing a high-resolution image of its architecture including the position of the voltage sensing N-terminal α-helix segment- oriented against the interior wall causing a partial narrowing at the center of the pore.

Rational design of synthetic peptides to generate antibodies that recognize in situ CD11c + putative dendritic cells in horse lymph nodes

A three-dimensional model of the αX I-domain of the horse integrin CD11c from dendritic cells provided information for selecting two segments of the primary structure for peptide synthesis. Peptide 1 contains 20 amino acids and peptide 2 has 17 amino acid residues. The first spans from position Thr229 to Arg248 of an α-helix segment of the structure, whereas peptide 2 goes from Asp158 to Phe174 and corresponds to an exposed segment of the loop considered to be the metal ion-dependent adhesion site. Murine polyclonal antisera against both peptides were generated and assayed in peripheral blood cell suspensions and in cryosections of horse lymph nodes. Only the serum against peptide 2 was capable of identifying cells in suspension and in situ by immunohistochemistry, some with evident dendritic morphology. Using this approach, an immunogenic epitope exposed in CD11c was identified in cells from horse lymph node in situ.

Modèle topologique de la structure d’un antiport vacuolaire de type NHX chez la vigne cultivée (Vitis vinifera)

After identifying and isolating a grapevine ( Vitis vinifera L.) NHX vacuolar antiporter and before initializing functional genomic studies, we juged necessary to acquire a minimum of knowledge about the VvNHX1 protein. Thus, we realized a bioinformatic analysis to determine its basic characteristics and to get structural informations that could guide us through the functional characterization. We have determined important physico-chemical parameters (molecular mass, isoelectric point, hydrophobic regions, etc.) and obtained interesting structural data (primary, secondary, and tertiary structures; conserved domains and interaction motives; etc.). The VvNHX1 gene, which encodes this 541 amino-acid protein with a predicted molecular mass of 60 kDa, is made of 14 exons and measures 6.5 kb. The amino-acidic composition of this protein is very important, in particular, for the establishment of the α-helix structure, which represents more than 50% of the protein, but also for charge distribution, which generates critical electrostatic interactions for the ionic flux. The secondary structure of VvNHX1 contains multiple transmembrane α-helix segments that are made of hydrophobic amino-acid residues, thus facilitating its insertion in the membrane. Globally, VvNHX1 has one hydrophobic N-terminal region, made of 10 transmembrane segments with 440 amino-acid residues, and one hydrophilic C-terminal region, made of 100 residues. The region located between the fourth and fifth transmembrane segments represents, with its structure mainly helicoidal and the presence of a favourable electrostatic environment, the pore where cation flux is performed across the membrane. VvNHX1 contains various interaction domains as well as several putative posttranslational modification sites, mainly at the C-terminus but also at the N-terminus, that play an important part in regulating protein activities, influence protein structural stability, or interact with other proteins or signalling molecules.

Disruption of a Plasmodium falciparum Multidrug Resistance-associated Protein (PfMRP) Alters Its Fitness and Transport of Antimalarial Drugs and Glutathione

ATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC50 levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRPΔ) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPΔ parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite.

Ab initio folding simulation of Trpcage by replica exchange with hybrid Hamiltonian

Replica-exchange molecular dynamics simulations with hybrid Hamiltonian in explicit solvent were performed to study the folding of a designed 20-residue miniprotein, Trpcage, from a fully extended structure. During the simulations several folding/unfolding events happened. In the folded states the majority of experimentally observed NMR NOE restraints are satisfied. The folded structures have root mean squared deviation of 2.0 Å with respect to the NMR structures considering all heavy atoms. The free-energy surface constructed by the hybrid Hamiltonian simulations is similar to the one built by a standard replica-exchange simulation which started from the native structure. Consistent with previous experimental observation, a pre-existing hydrophobic collapse in the unfolded state is detected by investigating the desolvation behavior of Trpcage. At room temperature, an intermediate state featured by a misfolded core, a nearly formed α-helix segment and an absence of 3 10-helix is found. The replica exchange with hybrid Hamiltonian method is shown here to be capable of resolving the folding picture of the miniprotein.