Α-helical State Research Articles

Latarcins: Antimicrobial and cell-penetrating peptides from spider venom.

The venom of the ant spider Lachesena tarabaevi is reported to be rich in linear membrane active peptides[1]. Latarcins is a group of seven linear antimicrobial peptides found in the venom of this spider. We have recently studied this entire group of peptides to address the secondary state, membrane orientation and toxicity using circular dichroism (CD) spectroscopy, oriented CD (OCD), solid-state 31P-/15N-NMR and fluorescence spectroscopy[2]. Our results show that all peptides fold into an α-helical state in presence of lipids. OCD and NMR experiments show that these helices are either surface bound, tilted in the membrane or tend to aggregate in the lipid bilayer. The most tilted peptide, Ltc2a, shows indiscriminate membrane-toxicity and damages natural membranes (antimicrobial and hemolytic activity) as well as artificial membranes (vesicle leakage) where as the aggregating peptide, Ltc6a, shows no toxicity towards natural membranes but instead induces leakage of artificial lipid vesicles. Other latarcins (Ltc1, Ltc3a, Ltc4a and Ltc5a) show high activity and are selective in bacterial killing without causing bilayer disturbances. This result indicates that latarcins may have intracellular targets, and thus the ability of these peptides to enter cancer cells was also studied. In order to reduce the membrane toxicity, truncated latarcins (short latarcins, Sltc) were synthesized. Shorter latarcins turned out to be even less cytotoxic and highly cell-penetrating. These biophysical studies of latarcins and their shorter analogues on model membranes along with their influence on natural membranes will be presented.

Physicochemical characterization of the G51D mutation of α-synuclein that is responsible for its severe cytotoxicity

Fibril formation and aggregation of α-synuclein are important for the pathogenesis of neurodegenerative disorders including Parkinson’s disease. In familial Parkinson’s disease, the G51D mutation of α-synuclein causes severe symptoms and rapid progression. α-Synuclein, an intrinsically disordered protein, was shown to adopt an α-helical tetrameric state that resists fibrillation and aggregation. Here, we isolated the stable dimeric state of recombinant wild-type (WT) α-synuclein and G51D α-synuclein protein. Using circular dichroism spectroscopy, we determined that the α-synuclein dimer and monomer structures were unfolded. The WT α-synuclein dimer was more resistant to fibril formation than the monomer. However, the fibril formation rate of the G51D α-synuclein dimer was similar to that of the G51D α-synuclein monomer. The fibril morphology and properties of the G51D α-synuclein monomer were different from those of the WT α-synuclein monomer and dimer and G51D α-synuclein dimer. Additionally, G51D α-synuclein monomer fibrils were more cytotoxic than other fibrils. Our findings indicate that the structural differences between G51D α-synuclein monomer fibrils and other fibrils are critically responsible for its severe neurotoxicity in familial Parkinson’s disease.

The C-terminal domain of transcription factor RfaH: Folding, fold switching and energy landscape.

We simulate the folding and fold switching of the C-terminal domain (CTD) of the transcription factor RfaH using an all-atom physics-based model augmented with a dual-basin structure-based potential energy term. We show that this hybrid model captures the essential thermodynamic behavior of this metamorphic domain, that is, a change in the global free energy minimum from an α-helical hairpin to a 5-stranded β-barrel upon the dissociation of the CTD from the rest of the protein. Using Monte Carlo sampling techniques, we then analyze the energy landscape of the CTD in terms of progress variables for folding toward the two folds. We find that, below the folding transition, the energy landscape is characterized by a single, dominant funnel to the native β-barrel structure. The absence of a deep funnel to the α-helical hairpin state reflects a negligible population of this fold for the isolated CTD. We observe, however, a higher α-helix structure content in the unfolded state compared to results from a similar but fold switch-incompetent version of our model. Moreover, in folding simulations started from an extended chain conformation we find transiently formed α-helical structure, occurring early in the process and disappearing as the chain progresses toward the thermally stable β-barrel state.

TAT-RasGAP317-326 kills cells by targeting inner-leaflet–enriched phospholipids

TAT-RasGAP317-326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317-326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317-326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317-326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317-326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.

The Kinetics of Amyloid Fibrillar Aggregation of Uperin 3.5 Is Directed by the Peptide's Secondary Structure.

Many peptides aggregate into insoluble β-sheet rich amyloid fibrils. Some of these aggregation processes are linked to age-related diseases, such as Alzheimer's disease and type 2 diabetes. Here, we show that the secondary structure of the peptide uperin 3.5 directs the kinetics and mechanism of amyloid fibrillar aggregation. Uperin3.5 variants were investigated using thioflavin T fluorescence assays, circular dichroism spectroscopy, and structure prediction methods. Our results suggest that those peptide variants with a strong propensity to form an α-helical secondary structure under physiological conditions are more likely to aggregate into amyloid fibrils than peptides in an unstructured or "random coil" conformation. This conclusion is in good agreement with the hypothesis that an α-helical transition state is required for peptide aggregation into amyloid fibrils. Specifically, uperin 3.5 variants in which charged amino acids were replaced by alanine were richer in α-helical content, leading to enhanced aggregation compared to that of wild type uperin 3.5. However, the addition of 2,2,2-trifluoroethanol as a major co-solute or membrane-mimicking phospholipid environments locked uperin 3.5 to the α-helical conformation preventing amyloid aggregation. Strategies for stabilizing peptides into their α-helical conformation could provide therapeutic approaches for overcoming peptide aggregation-related diseases. The impact of the physiological environment on peptide secondary structure could explain aggregation processes in a cellular environment.

Modulation of Measles Virus NTAIL Interactions through Fuzziness and Sequence Features of Disordered Binding Sites

In this paper we review our recent findings on the different interaction mechanisms of the C-terminal domain of the nucleoprotein (N) of measles virus (MeV) NTAIL, a model viral intrinsically disordered protein (IDP), with two of its known binding partners, i.e., the C-terminal X domain of the phosphoprotein of MeV XD (a globular viral protein) and the heat-shock protein 70 hsp70 (a globular cellular protein). The NTAIL binds both XD and hsp70 via a molecular recognition element (MoRE) that is flanked by two fuzzy regions. The long (85 residues) N-terminal fuzzy region is a natural dampener of the interaction with both XD and hsp70. In the case of binding to XD, the N-terminal fuzzy appendage of NTAIL reduces the rate of α-helical folding of the MoRE. The dampening effect of the fuzzy appendage on XD and hsp70 binding depends on the length and fuzziness of the N-terminal region. Despite this similarity, NTAIL binding to XD and hsp70 appears to rely on completely different requirements. Almost any mutation within the MoRE decreases XD binding, whereas many of them increase the binding to hsp70. In addition, XD binding is very sensitive to the α-helical state of the MoRE, whereas hsp70 is not. Thus, contrary to hsp70, XD binding appears to be strictly dependent on the wild-type primary and secondary structure of the MoRE.

The folding equilibrium of huntingtin exon 1 monomer depends on its polyglutamine tract

Expansion of the polyglutamine (polyQ) tract in exon 1 of the huntingtin protein (Httex1) leads to Huntington's disease resulting in fatal neurodegeneration. However, it remains poorly understood how polyQ expansions alter protein structure and cause toxicity. Using CD, EPR, and NMR spectroscopy, we found here that monomeric Httex1 consists of two co-existing structural states whose ratio is determined by polyQ tract length. We observed that short Q-lengths favor a largely random-coil state, whereas long Q-lengths increase the proportion of a predominantly α-helical state. We also note that by following a mobility gradient, Httex1 α-helical conformation is restricted to the N-terminal N17 region and to the N-terminal portion of the adjoining polyQ tract. Structuring in both regions was interdependent and likely stabilized by tertiary contacts. Although little helicity was present in N17 alone, each Gln residue in Httex1 enhanced helix stability by 0.03-0.05 kcal/mol, causing a pronounced preference for the α-helical state at pathological Q-lengths. The Q-length-dependent structuring and rigidification could be mimicked in proteins with shorter Q-lengths by a decrease in temperature, indicating that lower temperatures similarly stabilize N17 and polyQ intramolecular contacts. The more rigid α-helical state of Httex1 with an expanded polyQ tract is expected to alter interactions with cellular proteins and modulate the toxic Httex1 misfolding process. We propose that the polyQ-dependent shift in the structural equilibrium may enable future therapeutic strategies that specifically target Httex1 with toxic Q-lengths.

Myeloid Cell Leukemia 1 Inhibition: An in Silico Study Using Non-equilibrium Fast Double Annihilation Technology.

2017 , 57 , 1240 ]. As an important byproduct, this work constitutes hence one of the first and most challenging tryouts for the GAFF2 parameter set. Agreement with experimental measurements is found to be generally satisfactory, validating the GAFF2 parametrization of the ligands and foreseeing a possible role of FSDAM for industrial application in drug discovery.

Effect of the geometry of confining media on the stability and folding rate of -helix proteins.

Protein folding in confined media has attracted wide attention over the past 15 years due to its importance to both in vivo and in vitro applications. It is generally believed that protein stability increases by decreasing the size of the confining medium, if the medium's walls are repulsive, and that the maximum folding temperature in confinement is in a pore whose size D 0 is only slightly larger than the smallest dimension of a protein's folded state. Until recently, the stability of proteins in pores with a size very close to that of the folded state has not received the attention it deserves. In a previous paper [L. Javidpour and M. Sahimi, J. Chem. Phys. 135, 125101 (2011)], we showed that, contrary to the current theoretical predictions, the maximum folding temperature occurs in larger pores for smaller α-helices. Moreover, in very tight pores, the free energy surface becomes rough, giving rise to a new barrier for protein folding close to the unfolded state. In contrast to unbounded domains, in small nanopores proteins with an α-helical native state that contain the β structures are entropically stabilized implying that folding rates decrease notably and that the free energy surface becomes rougher. In view of the potential significance of such results to interpretation of many sets of experimental data that could not be explained by the current theories, particularly the reported anomalously low rates of folding and the importance of entropic effects on proteins' misfolded states in highly confined environments, we address the following question in the present paper: To what extent the geometry of a confined medium affects the stability and folding rates of proteins? Using millisecond-long molecular dynamics simulations, we study the problem in three types of confining media, namely, cylindrical and slit pores and spherical cavities. Most importantly, we find that the prediction of the previous theories that the dependence of the maximum folding temperature T f on the size D of a confined medium occurs in larger media for larger proteins is correct only in spherical geometry, whereas the opposite is true in the two other geometries that we study. Also studied is the effect of the strength of the interaction between the confined media's walls and the proteins. If the walls are only weakly or moderately attractive, a complex behavior emerges that depends on the size of the confining medium.

Effects of Cosolvents and Macromolecular Crowding on the Phase Transitions and Temperature-Pressure Stability of Chiral and Racemic Poly-Lysine

Abstract FTIR spectroscopy has been used to reveal the effects of different types of cosolvents (TMAO, urea) as well as macromolecular crowding (using the crowding agent Ficoll) on the temperature and pressure dependent structure of poly-L-lysine, poly-D-lysine and their racemic mixture. Compared to the effects of cosolvents on the unfolding transition of proteins, their effects on the α-helix to aggregated β-sheet transition of polylysine are quite small. High hydrostatic pressure has been found to favor the α-helical state over the aggregated β-sheet structure which is reflected in a volume decrease of ΔV=−32 mL mol−1, indicating that the packing mode is more efficient in the α-helical structure. Both, addition of urea and TMAO lead to a decrease in pressure stability of the aggregated β-sheet structure, which is accompanied by a three-fold decrease in ΔV, whereas the macromolecular crowder has little effect on the β-to-α transition. The more than 3 kbar higher β-to-α transition pressure of the racemic mixture compared with PLL confirms the drastic stabilization of β-sheet aggregates if the stereoisomers PLL and PDL are combined. Changes in hydration and packing of the polypeptide occurs upon interaction and fine packing of the polypeptide’s chains of opposed chirality, which are slightly modulated by the properties of cosolute and crowding, only. The underlying solvational and packing mechanisms observed here may be decisive factors responsible for the spontaneous protein aggregation in general and, as such, may shed additional light on the molecular basis of amyloid-associated diseases.

Identification of Key Interactions in the Initial Self-Assembly of Amylin in a Membrane Environment

Islet amyloid polypeptide, also known as amylin, forms aggregates that reduce the amount of insulin-producing cells in patients with type II diabetes mellitus. Much remains unknown about the process of aggregation and cytotoxicity, but it is known that certain cell membrane components can alter the rate of aggregation. Using atomistic molecular dynamics simulations combined with the highly mobile membrane mimetic model incorporating enhanced sampling of lipid diffusion, we investigate interaction of amylin peptides with the membrane components as well as the self-assembly of amylin. Consistent with experimental evidence, we find that an initial membrane-bound α-helical state folds into stable β-sheet structures upon self-assembly. Our results suggest the following mechanism for the initial phase of amylin self-assembly. The peptides move around on the membrane with the positively charged N-terminus interacting with the negatively charged lipid headgroups. When the peptides start to interact, they partly unfold and break some of the contacts with the membrane. The initial interactions between the peptides are dominated by aromatic and hydrophobic interactions. Oligomers are formed showing both intra- and interpeptide β-sheets, initially with interactions mainly in the C-terminal domain of the peptides. Decreasing the pH to 5.5 is known to inhibit amyloid formation. At low pH, His18 is protonated, adding a fourth positive charge at the peptide. With His18 protonated, no oligomerization is observed in the simulations. The additional charge gives a strong midpoint anchoring of the peptides to negatively charged membrane components, and the peptides experience additional interpeptide repulsion, thereby preventing interactions.

Conformational Fine-Tuning of Pore-Forming Peptide Potency and Selectivity.

To better understand the sequence–structure–function relationships that control the activity and selectivity of membrane-permeabilizing peptides, we screened a peptide library, based on the archetypal pore-former melittin, for loss-of-function variants. This was accomplished by assaying library members for failure to cause leakage of entrapped contents from synthetic lipid vesicles at a peptide-to-lipid ratio of 1:20, 10-fold higher than the concentration at which melittin efficiently permeabilizes the same vesicles. Surprisingly, about one-third of the library members are inactive under these conditions. In the negative peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that loss-of-function activity can be completely recapitulated by a single-residue change of the leucine at position 16 to glycine. Unlike the potently cytolytic melittin, the loss-of-function peptides, including the single-site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Loss of function is shown to result from a shift in the binding–folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, α-helical secondary structure, and potent activity of the “negative” peptides. While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes. The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.

Investigating the effect of structural transition on aggregation of β-lactoglobulin.

β-lactoglobulin (BLG), the major bovine whey protein, is a well-characterized globular protein. It is a model protein for studying the structural transition and aggregation. BLG unfolds and aggregates through chemical and physical processes. It is a predominantly β-sheet protein but, the non-native α-helical intermediate accumulates in its folding pathway. The present study aims to understand more about which stage of the protein folding is prone to aggregation. The intermediate states were trapped by TFE and their aggregation and structural changes evaluated, for this purpose. The experiments were carried out at various pH values, ionic strengths, protein concentrations and heating times by turbidity measurements, circular dichroism and fluorescence spectroscopy. Furthermore, the aggregated species at various molecular weights were detected by SDS-PAGE. Only a small change was observed in the secondary and tertiary structures of the protein at 10% TFE, but a further increase of TFE concentration results in induction of new α-helical structure and disruption of the rigid tertiary structure. The turbidity measurement indicated that the aggregation of BLG reaches a maximum level at 10% TFE on all experimental conditions and from this point forward, it decreases with increasing the amount of TFE. In conclusion, the results showed that the α-helical state is resistant to aggregation, in spite that its tertiary structure is partially unfolded. BLG becomes prone to aggregation, when its non-native α-helical structure converts to the β-sheet structure.

Impact of secondary structure and hydration water on the dielectric spectrum of poly-alanine and possible relation to the debate on slaved versus slaving water.

Using extensive molecular dynamics simulations of a single eight-residue alanine polypeptide in explicit water, we investigate the influence of α-helix formation on the dielectric spectrum. For this, we project long equilibrium trajectories into folded and unfolded states and thereby obtain dielectric spectra representative for disordered as well α-helical conformations without the need to change any other system parameter such as pH or temperature. The absorption spectrum in the α-helical state exhibits a feature in the sub-GHz range that is significantly stronger than in the unfolded state. As we show by an additional decomposition into peptide and water contributions, this slow dielectric mode, the relaxation time of which matches the independently determined peptide rotational relaxation time, is mostly caused by peptide polarization correlations, but also contains considerable contributions from peptide-water correlations. In contrast, the peptide spectral contribution shows no features in the GHz range where bulk water absorbs, not even in the peptide-water correlation part, we conclude that hydration water around Ala8 is more influenced by peptide polarization relaxation effects than the other way around. A further decomposition into water-self and water-collective polarization correlations shows that the dielectric response of hydration water is, in contrast to electrolyte solutions, retarded and that this retardation is mostly due to collective effects, the self relaxation of hydration water molecules is only slightly slowed down compared to bulk water. We find the dynamic peptide-water polarization cross correlations to be rather long-ranged and to extend more than one nanometer away from the peptide-water interface into the water hydration shell, in qualitative agreement with previous simulation studies and recent THz absorption experiments.

Analysis of Piscidin and Lipopolysaccharide Interactions: A Step Towards Characterizing Immunomodulation

Lipopolysaccharide (LPS) is one of the most extensively studied pathogen-associated molecular patterns (PAMPs), as it composes 90% of the membrane in Gram-negative bacteria. Recognition of micellar aggregates of LPS by the immune system subsequently mounts an inflammatory response against infection. However, if this process is over exaggerated, high levels of cytokines may become detrimental, leading to organ shutdown and septic shock. Various cationic antimicrobial peptides (AMPs) have been shown to decrease the transcription and release of pro-inflammatory cytokines associated with LPS recognition.Piscidin is a cationic antimicrobial peptide first isolated in the mast cells of fish. In the presence of a lipid bilayer, it folds into an amphipathic α-helix structure, which facilitates peptide-lipid bilayer binding. Based on the studies of other cationic AMPs, it was hypothesized that piscidin-1 (P1) and piscidin-3 (P3) would fold into their α-helical state and bind strongly to LPS, breaking apart its large aggregates. Using isothermal calorimetry to explore the thermodynamics of binding, it was shown that both peptides bound strongly to LPS through an exothermic reaction. Furthermore, fluorescence-dequenching studies were carried out using Fluorescein isothiocyanate labeled LPS (FITC-LPS), demonstrating piscidin's ability to separate FITC-LPS aggregates. Finally, both peptides were induced by LPS to be approximately 97% helical from their native random coil state, as shown by circular dichroism. Based upon these results, it is clear that significant interactions occur between LPS and piscidin, warranting future exploration of the effect of piscidin on LPS recognition by immune cells.

Thermodynamics of Deca-alanine Folding in Water

The determination of the folding dynamics of polypeptides and proteins is critical in characterizing their functions in biological systems. Numerous computational models and methods have been developed for studying structure formation at the atomic level. Due to its small size and simple structure, deca-alanine is used as a model system in molecular dynamics (MD) simulations. The free energy of unfolding in vacuum has been studied extensively using the end-to-end distance of the peptide as the reaction coordinate. However, few studies have been conducted in the presence of explicit solvent. Previous results show a significant decrease in the free energy of extended conformations in water, but the α-helical state is still notably favored over the extended state. Although sufficient in vacuum, we show that end-to-end distance is incapable of capturing the full complexity of deca-alanine folding in water. Using α-helical content as a second reaction coordinate, we deduce a more descriptive free-energy landscape, one which reveals a second energy minimum in the extended conformations that is of comparable free energy to the α-helical state. Equilibrium simulations demonstrate the relative stability of the extended and α-helical states in water as well as the transition between the two states. This work reveals both the necessity and challenge of determining a proper reaction coordinate to fully characterize a given process.

Interdomain contacts control folding of transcription factor RfaH

Escherichia coli RfaH activates gene expression by tethering the elongating RNA polymerase to the ribosome. This bridging action requires a complete refolding of the RfaH C-terminal domain (CTD) from an α-helical hairpin, which binds to the N-terminal domain (NTD) in the free protein, to a β-barrel, which interacts with the ribosomal protein S10 following RfaH recruitment to its target operons. The CTD forms a β-barrel when expressed alone or proteolytically separated from the NTD, indicating that the α-helical state is trapped by the NTD, perhaps co-translationally. Alternatively, the interdomain contacts may be sufficient to drive the formation of the α-helical form. Here, we use functional and NMR analyses to show that the denatured RfaH refolds into the native state and that RfaH in which the order of the domains is reversed is fully functional in vitro and in vivo. Our results indicate that all information necessary to determine its fold is encoded within RfaH itself, whereas accessory factors or sequential folding of NTD and CTD during translation are dispensable. These findings suggest that universally conserved RfaH homologs may change folds to accommodate diverse interaction partners and that context-dependent protein refolding may be widespread in nature.

Formation of Dynamic Soluble Surfactant-induced Amyloid β Peptide Aggregation Intermediates

Intermediate amyloidogenic states along the amyloid β peptide (Aβ) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aβ aggregation, we here investigate surfactant-induced Aβ aggregation. This process leads to co-aggregates featuring a β-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aβ in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via β-structure to the fully formed α-helical state at high surfactant concentration. The β-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aβ co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (k(ex) ∼1100 s(-1)) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with β-structure promoting substances, such as surfactants, Aβ is kinetically driven toward an aggregation-prone state.

Simultaneous prediction of protein secondary structure and transmembrane spans

Prediction of transmembrane spans and secondary structure from the protein sequence is generally the first step in the structural characterization of (membrane) proteins. Preference of a stretch of amino acids in a protein to form secondary structure and being placed in the membrane are correlated. Nevertheless, current methods predict either secondary structure or individual transmembrane states. We introduce a method that simultaneously predicts the secondary structure and transmembrane spans from the protein sequence. This approach not only eliminates the necessity to create a consensus prediction from possibly contradicting outputs of several predictors but bears the potential to predict conformational switches, i.e., sequence regions that have a high probability to change for example from a coil conformation in solution to an α-helical transmembrane state. An artificial neural network was trained on databases of 177 membrane proteins and 6048 soluble proteins. The output is a 3 × 3 dimensional probability matrix for each residue in the sequence that combines three secondary structure types (helix, strand, coil) and three environment types (membrane core, interface, solution). The prediction accuracies are 70.3% for nine possible states, 73.2% for three-state secondary structure prediction, and 94.8% for three-state transmembrane span prediction. These accuracies are comparable to state-of-the-art predictors of secondary structure (e.g., Psipred) or transmembrane placement (e.g., OCTOPUS). The method is available as web server and for download at www.meilerlab.org.

Effect of artemin on structural transition of β-lactoglobulin

Encysted embryos of Artemia are exceptionally resistant to severe environmental stress. This resistance is thought to depend in part on the existence of a protein termed artemin. There is only little information about the function of artemin. It has been reported artemin is a thermostable protein with RNA-binding ability. In addition, it reduces the extent of aggregation significantly and enhances the efficiency of refolding and activity recovery of carbonic anhydrase and horseradish peroxidase. In this study, the effect of artemin purified from Artemia urmiana on bovine β-lactoglobulin (BLG) and its α-helical intermediate state has been evaluated by circular dichroism and intrinsic and extrinsic fluorescence spectroscopy. The results obtained in aqueous buffer show, artemin decreases the compactness of BLG structure and causes to the exposure of some hydrophobic groups. The results also indicate artemin has an inhibitory effect on β-sheet→α-helix transition in the secondary structure of β-lactoglobulin. Since this transition occurs during unfolding of β-lactoglobulin, it seems artemin influences on the folding pathway of β-lactoglobulin. This structural effect of artemin can result from its high surface hydrophobicity. Consequently, it is expected that artemin has chaperoning potency because of its effect on the folding of BLG.