Increase In Α-helical Content Research Articles

Exploring the effect of protein secondary structure on the solid state and physical stability of protein-based amorphous solid dispersions

Amorphous solid dispersions (ASDs) using proteins as carriers have emerged as a promising strategy for stabilizing amorphous drug molecules. Proteins possess diverse three-dimensional structures that significantly influence their own properties and may also impact the properties of ASDs. We prepared β-lactoglobulin (BLG) with different contents of β-sheet and α-helical secondary structures by initially dissolving BLG in different mixed solvents, containing different ratios of water, methanol/ethanol, and acetic acid, followed by spray drying of the solutions. Our findings revealed that an increase in α-helical content resulted in a decrease in the glass transition temperature (Tg) of the protein. Subsequently, we utilized the corresponding mixed solvents to dissolve both BLG and the model drug celecoxib (CEL), allowing the preparation of ASDs containing either β-sheet-rich or α-helix/random coil-rich BLG. Using spray drying, we successfully developed BLG-based ASDs with drug loadings ranging from 10 wt% to 90 wt%. At drug loadings below 40 wt%, samples prepared using both methods exhibited single-phase ASDs. However, heterogeneous systems formed when the drug loading exceeded 40 wt%. At higher drug loadings, physical stability assessments demonstrated that the α-helix/random coil-rich BLG structure exerted a more pronounced stabilizing effect on the drug-rich phase compared to the β-sheet-rich BLG. Overall, our results highlight the importance of considering protein secondary structure in the design of ASDs.

Orexin A, an amphipathic α-helical neuropeptide involved in pleiotropic functions in the nervous and immune systems: Synthetic approach and biophysical studies of the membrane-bound state

This research reports on the membrane interactions of orexin A (OXA), an α-helical and amphipathic neuropeptide that contains 33 residues and two disulfide bonds in the N-terminal region. OXA, which activates the orexins 1 and 2 receptors in neural and immune cell membranes, has essential pleiotropic physiological effects, including at the levels of arousal, sleep/wakefulness, energy balance, neuroprotection, lipid signaling, the inflammatory response, and pain. As a result, the orexin system has become a prominent target to treat diseases such as sleep disorders, drug addiction, and inflammation. While the high-resolution structure of OXA has been investigated in water and bound to micelles, there is a lack of information about its conformation bound to phospholipid membranes and its receptors. NMR is a powerful method to investigate peptide structures in a membrane environment. To facilitate the NMR structural studies of OXA exposed to membranes, we present a novel synthetic scheme, leading to the production of isotopically-labeled material at high purity. A receptor activation assay shows that the 15N-labeled peptide is biologically active. Biophysical studies are performed using surface plasmon resonance, circular dichroism, and NMR to investigate the interactions of OXA with phospholipid bilayers. The results demonstrate a strong interaction between the peptide and phospholipids, an increase in α-helical content upon membrane binding, and an in-plane orientation of the C-terminal region critical to function. This new knowledge about structure-activity relationships in OXA could inspire the design of novel therapeutics that leverage the anti-inflammatory and neuro-protective functions of OXA, and therefore could help address neuroinflammation, a major issue associated with neurological disorders such as Alzheimer's disease.

Characterization of the Conformations of Amyloid Beta 42 in Solution That May Mediate Its Initial Hydrophobic Aggregation.

Intrinsically disordered peptides, such as amyloid β42 (Aβ42), lack a well-defined structure in solution. Aβ42 can undergo abnormal aggregation and amyloidogenesis in the brain, forming fibrillar plaques, a hallmark of Alzheimer's disease. The insoluble fibrillar forms of Aβ42 exhibit well-defined, cross β-sheet structures at the molecular level and are less toxic than the soluble, intermediate disordered oligomeric forms. However, the mechanism of initial interaction of monomers and subsequent oligomerization is not well understood. The structural disorder of Aβ42 adds to the challenges of determining the structural properties of its monomers, making it difficult to understand the underlying molecular mechanism of pathogenic aggregation. Certain regions of Aβ42 are known to exhibit helical propensity in different physiological conditions. NMR spectroscopy has shown that the Aβ42 monomer at lower pH can adopt an α-helical conformation and as the pH is increased, the peptide switches to β-sheet conformation and aggregation occurs. CD spectroscopy studies of aggregation have shown the presence of an initial spike in the amount of α-helical content at the start of aggregation. Such an increase in α-helical content suggests a mechanism wherein the peptide can expose critical non-polar residues for interaction, leading to hydrophobic aggregation with other interacting peptides. We have used molecular dynamics simulations to characterize in detail the conformational landscape of monomeric Aβ42 in solution to identify molecular properties that may mediate the early stages of oligomerization. We hypothesized that conformations with α-helical structure have a higher probability of initiating aggregation because they increase the hydrophobicity of the peptide. Although random coil conformations were found to be the most dominant, as expected, α-helical conformations are thermodynamically accessible, more so than β-sheet conformations. Importantly, for the first time α-helical conformations are observed to increase the exposure of aromatic and hydrophobic residues to the aqueous solvent, favoring their hydrophobically driven interaction with other monomers to initiate aggregation. These findings constitute a first step toward characterizing the mechanism of formation of disordered, low-order oligomers of Aβ42.

TFE-induced local unfolding and fibrillation of SOD1: bridging the experiment and simulation studies.

Misfolding and aggregation of Cu, Zn Superoxide dismutase (SOD1) is involved in the neurodegenerative disease, amyotrophic lateral sclerosis. Many studies have shown that metal-depleted, monomeric form of SOD1 displays substantial local unfolding dynamics and is the precursor for aggregation. Here, we have studied the structure and dynamics of different apo monomeric SOD1 variants associated with unfolding and aggregation in aqueous trifluoroethanol (TFE) through experiments and simulation. TFE induces partially unfolded β-sheet-rich extended conformations in these SOD1 variants, which subsequently develops aggregates with fibril-like characteristics. Fibrillation was achieved more easily in disulfide-reduced monomeric SOD1 when compared with wild-type and mutant monomeric SOD1. At higher concentrations of TFE, a native-like structure with the increase in α-helical content was observed. The molecular dynamics simulation results illustrate distinct structural dynamics for different regions of SOD1 variants and show uniform local unfolding of β-strands. The strands protected by the zinc-binding and electrostatic loops were found to unfold first in 20% (v/v) TFE, leading to a partial unfolding of β-strands 4, 5, and 6 which are prone to aggregation. Our results thus shed light on the role of local unfolding and conformational dynamics in SOD1 misfolding and aggregation.

The effect of dense phase carbon dioxide on the conformation of hemoglobin

Dense phase carbon dioxide (DPCD) sterilization is a non-thermal sterilization technology used to process heat-sensitive foods. Although nutritional and sensorial quality of food is preserved while unwanted microbial activity is reduced during DPCD sterilization, the effect on protein structure remains unclear. In this work, the effect of DPCD on the higher order structure and fluorescence properties of Hemoglobin (Hb) was investigated. The different conditions assessed during DPCD processing included variation in pressure, pH and heating conditions. Results from this study showed an inversely proportional correlation between α-helical content of Hb and pressure. As the pressure was lowered, the levels of α-helical content increased. The increased levels of α-helix correlated with a lower fluorescence intensity and a limited redshift in the fluorescence emission wavelength. TEM imaging showed that DPCD processing resulted in Hb with larger molecular diameters, which became smaller as the pressure increased. Interestingly, after 7-day storage at 4 °C, an increase in α-helical content was observed. Results from this work show that DPCD sterilization does impact the conformation of hemoglobin, with a notable impact on secondary and tertiary structure.

Biophysical insight into the interaction mechanism of plant derived polyphenolic compound tannic acid with homologous mammalian serum albumins

Numerous phenolic compounds have been reported in the last decade that have a good antioxidant property and interaction affinity towards mammalian serum albumins. In the present study, we have utilized mammalian serum albumins as a model protein to examine their comparative interaction property with polyphenolic compound tannic acid (TA) by using various spectroscopic and calorimetric methods We have also monitored the esterase and antioxidant activity of mammalian serum albumins in the absence and presence of TA. The obtain results recommended that the TA have a good binding affinity (∼104 to 106M−1) towards mammalian serum albumins and shows double sequential binding sites, which depends on the concentration of TA that induced the conformational alteration which responsible for the thermal stability of proteins. Binding affinity, structural transition and thermodynamic parameters were calculated from spectroscopic and calorimetric method reveals that non-covalent interaction causes partial conformational alteration in the secondary structure of protein ie.; increase in α-helical content with decrease in β-sheet, random coil and other structure. Meanwhile, we have found that esterase activities of serum albumins were also stabilized against hydrolysis and shows higher antioxidant activity in the presence of TA because albumins its self have an immense antioxidant activity beside TA.

Mechanistic insight into interaction of Sodium Dodecyl Sulphate to asialylated form of glycoprotein: A mimic of membrane protein-lipid system

The SDS-glycoprotein system is mimic of membrane protein-lipid system. Fate of glycoprotein, conformation and the interactive forces involved in membrane milieu are expected to be decided by the net charge on glycoprotein that may change during acidic environment in a range of pathological states, including cancer, stroke, and ischemia. Asialofetuin (ASF; asialylated form of glycoprotein) and SDS interaction is studied when glycoprotein bears varying range of net charge (i.e. at different pH’s) by steady state and time-resolved spectroscopic, calorimetric and microscopic approaches. SDS interacts differently with ASF when protein is in cationic (at pH 2, 3 and 4) and in anionic states (pH 7.4). ASF undergo aggregation at pH 2, 3 and 4 whereas have enhancement in α-helical structure at pH 7.4 at sub-micellar concentrations of SDS. At pH 2, 3 and 4, the positively charged ASF interacts electrostatically with negatively charged head groups of SDS, leaving its hydrophobic tail free to interact with other protein-SDS complex and consequently lead to amyloid formation. However, at pH 7.4, the ASF interacts hydrophobically with SDS and an increase in α-helical content occurs that constrains the environment of Trp51 and consequently decreases movement of Trp conformers.

A hydrophobic patch surrounding Trp154 in human neuroserpin controls the helix F dynamics with implications in inhibition and aggregation

Neuroserpin (NS) mediated inhibition of tissue-type plasminogen activator (tPA) is important for brain development, synapse formation and memory. Aberrations in helix F and β-sheet A movement during inhibition can directly lead to epilepsy or dementia. Conserved W154 residue in a hydrophobic patch between helix F and β-sheet A is ideally placed to control their movement during inhibition. Molecular Dynamics (MD) simulation on wild type (WT) NS and its two variants (W154A and W154P) demonstrated partial deformation in helix F and conformational differences in strands 1A and 2A only in W154P. A fluorescence and Circular Dichroism (CD) analysis with purified W154 variants revealed a significant red-shift and an increase in α-helical content in W154P as compared to W154A and WT NS. Kinetics of tPA inhibition showed a decline in association rates (ka) for W154A as compared to WT NS with indication of complex formation. Appearance of cleaved without complex formation in W154P indicates that the variant acts as substrate due to conformational misfolding around helix F. Both the variants however showed increased rate of aggregation as compared to WT NS. The hydrophobic patch identified in this study may have importance in helix F dynamics of NS.

Impact of Histone Variant and Post-Translational Modification on Nucleosome

Eukaryotic genome DNA is compactly packaged into a nucleus of cell in a form of protein-DNA complex. This protein-DNA complex is called as nucleosome which is composed of a histone octamer formed by two copies each of the four core histones H3, H4, H2A and H2B and about 150 bp of DNA wrapping almost twice around the octamer. Nucleosome, however, changes its form by replacing histones with their variants and post-translational modifications to function in transcription, DNA duplication and DNA repair processes. To study the impact of histone variants and post-translational modifications on the structure and stability of nucleosome, we have performed molecular dynamics simulations with enhanced sampling methods. Free energy profiles obtained by nucleosomal DNA-unwrapping simulations exhibited that H3-containing nucleosome was stable in a fully wrapped state. It cost about 10 kcal/mol for unwrapping the first 8 bp from the DNA end. The cost much decreased for further unwrapping up to 20 bp-unwrapping. In contrast, CENP-A containing nucleosome was the most stable in a state where 5 to 10 bp of DNA were unwrapped from either end of DNA. For both nucleosomes, we observed that DNA was unwrapped asymmetrically.Next we evaluated a post-translational modification, K14 acetylation of H3 histone in a nucleosome structure. Our simulations indicated that H3 histone tail was almost always located nearby DNA regardless of the acetylation, however, K14 acetylation enhanced unwrapping of DNA at entry/exit regions. This can be explained by reduction of interactions between the histone tail and DNA due to the charge neutralization and increase in α-helical content in the histone tail. As a consequence, K14 acetylation increases the chance that DNA-binding proteins access DNA. These results give a new view of how acetylation affects chromatin conformation.

Oxidized antithrombin is a dual inhibitor of coagulation and angiogenesis: Importance of low heparin affinity

Endogenous proteins that promote vascular endothelial cell based inhibition of angiogenesis are an attractive option for antitumor therapy. Inactive cleaved and latent conformations of antithrombin (AT) are antiangiogenic, but not its native form which is an inhibitor of proteases involved in blood coagulation. Unlike native, the cleaved and latent conformations are reactive center loop inserted conformations which binds heparin with very low affinity. We use a sulfoxy modified AT to assess the role of reactive center loop insertion and heparin affinity in antiangiogenic function. Chorioallantoic membrane assay (CAM) shows that antiangiogenic activity of latent and oxidized AT are better than thalidomide, a potent antiangiogenic drug. Wound healing experiments suggest that latent and oxidized conformations can influence endothelial cell migration. Latent and cleaved conformations of AT shows an increase in α-helical content in the presence of unfractionated heparin, but not the oxidized AT. Unlike the loop inserted polymer, cleaved and latent conformations, oxidized AT has factor Xa inhibitory activity indicating that loop insertion is not necessary for antiangiogenic role. The results of our study establish that active conformation of AT can become antiangiogenic while maintaining its anticoagulant activity possibly through chelation of low affinity heparin in the vicinity of endothelial cell.

PH-dependent differential interacting mechanisms of sodium dodecyl sulfate with bovine serum fetuin: a biophysical insight.

Sodium dodecyl sulfate (SDS)-glycoprotein interaction serves as a model for a biological membrane. To get mechanistic insight into the interaction of SDS and glycoprotein, the effect of SDS on bovine serum fetuin (BSF) was studied in subcritical micellar concentrations at pH 7.4 and pH 2 using multiple approaches. SDS interacts electrostatically with BSF through its negatively charged head groups at pH 2 and hydrophobically via its alkyl chains at pH 7.4 up to a 1:20 molar ratio of BSF to SDS. However, at higher concentrations of SDS, BSF undergoes amyloid fibril formation at pH 2, as confirmed by enhanced ThT fluorescence, β-sheet formation, and TEM microscopy, whereas BSF undergoes induction of an α-helical structure in the presence of higher SDS concentration at pH 7.4. The increase in α-helical content with increasing SDS concentrations constrains the environment around tryptophan. As a consequence, the interconversion of tryptophan conformers decreases, resulting in a decrement of the fluorescence lifetime for BSF in the presence of SDS at pH 7.4.

Peripherin/rds co-distributes with putative binding partners in basal rod outer segment disks

The retinal degeneration slow protein peripherin/rds, (a.k.a. RDS), encoded by the rds gene, is essential for the formation, organization and maintenance of outer segments (Arikawa et al., 1992; Connell et al., 1991; Connell and Molday, 1990; Kedzierski et al., 2001; Molday, 1998). In humans, any one of over 160 mutations within this gene results in a broad variety of late onset progressive retinal dystrophies characterized by abnormal photoreceptor structure (Dryja et al., 1997; Keen and Inglehearn, 1996; Kohl et al., 1998). The pathogenic mechanism(s) underlying peripherin/rds mediated retinal dystrophies are unknown, however new insight has been gained in our understanding of peripherin/rds function based upon recent structural studies identifying the C-terminal domain of this protein as homologous to structures called intrinsically disordered domains (IDD) (Dyson, 2005; Edrington, 2007b; Ritter, 2005). IDDs are unstructured domains that become structurally stabilized by interacting with one or with several binding partners (Uversky et al., 2008). Structural flexibility imparts multi-functionality to the protein and binding partners act as regulators of its function (Lakoucheva et al., 2002; Vucetic et al., 2007). In humans, numerous mutations in the rds C-terminus result in rod/cone or cone/rod dystrophies. In a murine model of retinitis pigmentosa, deletion of codon 307 in the rds gene results in severely dysmorphic outer segments (OSs) in mice (McNally et al., 2002). The rds 307 del mouse provides the first in vivo evidence for a correlation between intrinsic disorder and retinopathy. In these mice disruption of the peripherin/rds C-terminus results in a gene product with a more intrinsically ordered C-terminal domain characterized by an increase in α-helical content.

Interactions of the Antimicrobial Peptide Maculatin 1.1 and Analogues with Phospholipid Bilayers

The interactions of the antimicrobial peptide, maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH2) and two analogues, with model phospholipid membranes have been studied using solid-state NMR and circular dichroism spectroscopy. Maculatin 1.1 and the P15G and P15A analogues displayed minimal secondary structure in water, but with zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles displayed a significant increase in α-helical content. In mixed phospholipid vesicles of DMPC and anionic dimyristoylphosphatidylglycerol (DMPG), each peptide was highly structured with ~80% α-helical content. In DMPC vesicles, the native peptide displayed moderate head group interaction and significant perturbation of the lipid acyl chains. In DMPC/DMPG vesicles, maculatin 1.1 promoted formation of a DMPG-enriched phase and moderately increased disorder towards acyl chain ends of DMPC in the mixed bilayer. Both analogues showed reduced phospholipid head group interactions with DMPC but displayed significant interactions with the mixed lipid system. These effects support the preferential activity of these antimicrobial peptides for bacterial membranes.

The Sodium Channel β3-Subunit Induces Multiphasic Gating in NaV1.3 and Affects Fast Inactivation via Distinct Intracellular Regions

Electrical excitability in neurons depends on the activity of membrane-bound voltage gated sodium channels (Na(v)) that are assembled from an ion conducting α-subunit and often auxiliary β-subunits. The α-subunit isoform Na(v)1.3 occurs in peripheral neurons together with the Na(v) β3-subunit, both of which are coordinately up-regulated in rat dorsal root ganglion neurons after nerve injury. Here we examine the effect of the β3-subunit on the gating behavior of Na(v)1.3 using whole cell patch clamp electrophysiology in HEK-293 cells. We show that β3 depolarizes the voltage sensitivity of Na(v)1.3 activation and inactivation and induces biphasic components of the inactivation curve. We detect both a fast and a novel slower component of inactivation, and we show that the β3-subunit increases the fraction of channels inactivating by the slower component. Using CD and NMR spectroscopy, we report the first structural analysis of the intracellular domain of any Na(v) β-subunit. We infer the presence of a region within the β3-subunit intracellular domain that has a propensity to form a short amphipathic α-helix followed by a structurally disordered sequence, and we demonstrate a role for both of these regions in the selective stabilization of fast inactivation. The complex gating behavior induced by β3 may contribute to the known hyperexcitability of peripheral neurons under those physiological conditions where expression of β3 and Na(v)1.3 are both enhanced.

The Influence of Vesicle Size and Composition on α-Synuclein Structure and Stability

Monomeric α-synuclein ( αSN), which has no persistent structure in aqueous solution, is known to bind to anionic lipids with a resulting increase in α-helix structure. Here we show that at physiological pH and ionic strength, αSN incubated with different anionic lipid vesicles undergoes a marked increase in α-helical content at a temperature dictated either by the temperature of the lipid phase transition, or (in 1,2-DilauroylSN-Glycero-3-[Phospho-rac-(1-glycerol)] (DLPG), which is fluid down to 0°C) by an intrinsic cold denaturation that occurs around 10–20°C. This structure is subsequently lost in a thermal transition around 60°C. Remarkably, this phenomenon is only observed for vesicles >100 nm in diameter and is sensitive to lipid chain length, longer chain lengths, and larger vesicles giving more cooperative unfolding transitions and a greater degree of structure. For both vesicle size and chain length, a higher degree of compressibility or permeability in the lipid thermal transition region is associated with a higher degree of αSN folding. Furthermore, the degree of structural change is strongly reduced by an increase in ionic strength or a decrease in the amount of anionic lipid. A simple binding-and-folding model that includes the lipid phase transition, exclusive binding of αSN to the liquid disordered phase, the thermodynamics of unfolding, and the electrostatics of binding of αSN to lipids is able to reproduce the two thermal transitions as well as the effect of ionic strength and anionic lipid. Thus the nature of αSN's binding to phospholipid membranes is intimately tied to the lipids' physico-chemical properties.

Amyloid Peptide Conformations In Soluble Oligomers

Fluorescence Resonance Energy Transfer (FRET) was used to study protein structures within soluble oligomer intermediates of amyloid and non-amyloid aggregates. Three protein aggregation systems were studied using fluorescence donor and acceptor near the N and C terminus:(1) Alzheimer's Aβ1-40 peptide.(2) 20-residue polyglutamine K2Q16K2.(3) 20-residue polyglutamic acid E20. (non-amyloid)The aggregation of each peptide showed different conformational changes: Aβ1-40 partially compacted into a structure consistent with solid-state NMR structures, K2Q16K2 extended to form β-sheets, and E20 compacted heavily into β-hairpins. However, based on donor-acceptor distances, soluble oligomers conformations of all three peptides show a remarkable degree of similarity to their monomeric precursors, albeit with a slightly expanded conformation for Aβ1-40 and E20. These findings support assembly models of small soluble oligomers which largely consist of monomer-like structures, with some increase in α-helical content for Aβ1-40. These donor-acceptor distances are used to directly assess the accuracy of different molecular dynamics force fields in the study of these soluble oligomers.

Design of a Modern Liposome and Bee Venom Formulation for the Traditional VIT-Venom Immunotherapy

Traditional venom immunotherapy uses injections of whole bee venom in buffer or adsorbed in Al (OH)3 in an expensive, time-consuming way. New strategies to improve the safety and efficacy of this treatment with a reduction of injections would, therefore, be of general interest. It would improve patient compliance and provide socio-economic benefits. Liposomes have a long tradition in drug delivery because they increase the therapeutic index and avoid drug degradation and secondary effects. However, bee venom melittin (Mel) and phospholipase (PLA2) destroy the phospholipid membranes. Our central idea was to inhibit the PLA2 and Mel activities through histidine alkylation and or tryptophan oxidation (with pbb, para-bromo-phenacyl bromide, and/or NBS- N-bromosuccinimide, respectively) to make their encapsulations possible within stabilized liposomes. We strongly believe that this formulation will be nontoxic but immunogenic. In this paper, we present the whole bee venom conformation characterization during and after chemical modification and after interaction with liposome by ultraviolet, circular dichroism, and fluorescence spectroscopies. The PLA2 and Mel activities were measured indirectly by changes in turbidity at 400n m, rhodamine leak-out, and hemolysis. The native whole bee venom (BV) presented 78.06% of α-helical content. The alkylation (A-BV) and succynilation (S-BV) of BV increased 0.44 and 0.20% of its α-helical content. The double-modified venom (S-A-BV) had a 0.74% increase of α-helical content. The BV chemical modification induced another change on protein conformations observed by Trp that became buried with respect to the native whole BV. It was demonstrated that the liposomal membranes must contain pbb (SPC:Cho:pbb, 26:7:1) as a component to protect them from aggregation and/or fusion. The membranes containing pbb maintained the same turbidity (100%) after incubation with modified venom, in contrast with pbb-free membranes that showed a 15% size decrease. This size decrease was interpreted as membrane degradation and was corroborated by a 50% rhodamine leak-out. Another fact that confirmed our interpretation was the observed 100% inhibition of the hemolytic activity after venom modification with pbb and NBS (S-A-BV). When S-A-BV interacted with liposomes, other protein conformational changes were observed and characterized by the increase of 1.93% on S-A-BV α-helical content and the presence of tryptophan residues in a more hydrophobic environment. In other words, the S-A-BV interacted with liposomal membranes, but this interaction was not effective to cause aggregation, leak-out, or fusion. A stable formulation composed by S-A-BV encapsulated within liposomes composed by SPC:Cho:pbb, at a ratio of 26:7:1, was devised. Large unilamellar vesicles of 202.5 nm with a negative surface charge (–24.29 mV) encapsulated 95% of S-A-BV. This formulation can, now, be assayed on VIT.

Photoinduced electron transfer between hen egg white lysozyme and anticancer drug menadione

The protein, hen egg white lysozyme, on photoexcitation undergoes electron transfer with menadione (2-methyl-1,4-naphthoquinone), a widely known anticancer drug. With the addition of menadione the fluorescence of lysozyme is quenched with the simultaneous formation of an excited state charge-transfer complex in the longer wavelength and a ground state complex. The former is further evident from laser flash photolysis studies, which indicate a tryptophan to menadione electron transfer. From fluorescence quenching studies the binding constant is found to be ∼1.7×10 4 M −1 with the corresponding changes in enthalpy (Δ H°) and entropy (Δ S°) as 12.24 kJ mol −1 and 124.12 J mol −1 K −1, respectively, indicative of an entropy-driven process. The circular dichroism studies also show some structural changes with increase in α-helical content in the protein on interaction with menadione. Finally, docking studies give some insight into the role of Trp 108 of lysozyme in the interaction.

A predicted N-terminal helical domain of a Group 1 LEA protein is required for protection of enzyme activity from drying

Late embryogenesis abundant (LEA) proteins have been repeatedly implicated in the acquisition of desiccation tolerance in angiosperm seed embryos. However, the mechanism(s) by which protection occurs is not well understood. While the Group 1 LEA proteins are predicted to be largely unordered in solution, there is strong evidence that upon drying these proteins undergo a structural transition that leads to an increase in α-helical content. Several studies also suggest there is a direct interaction between Group 1 LEA proteins and other molecules in the cytoplasm that may be critical for the establishment of desiccation tolerance during embryo maturation. We have produced a recombinant Group 1 LEA protein and show that it is capable of protecting the enzyme lactate dehydrogenase from the deleterious effects of drying. We have also evaluated the ability of various altered recombinant Group 1 LEA proteins to protect in the same assay. Our results suggest that the highly conserved 20 amino acid Group 1 LEA signature motif is not required for protection in our in vitro assay. However, introduction of two juxtaposed proline residues into an N-terminal helical domain predicted to exist in the hydrated structure significantly compromises the ability of the recombinant protein to provide protection from drying. These results suggest that the N-terminal domain of Group 1 LEA proteins may be important for proper folding during dehydration.

Analogs of Insulin-like Peptide 3 (INSL3) B-chain Are LGR8 Antagonists in Vitro and in Vivo

Insulin-like peptide 3 (INSL3) is a member of the insulin superfamily that plays an important role in mediating testes descent during fetal development. More recently, it has also been demonstrated to initiate oocyte maturation and suppress male germ cell apoptosis. These actions are mediated via a specific G-protein-coupled receptor, LGR8. Little is known regarding the structure and function relationship of INSL3, although it is believed that the principal receptor binding site resides within its B-chain. We subsequently observed that the linear B-chain alone (INSL3B-(1-31)) bound to LGR8 and was able to antagonise INSL3 stimulated cAMP accumulation in HEK-293T cells expressing LGR8. Sequentially N- and C-terminally shortened linear analogs were prepared by solid phase synthesis and subsequent assay showed that the minimum length required for binding was residues 11-27. It was also observed that increased binding affinity correlated with a corresponding increase in alpha-helical content as measured by circular dichroism spectroscopy. Molecular modeling studies suggested that judicious placement of a conformational constraint within this peptide would increase its alpha-helix content and result in increased structural similarity to the B-chain within native INSL3. Consequently, intramolecularly disulfide-linked analogs of the B-chain showed a potentiation of INSL3 antagonistic activity, as well as exhibiting increased proteolytic stability, as assessed in rat serum in vitro. Administration of one of these peptides into the testes of rats resulted in a substantial decrease in testis weight probably due to the inhibition of germ cell survival, suggesting that INSL3 antagonists may have potential as novel contraceptive agents.