Alpha-helix Secondary Structure Research Articles

Antibacterial Peptide from Chymotrypsin Hydrolysate of Jatropha Seeds with RP-HPLC Fractionation

Antimicrobial peptide (AMP), as a new antibiotic agent, has promising prospects in overcoming the problem of resistance. AMP production can be carried out by proteolytic enzymes. Protein from castor bean (R. communis) is toxic, so it can potentially be a source of AMP. This study aims to obtain protein hydrolysate from the castor bean (R. communis) using several extraction methods, including SDS dialysis, SDS-gel filtration, trifluoroacetic acid (TFA), and acetone precipitation. Chymotrypsin enzyme was used to hydrolyze the protein, and the peptide hydrolysate was fractionated using RP-HPLC. The peptide fraction was tested for its antibacterial activity by agar diffusion and microdilution methods, and the most active fraction was identified for its amino acid sequence by LC-HRMS. The results showed that the acetone precipitation extraction method was the best method, with a degree of hydrolysis of 83.9%. The active fractions 6 and 10 of RP-HPLC had IC50 values of 14.1 and 14.5 µg/mL for E. coli and 13.3 and 14.4 µg/mL for S. aureus, respectively. NVLRGKGMASL peptides were found in fraction 10, and GIILLSSK, NMIAKR, and LLDILTKK peptides were found in fraction 6 with an alpha helix secondary structure that can cause membrane damage. The peptides NVLRGKGMASL, GIILLSSK, NMIAKR, and LLDILTKK are thought to have potential as antibacterial compounds.

Optimal transport technique to understand peptide conformations

Using Protein Data Bank (PDB), the distribution of backbone torsional angle for dipeptides and tripeptides has been obtained and analyzed in several past works. However, the more extended peptides distributions are difficult to obtain due to the data sparsity. In this paper, we introduce a technique based on Optimal Transport theory to understand peptide conformations. This technique renders a computational method to determine the multi-point distribution of the backbone torsional angles of the peptide. Applying this technique, we study tetrapeptide conformations, presenting a detailed analysis of tetrapeptides distributions composed of Ala and Gly amino acids, and compare it with PDB data. Our study shows that tetrapeptides such as AAAA, AAAG, AGAA, and GAAA prefer right-handed alpha-helix secondary structure formations and beta-turns in GGGG, GAGG, AAGG, and AAGA.

The Effect of Enzymatic-TGase Cross-Linking on the Structure of Soy Protein Isolate Based on Multiplex Spectroscopy

The functional properties of soy protein isolate (SPI) after alkaline protease digestion and transglutaminase (TGase) catalyzed polymerization is correlated with its internal molecule structure. The structure of SPI modified by alkaline protease digestion-TGase by using ultraviolet (UV) and Fourier transform infrared (FT-IR) and circular dichroism spectroscopy were characterized. The UV spectroscopy analysis showed that the cross-linking of SPI by alkaline protease digestion-TGase induced the reorganization and polymerization of small polypeptide chains, and the absorption intensity of Tyr and Trp increased, which demonstrated that the side chain structure of the SPI polypeptide chains modified by TGase was changed. The results of FT-IR showed that the absorption peaks of amide I and II segments shifted after TGase cross-linked SPI, and part of beta-sheet secondary structures was transformed into alpha-helix, indicated that the intramolecular hydrogen bonds of the modified SPI were changed. By circular dichroism spectroscopy showed that the content of random coil and alpha-helix secondary structure of SPI modified by TGase increased and the content of beta-sheet structure decreased.

Octominin: A Novel Synthetic Anticandidal Peptide Derived from Defense Protein of Octopus minor

The rapid emergence of multidrug-resistant pathogens makes an urgent need for discovering novel antimicrobial agents as alternatives to conventional antibiotics. Towards this end, we designed and synthesized a synthetic peptide of 23 amino acids (AAs) (1GWLIRGAIHAGKAIHGLIHRRRH23) from a defense protein 3 cDNA sequence of Octopus minor. The sequence of the peptide, which was named Octominin, had characteristic features of known antimicrobial peptides (AMPs) such as a positive charge (+5), high hydrophobic residue ratio (43%), and 1.86 kcal/mol of Boman index. Octominin was predicted to have an alpha-helix secondary structure. The synthesized Octominin was 2625.2 Da with 92.5% purity. The peptide showed a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 50 and 200 μg/mL, respectively, against Candida albicans. Field emission scanning electron microscopy observation confirmed that Octominin caused ultrastructural cell wall deformities in C. albicans. In addition, propidium iodide penetrated the Octominin-treated C. albicans cells, further demonstrating loss of cell membrane integrity that caused cell death at both MIC and MFC. Octominin treatment increased the production of intracellular reactive oxygen species and decreased cell viability in a concentration dependent manner. Cytotoxicity assays revealed no significant influence of Octominin on the viability of human embryonic kidney 293T cell line, with over 95% live cells in the Octominin-treated group observed up to 100 µg/mL. Moreover, we confirmed the antifungal action of Octominin in vivo using a zebrafish experimental infection model. Overall, our results demonstrate the Octominin is a lead compound for further studies, which exerts its effects by inducing cell wall damage, causing loss of cell membrane integrity, and elevating oxidative stress.

A Biomphalaria glabrata peptide that stimulates significant behaviour modifications in aquatic free-living Schistosoma mansoni miracidia.

The human disease schistosomiasis (or bilharzia) is caused by the helminth blood fluke parasite Schistosoma mansoni, which requires an intermediate host, the freshwater gastropod snail Biomphalaria glabrata (the most common intermediate host). The free-swimming parasite miracidia utilise an excellent chemosensory sense to detect and locate an appropriate host. This study investigated the biomolecules released by the snail that stimulate changes in the behaviour of the aquatic S. mansoni miracidia. To achieve this, we have performed an integrated analysis of the snail-conditioned water, through chromatography and bioassay-guided behaviour observations, followed by mass spectrometry. A single fraction containing multiple putative peptides could stimulate extreme swimming behaviour modifications (e.g. velocity, angular variation) similar to those observed in response to crude snail mucus. One peptide (P12;—R-DITSGLDPEVADD-KR—) could replicate the stimulation of miracidia behaviour changes. P12 is derived from a larger precursor protein with a signal peptide and multiple dibasic cleavage sites, which is synthesised in various tissues of the snail, including the central nervous system and foot. P12 consists of an alpha helix secondary structure as indicated by circular dichroism spectroscopy. This information will be helpful for the development of approaches to manipulate this parasites life cycle, and opens up new avenues for exploring other parasitic diseases which have an aquatic phase using methods detailed in this investigation.

Biochemical and biophysical characterization of cytokine-like protein 1 (CYTL1)

Cytokine-like protein 1 (CYTL1) is a small widely expressed secreted protein lacking significant primary sequence homology to any other known protein. CYTL1 expression appears to be highest in the hematopoietic system and in chondrocytes; however, maintenance of cartilage in mouse models of arthritis is its only reported function in vivo. Despite lacking sequence homology to chemokines, CYTL1 is predicted by computational methods to fold like a chemokine, and has been reported to function as a chemotactic agonist at the chemokine receptor CCR2 in mouse monocyte/macrophages. Nevertheless, since chemokines are defined by structure and chemokine receptors are able to bind many non-chemokine ligands, direct determination of the CYTL1 tertiary structure will ultimately be required to know whether it actually folds as a chemokine and therefore is a chemokine. Towards this goal, we have developed a method for producing functional recombinant human CYTL1 in bacteria, and we provide new evidence about the biophysical and biochemical properties of recombinant CYTL1. Circular dichroism analysis showed that, like chemokines, CYTL1has a higher content of beta-sheet than alpha-helix secondary structure. Furthermore, recombinant CYTL1 promoted calcium flux in chondrocytes. Nevertheless, unlike chemokines, CYTL1 had limited affinity to proteoglycans. Together, these properties further support cytokine-like properties for CYTL1 with some overlap with the chemokines.

Preparation of protein nanoparticle by dynamic aggregation and ionizing-induced crosslinking

Abstract Globular proteins can be considered as nanotools useful to perform a variety of chemical reactions. These biological macromolecules have very complex and detailed topological characteristics responsible for their several functions, which can be used for technological applications. Albumin is one of the few commercially available proteins with potential use in the preparation of nanomaterials by the bottom-up strategy. Albumin nanoparticles (NPs) can be prepared by several methods, but the preservation of the structure of the native protein in the final structure has not yet received too much attention. Our research group has recently reported the use of ionizing radiation to obtain albumin NPs. The irradiation of an ethanol solution of bovine serum albumin (BSA) allows obtaining protein NPs in the size range of 20–40 nm. However, a plausible mechanism to explain the process of preparation of protein-based NPs has not yet been described. In this work, we performed a series of experiments to prepare protein based NPs in two independent steps: (i) dynamic aggregation of BSA by ethanol and (ii) radiation-induced cross-linking by gamma or electron beam irradiation. Dynamic light scattering (DLS), circular dichroism (CD), Fourier Transform Infrared Spectroscopy (FT-IR) and UV spectroscopy measurements provided additional information about the conformation of BSA. No spectroscopy signal changes of aromatic amino acids were detected by UV and a loss of 20% of the alpha helix secondary structure was determined by CD. Drug-carrier functions were studied by binding and releasing assays of Merocyanine 540. BSA-NPs showed a drug-carrier behavior similar to that of BSA. Finally, we evaluated the possibility to prepare protein NPs containing more than one protein using the same procedure. Bi-protein NPs were prepared from lysozyme and BSA. The bi-protein NP showed enzymatic activity of lysozyme, which confirms the functionality of the NP prepared by this novel method.

Impact of cysteine variants on the structure, activity, and stability of recombinant human α‐galactosidase A

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.

Effect of pressure on secondary structure of proteins under ultra high pressure liquid chromatographic conditions

There are several spectroscopic techniques such as IR and CD, that allow for analyzing protein secondary structure in solution. However, a majority of these techniques require using purified protein, concentrated enough in the solution, to produce a relevant spectrum. Fundamental principles for the usage of reversed-phase ultra high pressure liquid chromatography (UHPLC) as an alternative technique to study protein secondary structures in solution were investigated. Several “model” proteins, as well as several small ionizable and neutral molecules, were used for these studies. The studies were conducted with UHPLC in isocratic mode, using premixed mobile phases at constant flow rate and temperature. The pressure was modified by a backpressure regulator from about 6000psi to about 12,000psi. It was found that when using a mobile phase composition at which proteins were fully denatured (loss of alpha-helix secondary structure), the retention factors of the proteins increased upon pressure increase in the same manner as non-proteins. When using a mobile phase composition in which proteins were not fully denatured, it was observed that the retention factors of the proteins displayed a much steeper (by one order of magnitude) increase in retention upon pressure increase. It was concluded that in a mobile phase in which the protein is not initially fully denatured, the increase of pressure may facilitate the folding back of the protein to its native state (alpha-helix secondary structure). The impact of different mobile phase compositions on the denaturation of the proteins was studied using CD (Circular Dichroism). Moreover, the effect of flow rate on retention of proteins and small molecules was studied at constant pressure on the different pore size silicas and the impact of internal frictional heating was evaluated.

Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences

Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.

The Carboxyl-Terminal Segment of Apolipoprotein A-V Undergoes a Lipid-Induced Conformational Change

Apolipoprotein (apo) A-V is a 343-residue, multidomain protein that plays an important role in regulation of plasma triglyceride homeostasis. Primary sequence analysis revealed a unique tetraproline sequence (Pro293-Pro296) near the carboxyl terminus of the protein. A peptide corresponding to the 48-residue segment beyond the tetraproline motif was generated from a recombinant apoA-V precursor wherein Pro295 was replaced by Met. Cyanogen bromide cleavage of the precursor protein, followed by negative affinity chromatography, yielded a purified peptide. Nondenaturing polyacrylamide gel electrophoresis verified that apoA-V(296-343) solubilizes phospholipid vesicles, forming a relatively heterogeneous population of reconstituted high-density lipoprotein with Stokes' diameters >17 nm. At the same time, apoA-V(296-343) failed to bind a spherical lipoprotein substrate in vitro. Far-UV circular dichroism spectroscopy revealed the peptide is unstructured in buffer yet adopts significant alpha-helical secondary structure in the presence of the lipid mimetic solvent trifluoroethanol (TFE; 50% v/v). Heteronuclear multidemensional NMR spectroscopy experiments were conducted with uniformly (15)N- and (15)N/(13)C-labeled peptide in 50% TFE. Peptide backbone assignment and secondary structure prediction using TALOS+ reveal the peptide adopts alpha-helix secondary structure from residues 309 to 334. In TFE, apoA-V(296-343) adopts an extended amphipathic alpha-helix, consistent with a role in lipoprotein binding as a component of full-length apoA-V.

Apolipoprotein A-V N-terminal Domain Lipid Interaction Properties in Vitro Explain the Hypertriglyceridemic Phenotype Associated with Natural Truncation Mutants

The N-terminal 146 residues of apolipoprotein (apo) A-V adopt a helix bundle conformation in the absence of lipid. Because similarly sized truncation mutants in human subjects correlate with severe hypertriglyceridemia, the lipid binding properties of apoA-V(1-146) were studied. Upon incubation with phospholipid in vitro, apoA-V(1-146) forms reconstituted high density lipoproteins 15-17 nm in diameter. Far UV circular dichroism spectroscopy analyses of lipid-bound apoA-V(1-146) yielded an alpha-helix secondary structure content of 60%. Fourier transformed infrared spectroscopy analysis revealed that apoA-V(1-146) alpha-helix segments align perpendicular with respect to particle phospholipid fatty acyl chains. Fluorescence spectroscopy of single Trp variant apoA-V(1-146) indicates that lipid interaction is accompanied by a conformational change. The data are consistent with a model wherein apoA-V(1-146) alpha-helices circumscribe the perimeter of a disk-shaped bilayer. The ability of apoA-V(1-146) to solubilize dimyristoylphosphatidylcholine vesicles at a rate faster than full-length apoA-V suggests that N- and C-terminal interactions in the full-length protein modulate its lipid binding properties. Preferential association of apoA-V(1-146) with murine plasma HDL, but not with VLDL, suggests that particle size is a determinant of its lipoprotein binding specificity. It may be concluded that defective lipoprotein binding of truncated apoA-V contributes to the hypertriglyceridemia phenotype associated with truncation mutations in human subjects.

Characterization of the In Situ Structural and Interfacial Properties of the Cationic Hydrophobic Heteropolypeptide, KL4, in Lung Surfactant Bilayer and Monolayer Models at the Air−Water Interface: Implications for Pulmonary Surfactant Delivery

This study examines the various equilibrium in situ secondary structures of the pharmaceutical heteropolypeptide, KL 4, in the solid state, in solution, and in the monolayer state alone and mixed with dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylglycerol (POPG). In situ surface circular dichroism spectroscopy, using a method first reported by Damodaran (Damodaran, S. Anal. Bioanal. Chem. 2003, 376, 182-188), of equilibrated KL 4, DPPC/KL 4, POPG/KL 4, and DPPC/POPG/KL 4 monolayers at the air-water interface was used to examine the in situ two-dimensional conformation of KL 4. Gravimetric vapor sorption by solid KL 4 was used to analyze the effects of water molecules on the conformation of KL 4 when confined as a monolayer at the surface of water. Solid-state KL 4 conformation was determined by X-ray powder diffraction (XRPD). The equilibrium interfacial and spreading properties were measured at 25 degrees C, 37 degrees C, and 45 degrees C using the Wilhelmy plate method and Langmuir film balance. Equilibrium phase transition temperatures were measured using differential scanning calorimetry (DSC). It was found that solid-state KL 4, which takes up very little water, exhibits beta-sheet and alpha-helix secondary structures, whereas KL 4 in solution appears to exist only as an alpha-helix. KL 4 forms a stable, insoluble monolayer, exhibiting beta-sheet and aperiodic structures. These structures provide KL 4, when confined in two-dimensions, the structural flexibility to maximize favorable cationic lysine-water interactions and favorable leucine-leucine hydrophobic and van der Waals interactions; while effectively "shielding" the leucine residues away from water. In DPPC/KL 4 monolayers, KL 4 retains its native beta-sheet and aperiodic structures, consistent with phase separation of DPPC and KL 4 in bilayers and monolayers. In POPG/KL 4 monolayers, KL 4 exhibits an increase in aperiodic secondary structures (loss of beta-sheet) to maximize favorable electrostatic interactions, consistent with the observed negative deviations from ideal monolayer mixing.

“Schizophrenic” Micellization Associated with Coil-to-Helix Transitions Based on Polypeptide Hybrid Double Hydrophilic Rod–Coil Diblock Copolymer

A polypeptide hybrid double hydrophilic diblock copolymer (DHBC), poly( N-isopropylacrylamide)- b-poly( l-glutamic acid) (PNIPAM- b-PLGA), was synthesized via the ring-opening polymerization of gamma-benzyl- l-glutamate N-carboxyanhydride (BLG-NCA) using monoamino-terminated PNIPAM as the macroinitiator, followed by deprotection of benzyl groups under alkaline conditions. Containing a thermoresponsive PNIPAM block and a pH-responsive PLGA block, the obtained polypeptide hybrid diblock copolymer molecularly dissolves in aqueous solution at alkaline pH and room temperature but supramolecularly self-assembles into PNIPAM-core micelles at alkaline pH and elevated temperatures and PLGA-core micelles at acidic pH and room temperature accompanied with coil-to-helix transition of the PLGA sequence. The pH- and thermoresponsive "schizophrenic" micellization behavior of PNIPAM- b-PLGA diblock copolymer has been investigated by (1)H NMR, optical transmittance, fluorescence probe measurement, transmission electron microscopy (TEM), dynamic and static laser light scattering (LLS), and circular dichroism (CD) spectroscopy. Moreover, the micellization process was investigated employing stopped-flow light scattering technique. The pH-induced micelle growth of PNIPAM- b-PLGA in aqueous solution exhibits drastically different kinetics compared to that of conventional pH-responsive DHBCs, probably due to the stabilization effects exerted by the formed alpha-helix secondary structures within the PLGA core at low pH. Exhibiting "schizophrenic" micellization, the polypeptide sequence of PNIPAM- b-PLGA can either locate within micelle cores or stabilizing coronas. The incorporation of polypeptide block into DHBCs can endow them with structural versatility, tunable spatial arrangement of chain segments within self-assembled nanostructures, and broader applications in the field of biomedicines.

The Lysozyme from Insect (Manduca sexta) Is a Cold-Adapted Enzyme

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of alpha-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 degrees C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.

Fingerprinting fission yeast: polymorphic markers for molecular genetic analysis of Schizosaccharomyces pombe strains

The fission yeast Schizosaccharomyces pombe is widely used as a model eukaryote for cell and molecular studies but little is known of natural genetic variation in this species. In order to obtain informative molecular markers, imperfect tandem repeats, identified through bioinformatic methods, were tested for length polymorphism in six wild-type strains of Sch. pombe isolated from different substrates and geographical locations in Africa, America, Asia and Europe. Of 26 loci tested, 21 were multi-allelic, consistent with tandem repeat copy number variation. Eleven of these polymorphic tandem repeats are in regions encoding intracellular proteins. Most of the protein-coding repeats are not sited within structured domains but have non-regular predicted structure; one has a repeat unit length corresponding to integer turns of a predicted amphipathic alpha-helix secondary structure, suggesting that this repeat may be tolerated because copy number mutations change alpha-helix length but not orientation within the protein structure. In contrast to the differences observed between natural isolates of Sch. pombe, genetic strains were found to be essentially isogenic: only two polymorphic loci were detected out of 26 minisatellites and five microsatellites tested in 16 strains, including a hypervariable microsatellite in the med15 gene. The polymorphic tandem repeat markers identified in this study will prove useful for DNA fingerprinting and molecular analysis of natural genetic variation in Sch. pombe isolates.

Interplay of Secondary Structure and Charge on the Diffusion of a Polypeptide through Negatively Charged Aqueous Pores

This study was conducted to investigate the interplay of secondary structure and charge of a polypeptide on its permeability through negatively charged pores of synthetic porous membranes and Caco-2 cell monolayers. Poly(D-glutamic acid) [Poly(D-Glu)] was used as a model polypeptide. Transport studies were conducted at 37 degrees C through both track-etched polycarbonate membranes (using side-by-side diffusion cells) and Caco-2 cell monolayers. Apparent permeability coefficients and diffusion coefficients were calculated. When diffusion was unhindered, poly(D-Glu) appeared to be transported at the same rate regardless of whether it existed in the random coil or the alpha-helix secondary structure. When moderately hindered diffusion was evaluated, poly(D-Glu) with partial alpha-helix secondary structure, exhibited significantly greater transport than when the polypeptide predominantly existed as the highly negatively charged random coil. This trend was reversed when the diffusion was severely hindered by the tight junctions of the Caco-2 cell monolayers. Neither charge, nor secondary structure, played a significant role in the unhindered diffusion of poly(D-Glu). When the molecules were moderately hindered, polypeptide/membrane charge interactions significantly influenced the rate of aqueous diffusion. As the overall molecular dimensions of the polypeptide approached the pore size, the inherent molecular flexibility of the random coil secondary structure overcame the effect of charge repulsion.

Pro‐apoptotic bax‐α1 synthesis and evidence for β‐sheet to α‐helix conformational change as triggered by negatively charged lipid membranes

Solid phase synthesis of Bax-alpha1, the 25 amino acids domain (14TSSEQIMKTGALLLQGFIQDRAGRM38) of the pro-apoptotic Bax protein has been accomplished using Fmoc chemistry. A new fast and harmless protocol is described for complete TFA removal from the purified peptide powder leading to a final purity greater than 98% as controlled by 19F-NMR, UV and MALDI-TOF mass spectrometry. Secondary structure was determined in various solution and membrane media using UV Circular Dichroism. In water solution, Bax-alpha1 is present as a mixture of beta-sheet and unstructured (random coil) conformations. A marked change from beta-sheet to alpha-helix secondary structures is observed upon interaction with negatively charged phospholipids vesicles whereas neutral lipid membranes have no significant effect on the aqueous peptide conformation. Results are discussed in terms of Bax binding to mitochondrial membranes.

Combined N- and C-Terminal Truncation of Human Apolipoprotein A-I Yields a Folded, Functional Central Domain

A combined N- and C-terminal truncation variant of human apolipoprotein A-I (apoA-I) was designed, expressed in Escherichia coli, isolated, and characterized. Hydrodynamic experiments yielded a weight average molecular weight of 34000, indicating apoA-I-(44-186) exists in solution predominantly as a dimer. An axial ratio of 4.2 was calculated for the dimer based on sedimentation velocity experiments. Far-UV circular dichroism spectroscopy of apoA-I-(44-186) in buffer indicated the presence of 65% alpha-helix secondary structure. Guanidine hydrochloride denaturation experiments yielded a transition midpoint of 0.5 M for apoA-I-(44-186). ApoA-I-(44-186) induced solubilization of dimyristoylphosphatidylcholine vesicles at a rate comparable to that of full-length apoA-I, displayed lipoprotein binding ability, and was an acceptor of ABCA1-mediated cholesterol efflux from cultured macrophages. Fluorescence quenching studies with KI indicate that the three Trp residues in apoA-I-(44-186) are shielded from the aqueous environment. Taken together, the data indicate that lipid-free apoA-I-(44-186) adopts a folded conformation in solution that possesses lipid binding capability. The central region of apoA-I appears to adopt a globular amphipathic alpha-helix bundle organization that is stabilized by intramolecular and/or intermolecular helix-helix interactions. Lipid association likely results in a conformational adaptation wherein helix-helix contacts are substituted for helix-lipid interactions.

A Randomly Coiled, High-Molecular-Weight Polypeptide Exhibits Increased Paracellular Diffusion in Vitro and in Situ Relative to the Highly Ordered ?-Helix Conformer

The current investigation was conducted to examine the effect of secondary structure of model polypeptides on their hindered paracellular diffusion. Poly-D-glutamic acid (PDGlu) was selected as one of the model polypeptides because of its ability to form two secondary structures; a negatively charged random coil and an alpha-helix with partial negative charge at pH 7.4 and 4.7, respectively. Poly-D-lysine (PDL) was selected as a positively charged random coil conformation at pH 7.4. Transport experiments were conducted across both a Caco-2 cell monolayer and the intestinal membrane of Sprague-Dawley rats. Additionally, using NMR, an estimation for the diffusion coefficient and the equivalent hydrodynamic radius for each model polypeptide was obtained. PDGlu in the randomly coiled conformation exhibited greater paracellular transport when compared to either the same polypeptide having an alpha-helix secondary structure or the positively charged, randomly coiled PDL. Randomly coiled PDGlu was able to permeate through the negatively charged tight junctions of both biological membranes to a greater extent than PDGlu having an alpha-helix structure and suggests that molecular flexibility associated with the random coil conformation may play a more important role than overall charge and hydrodynamic radius on its hindered paracellular diffusion.