Trichotoxin A-40, a new membrane-exciting peptide. Part B. Voltage-dependent pore formation in bilayer lipid membranes and comparison with other alamethicin analogues

Trichotoxin A-40 induces voltage-dependent pores in bilayer lipid membranes comparable to those formed by alamethicin and suzukacillin. The conductance values of the trichotoxin A-40 pores are of similar magnitude and show the same characteristic pattern sequence of non-integral multiples of a unit-conductance step as alamethicin and suzukacillin.However, voltage-jump current-relaxation experiments show significant differences between trichotoxin A-40 and alamethicin and suzukacillin. With trichotoxin A-40 three different relaxation processes could be observed, whereas with alamethicin and suzukacillin only two processes had been distinguished. The fast process in each case is related to pore state transitions and the slower (medium) process to the decay rate of pores. The third very slow process, which is not found with alamethicin and suzukacillin, could not clearly be assigned to a molecular mechanism. Whereas in the case of alamethicin and suzukacillin the relaxation amplitude of the slow process is considerably larger than the relaxation amplitude of the fast process, the reverse is true for trichotoxin A-40, where the largest relaxation amplitude is that of the fast process.Contrary to alamethicin and suzukacillin, trichotoxin A-40 is soluble in the lipid/decane membrane-forming solution, when added from an ethanolic stock solution. Its bilayer-modifying properties are not changed, whether the antibiotic is added to the aqueous salt solution or to the membrane-forming solution.Several different analogues of alamethicin, suzukacillin and trichotoxin A-40 have been investigated and compared with respect to the induced current-voltage characteristics in lipid bilayers. A suzukacillin A-derivative where phenylalaninol had been split off is active as well as trichotoxin A-40 which lacks the phenylalaninol group by nature. Different C-terminal groups like -COOH, -CONH₂, -COOCH₃ and -CO-Ala-Ala-OCH₃ cause qualitative changes but not the loss of the pore-formation property.     

Orientation and helical conformation of a tissue-specific hunter-killer peptide in micelles

Hunter-killer peptides are chimeric synthetic peptides that selectively target specific cell types for an apoptotic death. These peptides, which are models for potential therapeutics, contain a homing sequence for receptor-mediated interactions and a pro-apoptotic sequence. Homing domains have been designed to target angiogenic tumor cells, prostate cells, arthritic tissue and, most recently, adipose tissue. After a receptor-mediated internalization, the apoptotic sequence, which contains D-enantiomer amino acids, initiates apoptosis through mitochondrial membrane disruption. We have begun structure and functional studies on a peptide (HKP1) that specifically targets angiogenic tumor cells for apoptosis. As a model for mitochondrial membrane disruption, we have examined peptide-induced leakage of a calcein fluorophore from large unilamellar vesicles. These experiments demonstrate more potent leakage activity by HKP1 than the peptide lacking the homing domain. Circular dichroism and 2D homonuclear NMR experiments demonstrate that this tumor-specific HKP adopts a left-handed amphipathic helix in association with dodecylphosphorylcholine micelles in a parallel orientation to the lipid-water interface with the homing domain remaining exposed to solvent. The amphipathic helix of the apoptotic domain orients with nonpolar leucine and alanine residues inserting most deeply into the lipid environment.     

Isolation and primary structure of human PHI (peptide HI)

The isolation of the human form of PHI (peptide HI) is described. The peptide was purified from human colonic extracts by using a chemical method for the detection of its C-terminal amidated structure. Human PHI consists of 27 amino acid residues and the complete amino acid sequence is: His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Phe-Ser-Lys-Leu-Leu-Gly-Gln-Leu-Ser- Ala-Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Met-NH2. The differences between the structures of porcine and human PHI are at position 12 (Arg/Lys replacement) and at position 27 (Ile/Met).     

Corynebacterium sarcosine oxidase: a unique enzyme having covalently-bound and noncovalently-bound flavins

A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC, 1.5.3.1) was purified to homogeneity from Corynebacterium sp. U-96 by the use of ionic exchange chromatographies and gel filtrations. The enzyme contained two mol FAD per mol enzyme (one covalently-bound and one noncovalently-bound; mol. wt., 174,000). The “semiapoenzyme”, which contains the covalently-bound FAD alone, was prepared by the acid-ammonium sulfate treatment. The semiapoenzyme had practically no activity for sarcosine oxidation, but retained intact back-bone structure judging from the circular dichroic spectrum in the far ultraviolet region. On the contrary, the circular dichroic spectrum of the semiapoenzyme in the visible region (a large negative band around 443 nm) was quite distinct from that of the holoenzyme (positive bands at 387, 456 and 489 nm).     

FtsZ polymer-bundling by the Escherichia coli ZapA orthologue, YgfE, involves a conformational change in bound GTP

Cell division is a fundamental process for both eukaryotic and prokaryotic cells. In bacteria, cell division is driven by a dynamic, ring-shaped, cytoskeletal element (the Z-ring) made up of polymers of the tubulin-like protein FtsZ. It is thought that lateral associations between FtsZ polymers are important for function of the Z-ring in vivo, and that these interactions are regulated by accessory cell division proteins such as ZipA, EzrA and ZapA. We demonstrate that the putative Escherichia coli ZapA orthologue, YgfE, exists in a dimer/tetramer equilibrium in solution, binds to FtsZ polymers, strongly promotes FtsZ polymer bundling and is a potent inhibitor of the FtsZ GTPase activity. We use linear dichroism, a technique that allows structure analysis of molecules within linear polymers, to reveal a specific conformational change in GTP bound to FtsZ polymers, upon bundling by YgfE. We show that the consequences of FtsZ polymer bundling by YgfE and divalent cations are very similar in terms of GTPase activity, bundle morphology and GTP orientation and therefore propose that this conformational change in bound GTP reveals a general mechanism of FtsZ bundling.     

Circular dichroism studies on the binding of type I substrates and reverse type I compounds to rabbit liver microsomal cytochrome P-450.

Liver microsomes from control, 3-methylcholanthrene-treated, and phenobarbital-treated New Zealand White rabbits were examined for differences detectable by circular dichroism (CD) spectroscopy. Addition of the Type I substrate cyclohexane to phenobarbital microsomes decreases the negative ellipticity at about 418 nm and concomitantly increases the negative ellipticity at about 395 nm. Cyclohexane added to microsomes from control or 3-methylcholanthrene-treated animals shows little or no CD changes in these wavelength regions. The effect by cyclohexane is completely reversed by the subsequent addition of butanol-1. Addition of benzo[a]pyrene to phenobarbital microsomes also decreases the negative ellipticity at about 418 nm, and this effect can be completely reversed with the subsequent addition of butanol-1. The ellipticity at about 395 nm is reversed in sign and is markedly increased by benzo[a]pyrene, however, and this effect is not changed with the subsequent addition of butanol-1. Restoring the cyclohexane- or benzo[a]pyrene-induced changes by the subsequent addition of alcohol is proportional to the aliphatic chain length, with 4 or more carbon atoms being maximally effective. Primary alcohols inhibit aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2) activity of phenobarbital microsomes, and the inhibitory effect is enhanced with increasing chain length of the alcohols; 4 or more carbon atoms being maximally effective. Stimulation of monooxygenase metabolism of cyclohexane or benzo[a]pyrene by NADPH results in restoration of the negative ellipticity band at about 418 nm, whereas the ellipticity peak at about 395 nm remains unchanged. More negative ellipticity at about 210 and 222 nm is found in phenobarbital microsomes than in control or 3-methylcholanthrene microsomes and cyclohexane addition in vitro increases these negative ellipticity peaks in phenobarbital microsomes but not in control or 3-methylcholanthrene microsomes.These results show that with CD studies one can detect directly both high spin (negative ellipticity peak at 385–395 nm) and low spin (negative ellipticity peak at about 418 nm) P-450 iron in liver microsomes from control, 3-methylcholanthrene-treated, or phenobarbital-treated rabbits. These data are consistent with a weak ligand such as oxygen, rather than a stronger ligand such as nitrogen, in the sixth position of 6-coordinated (low spin) ferric iron in P-450 in vivo. Type I substrates such as cyclohexane or benzo[a]pyrene, when bound to P-450, change low spin P-450 iron to the high spin state. Cyclohexane-bound high spin P-450 iron in vitro is more easily converted to low spin iron by butanol-1 than is benzo[a]pyrene-bound high spin P-450 iron. Liver microsomal proteins from phenobarbital-treated rabbits have a higher helical content than those from either control or 3-methylcholanthrene-treated rabbits. Cyclohexane addition in vitro increases this helical character only in phenobarbital microsomes, indicating that one or more forms of phenobarbital-induced P-450 apoproteins is (are) more specific for cyclohexane binding and metabolism than control or 3-methylcholanthrene-induced forms of P-450.     

Bacteriorhodopsin vesicles. An outline of the requirements for light-dependent H+ pumping

A systematic study was performed to determine under which conditions bacteriorhodopsin can be applied as an energy generator in reconstituted systems. It is concluded that reconstitution of an active light-driven proton pump is possible over a wide range of conditions.High extents (per bacteriorhodopsin molecule) of proton uptake by reconstituted vesicles are found at a high lipid to protein ratio, after long sonication and at high pH. No active proton pump is obtained if reconstitution is attempted at high pH with neutral phospholipids or at low ionic strength with negatively charged lipids. Attention was also paid to the requirement of a crystalline array for active pumping; most likely, monomeric bacteriorhodopsin molecules can effectively pump protons.     

Functional recombination of dynein 1 with demembranated sea urchin sperm partially extracted with KC1.

Demembranated sea urchin sperm were extracted with 0.5 M KCl as described earlier and reactivated in a solution containing 1 mM ATP. Their flagellar beat frequency was approximately 13 Hz, while that of standard reactivated sperm which had not been extracted with KCl was approximately 31 Hz at 23°C. Addition of soluble dynein 1 caused a gradual increase in the flagellar beat frequency to approximately 25 Hz after 10 min at room temperature. This restoration of frequency occurred in the absence or presence of ATP. Examination by electron microscopy showed that, whereas KCl-extracted sperm were lacking the majority of the outer arms on the doublet tubules, they had regained most of their outer arms following incubation with soluble dynein 1.     

Optical studies of a conformational change in DNA before melting

The circular dichroism of double-stranded DNA is temperature dependent prior to its melting. As the temperature is increased the spectrum becomes more nonconservative. This is certainly due to a conformational change within the framework of the double helix. To ascertain the nature of the conformational change, a series of synthetic and natural DNA's from a variety of sources was investigated. The same qualitative changes were seen for all the DNA samples, independent of base composition. However, there were definite quantitative differences, with poly [d(A-T)] manifesting the largest effect. Oligomers of the form [d(A-T)]_n with n = 10 to 21 behaved in a manner similar to the polymer. There is no observed chain-length dependence. The breadth of the pre-melt transition indicates a low ΔH (less than 5 kcal./mole); the lack of dependence on chain length indicates that the co-operative unit is smaller than eight base pairs.     

Identification and biochemical characterization of Asp t 36, a new fungal allergen from Aspergillus terreus

Aspergillus terreus is an allergenic fungus, in addition to causing infections in both humans and plants. However, the allergens in this fungus are still unknown, limiting the development of diagnostic and therapeutic strategies. We used a proteomic approach to search for allergens, identifying 16 allergens based on two-dimensional immunoblotting with A. terreus susceptible patient sera. We further characterized triose-phosphate isomerase (Asp t 36), one of the dominant IgE (IgE)-reactive proteins. The gene was cloned and expressed in Escherichia coli. Phylogenetic analysis showed Asp t 36 to be highly conserved with close similarity to the triose-phosphate isomerase protein sequence from Dermatophagoides farinae, an allergenic dust mite. We identified four immunodominant epitopes using synthetic peptides, and mapped them on a homology-based model of the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure, rendering it an IgE-binding hotspot. Biophysical analysis indicated that Asp t 36 shows similar secondary structure content and temperature sensitivity with other reported triose-phosphate isomerase allergens. In vivo studies using a murine model displayed that the recombinant Asp t 36 was able to stimulate airway inflammation, as demonstrated by an influx of eosinophils, goblet cell hyperplasia, elevated serum Igs, and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus, and define a new fungal allergen more broadly. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.     

An ochre suppressor of bacteriophage T4 that is associated with a transfer RNA

Ultraviolet circular dichroism spectra have been obtained for native and heat-denatured Drosophila virilis satellite DNAs I, II and III. Gall &; Atherton (1974) have found that these DNAs have simple, unique sequences. We compare here the circular dichroism spectra of these satellite sequences with the circular dichroism spectra of synthetic DNAs of simple sequences which are combined in first-neighbor calculations. We also apply an analytical procedure for determining nearest-neighbor frequencies from the DNA spectra (Allen et al., 1972). The results are an indication of the potential usefulness and present limitations of circular dichroism measurements in confirming or determining the nearestneighbor frequencies of satellite DNAs of simple sequences.     

Dissecting a role of a charge and conformation of Tat2 peptide in allosteric regulation of 20S proteasome

Proteasome is a ‘proteolytic factory’ that constitutes an essential part of the ubiquitin‐proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiology makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti‐cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV‐1 Tat protein: R⁴⁹KKRRQRR⁵⁶, supplemented with Q⁶⁶DPI⁶⁹ fragment, inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biological activity measurements and FTIR and CD spectroscopy to dissect contribution of a charge and conformation of Tat2 to its capability to influence peptidase activity of the proteasome. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their capability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their positive charge. The data provide valuable clues for the future optimization of the Tat2‐based proteasome regulators. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Influence of Substrate Conformation on the Deglycosylation of Ribonuclease B by Recombinant Yeast Peptide：N-glycanase

Peptide:N-glycanase has been thought to be responsible for proteasome-dependent degradationof misfolded glycoproteins translocated from the endoplasmic reticulum (ER) to the cytosol.Therefore,theenzyme was supposed to be able to distinguish between native and non-native glycoproteins.In the presentstudy,a recombinant,yeast peptide:N-glycanase,Png lp, was expressed in Escherichia coli as inclusionbodies and was purified,refolded and characterized.The results showed that the recombinant enzymehas a broad pH range adaptation,from pH 4.0 to pH 10.0,and has an optimum temperature of 30 ℃.This enzyme is a zinc metalloenzyme.Its activity was abolished with the addition of EDTA and notrestored by adding metal ions.Furthermore,the deglycosylation efficiency of recombinant Pnglpfrom E.coli was investigated with respect to the substrate conformation in vitro.When ribonuclease B(RNase B) was denatured at 60-65 ℃ or by 40-60 mM dithiothreitol, indicated by its obvious structuralchange and sharpest activity change,its deglycosylation by Pnglp was most prominent.The deglycosylationefficiency of RNase B by Pnglp was found to be related to its structural conformation and enzymaticactivity.     

Studies of human transcortin at different pHs: circular dichroism, polymerisation and binding affinity.

The transcortin we have used in this work is extremely pure. This was shown by the polymerisation observed at pH 4. This polymerisation is never observed with an impure form of transcortin [4]. Moreover, since it is known that the presence of cortisol in the binding site is an essential condition to the activity of purified transcortin [5], it appears that a correlation between the secondary structure and the biological activity of the transcortin exists. The results we have obtained are summarized below: (1) The inhibition of the transcortin binding capacity essentially takes place between pH 5 and 4. (2) A reorganisation of the structure of the protein moiety is observed between pH 6.5 and 5.9. (3) A decrease of the helicity ratio is observed between pH 5 and 4. It appears therefore that, in the limits of experimental accuracy of CD measurements to determine the amount of beta-structure, no appreciable change of binding activity is taking place after the appearance of a large percentage of beta-structure between pH 6.5 and 6. On the other hand, the sudden decrease of protein activity at low pH is likely to be correlated with the disappearance of a well-defined helical region. Other biochemical and physical experiments would be of course necessary, in order to precise this first observation of a structure-function relationship in transcortin.     

Conformational studies of alanine-rich peptide using CD and FTIR spectroscopy.

The circular dichroism (CD) and Fourier transform infrared (FTIR) methods were applied to the conformational studies of alanine-rich peptide Ac-K-[A]11-KGGY-NH2 (where K is lysine, A is alanine, G is glycine and Y is tyrozyne) in water, methanol (MeOH) and trifluoroethanol (TFE). The analysis of CD-spectra of the peptide in water at different concentrations revealed that the secondary structure content depends on the peptide concentration and pH of the solution. The increase of the peptide concentration causes a decrease of alpha-helix content and, simultaneously, an increase of beta-sheet structure, while the unordered structure is the predominant one. Additional elements are discovered in MeOH and TFE but alpha-helix and beta-turns predominate. Moreover, in these solutions the percentage content of the secondary structure does not depend on the temperature. FTIR measurements, carried out at higher peptide concentration (about one order of magnitude) than these CD measurements mentioned above, revealed that in water solution the solid state beta-sheet, and aggregated structures, dominate. However, in TFE the most abundant are alpha-helix and beta-turns structures. The thioflavine T assay showed the tendency of the studied peptide for aggregate.     

Solubilisation of ferricytochrome c in methanol using a crown ether: absorption, circular dichroism and EPR spectral properties

Ferricytochrome c is normally insoluble in methanol, but its solution is facilitated by complexation with 18-crown-6. Absorption, circular dichroism and EPR spectroscopy indicate that the solubilised protein in MeOH exists in at least three conformational states, all different from the native state in neutral aqueous solution. In two states the haem iron (III) is low spin and in one state it is high spin, but it seems likely that all three forms are globular. The proportion in the high spin form increases at increasing crown ether concentration and on ageing the protein solution. The protein appears to return to its native conformation when it is restored to an aqueous environment.     

Determination of the absolute configuration and solution conformation of a novel disubstituted pyrrolidine acid A by vibrational circular dichroism

Compound A, a novel disubstituted pyrrolidine acid, is a member of a new class of agents that are potentially useful for the treatment of diabetes and dyslipidemia. The absolute configuration of this compound was determined by using vibrational circular dichroism (VCD). The results are in agreement with the assignments based on both X-ray analysis and the stereo-selective chemical synthesis. During VCD analysis, the solution conformation for a portion of compound A in CDCl(3) was also established. The compound is found to associate as an H-bonded carboxylic acid "dimer" in CDCl(3) solution, and VCD calculations on a model dimer fragment were required to establish the absolute configuration.