Modification of carboxyl groups converts α-lactalbumin into an active molten globule state with membrane-perturbing activity and cytotoxicity.

We investigated whether the modification of the negatively charged carboxyl groups with semicarbazide could confer membrane-disrupting and cytotoxic properties to bovine α-lactalbumin (LA). MALDI-TOF analysis revealed that eighteen of the twenty-one carboxyl groups in LA were coupled with semicarbazide molecules. Measurement of circular dichroism spectra and Trp fluorescence quenching studies showed that semicarbazide-modified LA (SEM-LA) had a molten globule-like conformation that retained the α-helix secondary structure but lost the tertiary structure of LA. Compared to LA, SEM-LA had a higher structural flexibility in response to trifluoroethanol- and temperature-induced structural transitions. In sharp contrast to LA, SEM-LA exhibited membrane-damaging activity and cytotoxicity. Furthermore, SEM-LA-induced membrane permeability promoted the uptake of daunorubicin and thereby its cytotoxicity. The microenvironment surrounding the Trp residues of SEM-LA was enriched in positive charges, as revealed by iodide quenching studies. The binding of SEM-LA with lipid vesicles altered the positively charged cluster around Trp residues. Although LA and SEM-LA displayed similar lipid-binding affinities, the membrane interaction modes of SEM-LA and LA differed. Collectively, these results suggest that blocking of negatively charged residues enables the formation of a molten-globule conformation of LA with structural flexibility and increased positive charge, thereby generating functional LA with membrane-disrupting activity and cytotoxicity.

Inhibiting Human Calcitonin Fibril Formation with Its Most Relevant Aggregation-Resistant Analog.

The most common obstacles to the development of therapeutic polypeptides are peptide stability and aggregation. Human calcitonin (hCT) is a 32-residue hormone polypeptide secreted from the C-cells of the thyroid gland and is responsible for calcium and phosphate regulation in the blood. hCT reduces calcium levels by inhibiting the activity of osteoclasts, which are bone cells that are mainly responsible for breaking down the bone tissue or decreasing the resorption of calcium from the kidneys. Thus, calcitonin injection has been used to treat osteoporosis and Paget's disease of bone. hCT is an aggregation-prone peptide with a high tendency to form amyloid fibrils. As a result, salmon calcitonin (sCT), which is different from hCT at 16-residue positions and has a lower propensity to aggregate, has been chosen as a clinical substitute for hCT. However, significant side effects, including immune reactions, have been shown with the use of sCT injection. In this study, we found that two residues, Tyr-12 and Asn-17, play key roles in inducing the fibrillization of hCT. Double mutation of hCT at these two crucial sites could greatly enhance its resistance to aggregation and provide a peptide-based inhibitor to prevent amyloid formation by hCT. Double-mutated hCT retains its ability to interact with its receptor in vivo. These findings suggest that this variant of hCT would serve as a valuable therapeutic alternative to sCT.

Interfacial properties and structural analysis of the antimicrobial peptide NK-2.

The structure of the antimicrobial peptide NK-2 has been studied at the air-water interface and in different solutions using spectroscopic methods such as circular dichroism (CD) and infrared reflection absorption spectroscopy (IRRAS) as well as specular X-ray reflectivity (XR). NK-2 adopts an unordered structure in water, buffer, and in the presence of monomeric cationic and noncharged amphiphiles. However, it forms a stable alpha-helix in 2,2,2-trifluoroethanol (TFE) and in micellar solutions of anionic, cationic as well as nonionic amphiphiles, whereas only in sodium dodecyl sulfonate solutions the alpha-helical structure can also be found below the critical micellar concentration (cmc). The amphiphilic molecule NK-2 is surface active and forms a Gibbs monolayer at the air-buffer interface. In contrast, no adsorption was observed if NK-2 is dissolved in water. During the adsorption process in buffer solutions, NK-2 undergoes a conformational transition from random coil in bulk to alpha-helix at the interface. This change of the peptide's secondary structure is known to be associated with its antimicrobial activity. A comparison of the experimental IRRA spectra with the simulated spectra indicates that the adsorbed NK-2 alpha-helix lies flat at the interface. This is confirmed by XR measurements which show that the thickness of the NK-2 layer is approximately 17 A, which is the average diameter of a alpha-helix, indicating that only a monomolecular adsorption layer is formed.

Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology.

As shown before, human stefin B (cystatin B) populates two partly unfolded species, a native-like state at pH 4.8 and a structured molten globule state at pH 3.3 (high ionic strength), from each of which amyloid fibrils grow. Here, we show that the fibrils obtained at pH 3.3 differ from those at pH 4.8 and that those obtained at pH 3.3 (protofibrils) do not transform readily to mature fibrils. In addition we show that amorphous aggregates are also a source of fibrils. The kinetics of amyloid fibril formation at different trifluoroethanol (TFE) concentrations were measured. TFE accelerates fibril growth at predenaturational concentrations of the alcohol. At concentrations higher than 10%, the fibrillar yield decreases proportionately as the population of an all alpha-helical, denatured form of the protein increases. At an optimum TFE concentration, the lag and the growth phases are observed, similarly to some other amyloidogenic proteins. Morphology of the protein species at the beginning and the end of the reactions was observed using atomic force microscopy and transmission electron microscopy. Final fibril morphologies differ depending on solvent conditions.

Characterization of the unique function of a reduced amide bond in a cytolytic peptide that acts on phospholipid membranes.

The incorporation of a reduced amide bond, psi(CH(2)NH), into peptide results in an increase in the net positive charge and the perturbation of alpha-helical structure. By using this characteristic of the reduced amide bond, we designed and synthesized novel pseudopeptides containing reduced amide bonds, which had a great selectivity between bacterial and mammalian cells. A structure-activity relationship study on pseudopeptides indicated that the decrease in alpha-helicity and the increase in net positive charge in the backbone, caused by the incorporation of a reduced amide bond into the peptide, both contributed to an improvement in the selectivity between lipid membranes with various surface charges. However, activity results in vitro indicated that a perturbation of alpha-helical structure rather than an increase in net positive charge in the backbone is more important in the selectivity between bacterial and mammalian cells. The present result revealed that the backbone of membrane-active peptides were important not only in maintaining the secondary structure for the interactions with lipid membranes but also in direct interactions with lipid membranes. The present study showed the unique function of a reduced amide bond in cytolytic peptides and a direction for developing novel anti-bacterial agents from cytolytic peptides that act on the lipid membrane of micro-organisms.

Structure of the cytoplasmic domain of p23 in solution: implications for the formation of COPI vesicles.

Coatomer, the coat protein complex of coat protein (COPI) vesicles, is involved in the budding of these vesicles. Its interaction with the cytoplasmic domains of some p24-family members, type I transmembrane proteins of the Golgi, has been shown to induce a conformational change of coatomer that initiates polymerization of the complex. From stoichiometrical data it is likely that interaction of coatomer with the small tail domains involves an oligomeric form of the p24 proteins. Here we present the structure of peptide analogs of the cytoplasmic domain of p23, a member of the p24 family, as determined by two-dimensional nuclear magnetic resonance spectroscopy in the presence of 2,2,2-trifluoroethanol. An improved strategy for structure calculation revealed that the tail domain peptides form alpha-helices and adopt a tetrameric state. Based on these results we propose an initial model for the binding of coatomer by p23 and the induced conformational change of coatomer that results in its polymerization, curvature of the Golgi membrane to form a bud, and finally a COPI-coated vesicle.

Conformation of the RNA polymerase II C-terminal domain: circular dichroism of long and short fragments.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandemly repeated copies of a heptapeptide with the Y(1)S(2)P(3)T(4)S(5)P(6)S(7) consensus sequence. This repeat contains two overlapping SPXX motifs that can adopt a beta-turn conformation. In addition, each CTD repeat contains the PXXP sequence characteristic of the left-handed helix of polyproline II (P(II)) found in SH3 domain ligands and the PXY sequence that is the target for WW domains. We have studied CTD fragments using circular dichroism (CD) to characterize the conformation of the CTD in water and in the hydrogen bond-promoting solvent trifluoroethanol (TFE). In water, an eight-repeat fragment is predominantly unordered, but at 32 degrees C has P(II) and beta-turn contents estimated to be about 15 % and less than 10 %, respectively. In 90 % TFE, the beta-turn fraction is estimated to be about 75 %, the remainder being unordered and P(II) conformations. The Tyr side-chains are ordered to a significant extent in 90 % TFE. Replacement of the fully conserved Pro residues by alpha-aminoisobutyric acid leads to a large increase in beta-turn. Replacement of Ser2 by Ala does not substantially alter the CTD conformation in water or TFE. Ser5 replacement by Ala increases the P(II) content in water and affects the conformation in TFE-rich solutions. Phosphorylation of Ser2 and Ser5 has little effect in water, but Ser2 affects the conformation in TFE-rich solution in much the same way as Ser5-->Ala substitution. The CD of the full-length murine CTD in water is similar to that of the eight-repeat fragment, indicating little difference in conformation with increasing chain length beyond eight repeats. The roles of P(II) and beta-turn in the interaction of CTD with its target proteins (mediator and RNA-processing components) are discussed. The most likely interactions are between P(II) and WW or SH3 domains, or with some unknown P(II)-binding motif.

Induction of helical conformation in all beta-sheet proteins by trifluoroethanol.

The effect of 2,2,2-Trifluoroethanol (TFE) on the structure of five all beta-sheet proteins, isolated from the venom of the Taiwan cobra (Naja naja atra), is studied. In all the toxins used, it is observed that significant amount of alpha-helix is induced at higher concentrations of TFE. In all these proteins, the induction of helical conformation and disruption of the tertiary structure seem to occur simultaneously. The structural transitions induced by TFE in reduced and denatured protein appear to be different from those observed in the native protein(s). In our opinion, the findings reported herein could have significant implications on research in the area of protein folding.

Acute toxicity of fluorinated ether anesthetics: role of 2,2,2-trifluoroethanol and other metabolites.

The fluorinated ethers 2,2,2-trifluoroethyl vinyl ether (TFVE), 2,2,2-trifluoroethyl ethyl ether (TFEE), and 2,2,2-trifluoroethyl allyl ether (TFAE) are lethal to rats pretreated with a variety of cytochrome P-450-inducing agents at doses not toxic to uninduced rats. The hepatic microsomal cytochrome P-450-catalyzed metabolic pathways have been elucidated: TFVE yields 2,2,2-trifluoroethanol (TFE) and glycolaldehyde; TFEE yields TFE and acetaldehyde; and TFAE yields TFE and acrolein. Time courses of metabolite concentrations in blood after administration of toxic doses of metabolites or anesthetics to variously induced rats were compared. For TFVE, phenobarbital (PB) or pregnenolone-16 alpha-carbonitrile (PCN) induction increased acute lethality 3.1- and 2.4-fold respectively, and blood TFE concentrations reached lethal levels. beta-Naphthoflavone (BNF) induction did not produce lethality and TFE concentrations did not reach lethal levels. Glycolaldehyde concentrations did not approximate lethal levels in any case. For TFEE, PB, BNF, or PCN induction increased lethality 3.4-, 2.6-, and 2.0-fold respectively, and TFE concentrations exceeded lethal levels in all cases. Acetaldehyde concentrations did not approximate lethal levels in any case. For TFAE, BNF, or PCN induction increased lethality 3.2- and 4.6-fold respectively, and TFE concentrations approached lethal levels. PB induction did not produce lethality nor did TFE concentrations reach lethal levels. Blood acrolein concentrations could not be determined. Thus the lethal effects of the three anesthetics arise from specific cytochrome P-450 isozyme-catalyzed metabolism to TFE. The relative rates of formation of TFE from the three anesthetics, catalyzed by microsomes from untreated and variously induced rats, supported this conclusion.