Cooperative alpha-helix formation of beta-lactoglobulin and melittin induced by hexafluoroisopropanol.

Alcohols denature the native state of proteins, and also stabilize the alpha-helical conformation in unfolded proteins and peptides. Among various alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) are often used because of their high potential to induce such effects. However, the reason why TFE and HFIP are more effective than other alcohols is unknown. Using CD, we studied the effects of TFE and HFIP as well as reference alcohols, i.e., methanol, ethanol, and isopropanol, on the conformation of bovine beta-lactoglobulin and the bee venom melittin at pH 2. Upon addition of alcohols, beta-lactoglobulin exhibited a transformation from the native state, consisting of beta-sheets, to the alpha-helical state, whereas melittin folded from the unfolded state to the alpha-helical state. In both cases, the order of effectiveness of alcohols was shown to be: HFIP > TFE > isopropanol > ethanol > methanol. The alcohol-induced transitions were analyzed assuming a two-state mechanism to obtain the m value, a measure of the dependence of the free energy change on alcohol concentration. Comparison of the m values indicates that the high potential of TFE can be explained by the additive contribution of constituent groups, i.e., F atoms and alkyl group. On the other hand, the high potential of HFIP is more than that expected from the additive effects, suggesting that the cooperative formation of micelle-like clusters of HFIP is important.     

Possible formation of the left-handed alpha-helix of poly-L-serine

A conformational study of poly-L -serine has shown that it can exist in the left-handed α-helical form. A study of a pair of peptide units with the serine sidegroup attached to the α carbon atom linking the two units showed that O? H ?O hydrogen bonds between the OH group of the side chain and a carbonyl oxygen of the first peptide group in the backbone can occur in two regions of ?, namely, ? = 15°–30° for χ₁ = 300° and for ? = 225°-230° for ? = 60°. The latter is close to a possible left-handed helix of poly-L -serine, stabilized by N? H ?O hydrogen bonds. From a study of contact criteria, the best conformation for this helix is found to be ? = 227°, Ψ = 238°, χ₁ = 65° which has n = 3.65, h = 1.51 A. The N? H ?O hydrogen bond has a length of 2.90 A. (6°) and the O? H ?O hydrogen bond is of length 2.60 A. (0°). There are no other bad short contacts in the structure. The cylindrical coordinates of the atoms, as well as a perspective view of the structure arc given in this paper.     

Folding of a peptide corresponding to the alpha-helix in bovine pancreatic trypsin inhibitor

A short peptide corresponding to the alpha-helical region of BPTI shows partial folding in aqueous solution (pH 7) as judged by circular dichroism (CD). Folding is temperature and denaturant sensitive, and the peptide is monomeric. The difference CD spectrum, obtained from spectra at two temperatures, indicates that the peptide folds as an alpha-helix. Difference CD spectroscopy provides a sensitive assay for helix formation in peptides exhibiting small amounts of structure. Helix stability in this peptide shows a marked pH dependence which is consistent with stabilizing charged side-chain interactions with the helix dipole and/or salt bridge formation.     

Effect of glycosaminoglycans on peptide bond formation in bacterial ribosomes.

1. A cell-free system derived from E. coli has been used in this study. The process of peptide bond formation was assessed with the aid of the puromycin reaction, which is catalyzed by peptidyltransferase. 2. This reaction is inhibited by heparin, in contrast, this reaction is activated by hyaluronic acid. 3. The presence of heparin decreases the percentage of formed initiation complex (complex C), but hyaluronic acid, chondroitin sulphate and keratan sulphate have no effect on the formation of complex C. 4. From other types of glycosaminoglycans, only hyaluronic acid increases the stability of active complex C.     

Kinetics of inhibition of peptide bond formation on bacterial ribosomes.

A cell-free system derived from Escherichia coli has been used in order to study the kinetics of inhibition of peptide bond formation with the aid of the puromycin reaction in solution. A similar study has been carried out earlier on a solid support matrix with the same inhibitors. We find that the overall pattern of the kinetics of inhibition is the same in the two systems. At low concentrations of inhibitor there is a competitive phase of inhibition, whereas at higher concentrations of inhibitor the type of inhibition becomes mixed noncompetitive. The values of Ki of the competitive phase in the system in solution are: 5.8 microM (amicetin), 0.2 microM (blasticidin S), 0.5 microM (chloramphenicol), and 0.5 microM (tevenel). The inhibitors amicetin, blasticidin S, and tevenel interact with the ribosome in a reaction which is slower than that of the substrate puromycin, showing clear-cut characteristics of slow-onset inhibition in both systems. Chloramphenicol, on the other hand does not easily show such a delay in solution. It interacts with the ribosome relatively faster than the other three antibiotics. Despite this, chloramphenicol too shows characteristics of time-dependent inhibition.     

Transformation of an alpha-helix peptide into a beta-hairpin induced by addition of a fragment results in creation of a coexisting state

Intrinsic rules of determining the tertiary structure of a protein have been unknown partly because physicochemical factors that contribute to stabilization of a protein structure cannot be represented as a linear combination of local interactions. To clarify the rules on the nonlinear term caused by nonlocal interaction in a protein, we tried to transform a peptide that has a fully helical structure (Target Peptide or TP) into a peptide that has a beta-hairpin structure (Designed Peptide or DP) by adding seven residues to the C terminus of TP. According to analyses of nuclear magnetic resonance measurements, while the beta-hairpin structure is stabilized in some DPs, it is evident that the helical structure observed in TP is also persistent and even extended throughout the length of the molecule. As a result, we have produced a peptide molecule that contains both the alpha-helix and beta-hairpin conformation at an almost equally populated level. The helical structures contained in these DPs were more stable than the helix in TP, suggesting that stabilizing one conformation does not result in destabilizing the other conformation. These DPs can thus be regarded as an isolated peptide version of the chameleon sequence, which has the capability of changing the secondary structure depending on the context of the surrounding environment in a protein structure. The fact that the transformation of one secondary structure caused stabilization of both the original and the induced structure would shed light on the mechanism of protein folding.     

Anionic phospholipids are essential for alpha-helix formation of the signal peptide of prePhoE upon interaction with phospholipid vesicles.

The conformational consequences of the interaction of the PhoE signal peptide with bilayers of different types of phospholipids was investigated using circular dichroism. It was found that interaction of the signal peptide with anionic phospholipid vesicles of dioleoylphosphatidylglycerol and dioleoylphosphatidylserine results in induction of high amounts of alpha-helical structure of 70% and 57%, respectively. Upon addition of the signal peptide to cardiolipin vesicles, less but still significant alpha-helical structure was induced (29%). In contrast, no alpha-helix formation was observed upon the interaction of the signal peptide with zwitterionic dioleoylphosphatidylcholine vesicles. In bilayers of dioleoylphosphatidylcholine with dioleoylphosphatidylglycerol, it was shown that in the presence of 100 mM NaCl a minimum amount of 50% of negatively charged lipid was required for induction of the maximal percentage of alpha-helix, whereas in the absence of salt a minimum amount of 35% of negatively charged lipid was necessary. Induction of alpha-helix structure appeared to be correlated with functionality, since, in a less functional analogue of the PhoE signal peptide, the PhoE-[Asp-19,20] signal peptide, less alpha-helix was induced than in the wild-type PhoE signal peptide. It is proposed that the interaction with anionic phospholipids is essential for a functional conformation of the PhoE signal sequence during protein translocation.     

A polyalanine-based peptide cannot form a stable transmembrane alpha-helix in fully hydrated phospholipid bilayers

The conformation and amide proton exchangeability of the peptide acetyl-K(2)-A(24)-K(2)-amide (A(24)) and its interaction with phosphatidylcholine bilayers were examined by a variety of physical techniques. When dissolved in or cast from methanol as a dried film, A(24) is predominantly alpha-helical. In aqueous media, however, A(24) exists primarily as a mixture of helical (though not necessarily alpha-helical) and random coiled structures, both of which allow rapid H-D exchange of all amide protons. When incorporated into phospholipids in the absence of water, A(24) also exists primarily as a transmembrane alpha-helix. However, upon hydration of that system, rapid exchange of all amide protons also occurs along with a marked change in the amide I absorption band of the peptide. Also, when dispersed with phosphatidylcholine in aqueous media, the conformation and thermal stability of A(24) are not significantly altered by the presence of the phospholipid or by its gel/liquid-crystalline phase transition. Differential scanning calorimetric and electron spin resonance spectroscopic studies indicate that A(24) has relatively minor effects on the thermodynamic properties of the lipid hydrocarbon chain-melting phase transition, that it does not abolish the lipid pretransition, and that its presence has no significant effect on the orientational order or rates of motion of the phospholipid hydrocarbon chains. We therefore conclude that A(24) has sufficient alpha-helical propensity, but insufficient hydrophobicity, to maintain a stable transmembrane association with phospholipid bilayers in the presence of water. Instead, it exists primarily as a dynamic mixture of helices and other conformers and resides mostly in the aqueous phase where it interacts weakly with the bilayer surface or with the polar/apolar interfacial region of phosphatidylcholine bilayers. Thus, polyalanine-based peptides are not good models for the transmembrane alpha-helical segments of natural membrane proteins.     

Simulation of alpha-helix and beta-hairpin formation in water-soluble proteins by the code physics method

One of the possible ways for complete and final solution of the problem of determination of three-dimensional structure of proteins on amino acid sequence is simulation of protein three-dimensional structure formation. The use of the code physics method developed by the author has been suggested to fulfill this task. The simulation of alpha-helix and beta-hairpin formation in water-soluble proteins as a start of realization of the plan is described here. The results of the simulation were compared with the experimental data for 14 proteins of no more than 50 amino acids and therefore with little number of alpha-helices and beta-strands (to meet limits of simulation process) and with secondary structure predictions by the best to data methods of protein secondary structure prediction, PSIpred, PORTER and PROFsec. Secondary structure of the proteins, obtained as a result of the simulation of alpha-helix and beta-hairpin formation using the code physics method, corresponded completely to experimental data while the secondary structure predicted by the PSIpred, PORTER and PROFsec methods differed from these data significantly.     

Blocking of bacterial biofilm formation by a fish protein coating

Bacterial biofilm formation on inert surfaces is a significant health and economic problem in a wide range of environmental, industrial, and medical areas. Bacterial adhesion is generally a prerequisite for this colonization process and, thus, represents an attractive target for the development of biofilm-preventive measures. We have previously found that the preconditioning of several different inert materials with an aqueous fish muscle extract, composed primarily of fish muscle alpha-tropomyosin, significantly discourages bacterial attachment and adhesion to these surfaces. Here, this proteinaceous coating is characterized with regards to its biofilm-reducing properties by using a range of urinary tract infectious isolates with various pathogenic and adhesive properties. The antiadhesive coating significantly reduced or delayed biofilm formation by all these isolates under every condition examined. The biofilm-reducing activity did, however, vary depending on the substratum physicochemical characteristics and the environmental conditions studied. These data illustrate the importance of protein conditioning layers with respect to bacterial biofilm formation and suggest that antiadhesive proteins may offer an attractive measure for reducing or delaying biofilm-associated infections.     

Beta-turn formation by a six-residue linear peptide in solution.

A model peptide AAGDYY-NH2 (B1), which is found to adopt a beta-turn conformation in the TEM-1 beta-lactamase inhibitor protein (BLIP) in the TEM-1/BLIP co-crystal, was synthesized to elucidate the mechanism of its beta-turn formation and stability. Its structural preferences in solution were comprehensively characterized using CD, FT-IR and 1H NMR spectroscopy, respectively. The set of observed diagnostic NOEs, the restrained molecular dynamics simulation, CD and FT-IR spectroscopy confirmed the formation of a beta-turn in solution by the model peptide. The dihedral angles [(phi3, phi3) (phi4, phi4)] of [(-52 degrees, -32 degrees ) (-38 degrees, -44 degrees )] of Gly-Asp fragment in the model peptide are consistent with those of a type III beta-turn. In a conclusion, the conformational preference of the linear hexapeptide B1 in solution was determined, and it would provide a simple template to study the mechanism of beta-turn formation and stability.     

Suppression of bacterial infection in rice by treatment with a sulfated peptide

The rice XA21 receptor kinase confers robust resistance to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). A tyrosine‐sulfated peptide from Xoo, called RaxX, triggers XA21‐mediated immune responses, including the production of ethylene and reactive oxygen species and the induction of defence gene expression. It has not been tested previously whether these responses confer effective resistance to Xoo. Here, we describe a newly established post‐inoculation treatment assay that facilitates investigations into the effect of the sulfated RaxX peptide in planta. In this assay, rice plants were inoculated with a virulent strain of Xoo and then treated with the RaxX peptide 2 days after inoculation. We found that post‐inoculation treatment of XA21 plants with the sulfated RaxX peptide suppresses the development of Xoo infection in XA21 rice plants. The treated plants display restricted lesion development and reduced bacterial growth. Our findings demonstrate that exogenous application of sulfated RaxX activates XA21‐mediated immunity in planta, and provides a potential strategy for the control of bacterial disease in the field.     

Alcohol-induced denaturation of beta-lactoglobulin: a close correlation to the alcohol-induced alpha-helix formation of melittin

Alcohols denature the native structure of proteins and induce alpha-helical structure. The potential of alcohols causing such effects varies substantially depending on the alcohol species. With beta-lactoglobulin as a model protein, we compared the effects of various alcohols and observed the additive contribution of each group constituting the alcohol molecules. Whereas the hydrophobic hydrocarbon group promotes the transition according to their size, hydrophilic hydroxyl group suppresses the transition. Halogen groups promote the transition depending on their type and number. It has been known that alcohols induce the alpha-helical structure on the short peptides such as melittin. There is a close correlation between the potentials of alcohol in denaturing beta-lactoglobulin and those in inducing the helical structure in melittin, indicating that the underlying mechanisms of the two phenomena are the same.     

The effect of charge-charge interactions on the kinetics of alpha-helix formation

The formation of the monomeric α-helix represents one of the simplest scenarios in protein folding; however, our current understanding of the folding dynamics of the α-helix motif is mainly based on studies of alanine-rich model peptides. To examine the effect of peptide sequence on the folding kinetics of α-helices, we studied the relaxation kinetics of a 21-residue helical peptide, Conantokin-T (Con-T), using time-resolved infrared spectroscopy in conjunction with a laser-induced temperature jump technique. Con-T is a neuroactive peptide containing a large number of charged residues that is found in the venom of the piscivorous cone snail Conus tulipa. The temperature-jump relaxation kinetics of Con-T is distinctly slower than that of previously studied alanine-based peptides, suggesting that the folding time of α-helices is sequence-dependent. Furthermore, it appears that the slower folding of Con-T can be attributed to the fact that its helical conformation is stabilized by charge-charge interactions or salt bridges. Although this finding contradicts an earlier molecular dynamics simulation, it also has implications for existing models of protein folding.     

Inhibition of bacterial peptide deformylase by biaryl acid analogs

Peptide deformylase is an essential eubacterial metalloenzyme involved in the maturation of proteins by cleaving the N-formyl group from N-blocked methionine polypeptides. Biaryl acid analogs containing tetrazole, acyl sulfonamide, or carboxylate pharmacophores were found to be potent inhibitors of recombinant Escherichia coli peptide deformylase. Two of these compounds, a biphenyl tetrazole, compound 1, and a biphenyl acyl sulfonamide, compound 4, were competitive inhibitors with K(i) values of 1.2 and 6.0 microM, respectively. By analogy to the binding of related compounds to other metalloenzymes such as Bacteroides fragilis metallo-beta-lactamase CcrA and human carbonic anhydrase, a mechanism of inhibition is proposed for these peptide deformylase inhibitors where the acidic moieties form direct ionic interactions with the active site metal cation.     

A model for the coupling of alpha-helix and tertiary contact formation

Peptides corresponding to excised alpha-helical segments of natural proteins can spontaneously form helices in solution. However, peptide helices are usually substantially less stable in solution than in the structural context of a folded protein, because of the additional interactions possible between helices in a protein. Such interactions can be thought of as coupling helix formation and tertiary contact formation. The relative energetic contributions of the two processes to the total energy of the folded state of a protein is a matter of current debate. To investigate this balance, an extended helix-coil model (XHC) that incorporates both effects has been constructed. The model treats helix formation with the Lifson-Roig formalism, which describes helix initiation and propagation through cooperative local interactions. The model postulates an additional parameter representing participation of a site in a tertiary contact. In the model, greater helix stability can be achieved through combinations of these short-range and long-range interactions. For instance, stronger tertiary contacts can compensate for helices with little intrinsic stability. By varying the strength of the nonlocal interactions, the model can exhibit behavior consistent with a variety of qualitative models describing the relative importance of secondary and tertiary structure. Moreover, the model is explicit in that it can be used to fit experimental data to individual peptide sequences, providing a means to quantify the two contributions on a common energetic basis.     

The role of context on alpha-helix stabilization: host-guest analysis in a mixed background peptide model.

The helix content of a series of peptides containing single substitutions of the 20 natural amino acids in a new designed host sequence, succinyl-YSEEEEKAKKAXAEEAEKKKK-NH2, has been determined using CD spectroscopy. This host is related to one previously studied, in which triple amino acid substitutions were introduced into a background of Glu-Lys blocks completely lacking alanine. The resulting free energies show that only Ala and Glu- prove to be helix stabilizing, while all other side chains are neutral or destabilizing. This agrees with results from studies of alanine-rich peptide modela, but not the previous Glu-Lys block oligomers in which Leu and Met also stabilize helix. The helix propensity scale derived from the previous block oligomers correlated well with the frequencies of occurrence of different side chains in helical sequences of proteins, whereas the values from the present series do not. The role of context in determining scales of helix propensity values is discussed, and the ability of algorithms designed to predict helix structure from sequence is compared.     

Catalysis of peptide bond formation by histidyl-histidine in a fluctuating clay environment

Summary The condensation of glycine to form oligoglycines during wet-dry fluctuations on clay surfaces was enhanced up to threefold or greater by small amounts of histidyl-histidine. In addition, higher relative yields of the longer oligomers were produced. Other specific dipeptides tested gave no enhancement, and imidazole, histidine, and N-acetylhistidine gave only slight enhancements. Histidyl-histidine apparently acts as a true catalyst (in the sense of repeatedly catalyzing the reaction), since up to 52 nmol of additional glycine were incorporated into oligoglycine for each nmol of catalyst added. This is the first known instance of a peptide or similar molecule demonstrating a catalytic turnover number greater than unity in a prebiotic oligomer synthesis reaction, and suggests that histidyl-histidine is a model for a primitive prebiotic protoenzyme. Catalysis of peptide bond synthesis by a molecule which is itself a peptide implies that related systems may be capable of exhibiting autocatalytic growth.