Effect of the Hydrophilic-Hydrophobic Balance of Antigen-Loaded Peptide Nanofibers on Their Cellular Uptake, Cellular Toxicity, and Immune Stimulatory Properties.

Recently, nanofibers (NFs) formed from antigenic peptides conjugated to ß-sheet-forming peptides have attracted much attention as a new generation of vaccines. However, studies describing how the hydrophilic-hydrophobic balance of NF components affects cellular interactions of NFs are limited. In this report, three different NFs were prepared by self-assembly of ß-sheet-forming peptides conjugated with model antigenic peptides (SIINFEKL) from ovalbumin and hydrophilic oligo-ethylene glycol (EG) of differing chain lengths (6-, 12- and 24-mer) to investigate the effect of EG length of antigen-loaded NFs on their cellular uptake, cytotoxicity, and dendritic cell (DC)-stimulation ability. We used an immortal DC line, termed JAWS II, derived from bone marrow-derived DCs of a C57BL/6 p53-knockout mouse. The uptake of NFs, consisting of the EG 12-mer by DCs, was the most effective and activated DC without exhibiting significant cytotoxicity. Increasing the EG chain length significantly reduced cellular entry and DC activation by NFs. Conversely, shortening the EG chain enhanced DC activation but increased toxicity and impaired water-dispersibility, resulting in low cellular uptake. These results show that the interaction of antigen-loaded NFs with cells can be tuned by the EG length, which provides useful design guidelines for the development of effective NF-based vaccines.

Morphological Transformation of Peptide Nanoassemblies through Conformational Transition of Core-forming Peptides.

Morphological control of nanostructures that are composed of amphiphilic di- or tri-block molecules by external stimuli broadens their applications for molecular containers, nanoreactors, and controlled release materials. In this study, triblock amphiphiles comprising oligo(ethylene glycol), oligo(l-lysine), and tetra(l-phenylalanine) were prepared for the construction of nanostructures that can transform accompanying α-to-ß transition of core-forming peptides. Circular dichroic (CD) measurements showed that the triblock amphiphiles adopted different secondary structures depending on the solvent environment: they adopt ß-sheet structures in aqueous solution, while α-helix structures in 25% 2,2,2-trifluoroethanol (TFE) solution under basic pH conditions. Transmission electron microscopic (TEM) observation revealed that the triblock amphiphiles formed vesicle structures in 25% TFE aq. Solvent exchange from 25% TFE to water induced morphological transformation from vesicles to arc-shaped nanostructures accompanying α-ß conformational transition. The transformable nanostructures may be useful as novel smart nanomaterials for molecular containers and micro reactors.

Fusion of polymeric material-binding peptide to cell-adhesion artificial proteins enhances their biological function.

Orientation-controlled protein immobilization on a solid substrate surface is important for the development of biomedical materials such as scaffolds used in tissue engineering. In this study, the authors demonstrated that the introduction of material-binding peptides (MBPs) in Arg-Gly-Asp (RGD)-fused artificial proteins called blocking peptide fragment (BPF), which are fragments (residues 419-607) of the molecular chaperone DnaK, enhances the oriented adsorption of proteins on the polymer surface and improves their cell adhesion capability. The authors used isotactic poly(methyl methacrylate) (it-PMMA) binding peptides (c02 peptide; ELWRPTR) as a model system. A quartz crystal microbalance study showed that the fusion of c02 peptide with BPF-RGD proteins slightly enhanced adsorption on it-PMMA surfaces. On the other hand, atomic force microscopic images of it-PMMA surfaces adsorbed with c02-BPF-RGD proteins showed a dotlike pattern, with the sizes of the dots comparable to those of BPF protein dimers, indicating that the immobilization of c02-BPF-RGD partially occurred in an oriented manner via specific interaction between the c02 peptide and it-PMMA. This is in sharp contrast to the random adsorption of BPF-RGD and BPF. These results were supported by results of the enzyme-linked immunosorbent assay using an antihistidine tag antibody. In addition, c02-BPF-RGD adsorbed on it-PMMA showed better cell attachment and spreading ability than BPF-RGD and BPF. This methodology can be applied to other MBP systems and cell-binding motifs. Thus, BPF-based artificial cell adhesion proteins fused with MBPs might be useful as surface modifiers of polymer materials for improving their cell adhesion ability.

The mechanism of fibril formation of a non-inhibitory serpin ovalbumin revealed by the identification of amyloidogenic core regions.

Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys(73) and Cys(120) in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4-5B are highly ß-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to ß-strands, thereby forming sequential intermolecular linkages.

Enzymatic conversion of atmospheric aldehydes into alcohol in a phospholipid polymer film.

We developed a unique method for converting atmospheric aldehyde into alcohol using formaldehyde dehydrogenase from Pseudomonas putida (PFDH) doped in a polymer film. A film of poly(2-methacryloyloxyethylphosphorylcholine-co-n-butyl methacrylate) (PMB), which has a chemical structure similar to that of a biological membrane, was employed for its biocompatibility. A water-incorporated polymer film entrapping PFDH and its cofactor NAD(+) was obtained by drying a buffered solution of PMB, PFDH, and NAD(+). The aldehydes in the air were absorbed into the polymer film and then enzymatically oxidized by PFDH doped in the PMB film. Interestingly, alcohol and carboxylic acid were produced by the enzymatic reaction, indicating that PFDH catalyzes dismutation of aldehyde in the PMB film. Importantly, a PFDH-PMB film catalyzes aldehyde degradation without consuming the nucleotide cofactor, thereby allowing repeated use of the film. The activity of PFDH in the PMB film was higher than that in other common water-soluble polymers, suggesting that the hydrational state in a phospholipid polymer matrix is suitable for enzymatic activity.

Effects of pressure on enzyme function of Escherichia coli dihydrofolate reductase.

To elucidate the effects of pressure on the function of Escherichia coli dihydrofolate reductase (DHFR), the enzyme activity and the dissociation constants of substrates and cofactors were measured at pressures up to 250 MPa at 25 degrees C and pH 7.0. The enzyme activity decreased with increasing pressure, accompanying the activation volume of 7.8 ml mol(-1). The values of the Michaelis constant (K(m)) for dihydrofolate and NADPH were slightly higher at 200 MPa than at atmospheric pressure. The hydride-transfer step was insensitive to pressure, as monitored by the effects of the deuterium isotope of NADPH on the reaction velocity. The dissociation constants of substrates and cofactors increased with pressure, producing volume reductions from 6.5 ml mol(-1) (tetrahydrofolate) to 33.5 ml mol(-1) (NADPH). However, the changes in Gibbs free energy with dissociation of many ligands showed different pressure dependences below and above 50 MPa, suggesting conformational changes of the enzyme at high pressure. The enzyme function at high pressure is discussed based on the volume levels of the intermediates and the candidates for the rate-limiting process.

Amyloid fibril formation and chaperone-like activity of peptides from alphaA-crystallin.

AlphaA-crystallin (alphaAC), a major component of eye lens, exhibits chaperone-like activity and is responsible for maintaining eye lens transparency. Synthetic peptides which corresponded to the putative substrate-binding site of alphaAC have been reported to prevent aggregation of proteins [Sharma, K. K., et al. (2000) J. Biol. Chem. 275, 3767-3771]. In this study, we found that these peptides, alphaAC(70-88), the peptide corresponding to amino acids 70-88 of alphaAC (KFVIFLDVKHFSPEDLTVK), and alphaAC(71-88), suppressed the amyloid fibril formation of amyloid beta protein (Abeta). On the other hand, while alphaAC(71-88) exhibited chaperone-like activity toward insulin, alphaAC(70-88) and alphaAC(70-88)K70D promoted rapid growth of aggregates consisting of insulin and these peptides in their solution mixtures. Interestingly, we found that alphaAC(71-88) itself can also form amyloid fibrils. It is possible that the chaperone-like activity of the alphaAC peptides is potentially related to their propensity for amyloid fibril formation. Analysis of variants of the alphaAC peptides suggested that F71 is important for amyloid formation, and interestingly, this same residue has previously been found to be essential for chaperone-like activity. Amyloid fibril formation was also observed with the shorter peptide, alphaAC(70-76)K70D, showing that the ability to form amyloid fibrils is maintained even with significant deletion of the C-terminal sequence. The formation of amyloid fibril was suppressed in alphaAC(70-88), suggesting that the K70 in the substrate binding site may play a role in suppressing the amyloid fibril formation of alphaAC, which agreed with recent proposals about the presence of an aggregation suppressor in the region flanking aggregation-prone hydrophobic sequences.

The roles of conserved amino acids on substrate binding and conformational integrity of ClpB N-terminal domain.

Escherichia coli heat shock protein ClpB disaggregates denatured protein in cooperation with the DnaK chaperone system. Several studies showed that the N-terminal domain is essential for the chaperone activity, but its role is still largely unknown. The N-terminal domain contains two structurally similar subdomains, and conserved amino acids Thr7 and Ser84 share the same position in two apparent sequence repeats. T7A and S84A substitutions affected chaperone activity of ClpB without significantly changing the native conformation [Liu, Z. et al. (2002) J. Mol. Biol. 321, 111-120]. In this study, we aimed to better understand the roles of several conserved amino acid residues, including Thr7 and Ser84, in the N-terminal domain. We investigated the effects of mutagenesis on substrate binding and conformational states of ClpB N-terminal domain fragment (ClpBN). Fluorescence polarization analysis showed that the T7A and S84A substitutions enhanced the interaction between ClpBN and protein aggregates. Interestingly, further analyses suggested that the mechanisms by which they do so are quite different. For T7A substitution, the increased substrate affinity could be due to a conformational change in the hydrophobic core as revealed by NMR spectroscopy. In contrast, for S84A, increased substrate binding would be explained by a unique conformational state of this mutant as revealed by pressure perturbation analysis. The thermal transition temperature of the S84A mutant, monitored by DSC, was 6.1 degrees C lower than that of wild-type. Our results revealed that conserved amino acids Thr7 and Ser84 both participated in maintaining the conformational integrity of the ClpB N-terminal domain.

Synergetic effects of pressure and chemical denaturant on protein unfolding: stability of a serine-type carboxyl protease, kumamolisin.

Kumamolisin, a serine carboxyl proteinase, is very stable and hardly denatured by single perturbation of a chemical denaturant (urea), pressure (<500 MPa) or temperature (<65 degrees C). In order to investigate the cooperative effects of these three denaturing agents, DSC, CD, intrinsic fluorescence, and fourth derivative UV absorbance were measured under various conditions. By application of pressure to kumamolisin in 8 M urea solution, substantial red-shift in the center of fluorescence emission spectral mass was observed, and the corresponding blue-shift was observed for two major peaks in fourth derivative UV absorbance, under the similar urea-containing conditions. The denaturation curves were analyzed on the basis of a simple two-state model in order to obtain thermodynamic parameters (DeltaV, DeltaG, and m values), and the combined effects of denaturing agents are discussed, with the special interest in the large cavity and neighboring Trp residue in kumamolisin.

Effect of the polypeptide binding on the thermodynamic stability of the substrate binding domain of the DnaK chaperone.

The effect of polypeptide binding on the stability of the substrate binding domain of the molecular chaperone DnaK has been studied by thermodynamic analysis. The calorimetric scan of the fragment of the substrate binding domain DnaK384-638, consisting of a beta-domain and an alpha-helical lid, showed two transitions centered at 56.2 and 76.0 degrees C. On the other hand, the thermal unfolding of the shorter fragment DnaK386-561, which lacks half of the alpha-helical lid, exhibited a single transition at 57.0 degrees C. Therefore, the transition of DnaK384-638 at 56.2 degrees C is mainly attributed to the unfolding of the beta-domain. The calorimetric scan of DnaK384-638D526N showed that the unfolding of the beta-domain was composed of two transitions. The polypeptide bound DnaK384-638 exhibited a symmetrical DSC peak at 58.6 degrees C, indicating that the substrate binding shifts the beta-domain toward a single cooperative unit. A low concentration of GdnHCl (<1.0 M) induced a conformational change in the beta-domain of DnaK384-638 without changes in the secondary structure. While the thermal unfolding of the beta-domain of DnaK384-638 was composed of two transitions in the presence of GdnHCl, the beta-domain of the substrate bound DnaK384-638 exhibited a single symmetrical DSC peak in the same condition. All together, our results indicate that complex between DnaK384-638 and substrate forms a rigid conformation in the beta-domain.

Interaction of the N-terminal domain of Escherichia coli heat-shock protein ClpB and protein aggregates during chaperone activity.

The Escherichia coli heat-shock protein ClpB reactivates protein aggregates in cooperation with the DnaK chaperone system. The ClpB N-terminal domain plays an important role in the chaperone activity, but its mechanism remains unknown. In this study, we investigated the effect of the ClpB N-terminal domain on malate dehydrogenase (MDH) refolding. ClpB reduced the yield of MDH refolding by a strong interaction with the intermediate. However, the refolding kinetics was not affected by deletion of the ClpB N-terminal domain (ClpBDeltaN), indicating that MDH refolding was affected by interaction with the N-terminal domain. In addition, the MDH refolding yield increased 50% in the presence of the ClpB N-terminal fragment (ClpBN). Fluorescence polarization analysis showed that this chaperone-like activity is explained best by a weak interaction between ClpBN and the reversible aggregate of MDH. The dissociation constant of ClpBN and the reversible aggregate was estimated as 45 muM from the calculation of the refolding kinetics. Amino acid substitutions at Leu 97 and Leu 110 on the ClpBN surface reduced the chaperone-like activity and the affinity to the substrate. In addition, these residues are involved in stimulation of ATPase activity in ClpB. Thus, Leu 97 and Leu 110 are responsible for the substrate recognition and the regulation of ATP-induced ClpB conformational change.

Mechanism for the phase transition of a genetically engineered elastin model peptide (VPGIG)40 in aqueous solution.

The concentration dependence of the pressure- and temperature-induced cloud point transition (Pc and Tc, respectively) of aqueous solutions of an elastin-like polypeptide with a repeating pentapeptide Val-Pro-Gly-Ile-Gly sequence (MGLDGSMG(VPGIG)40VPLE) was investigated by using apparent light scattering, differential scanning calorimetry, and circular dichroism methods. In addition, the effects of salts and surfactants on these properties were investigated. The Pc and Tc of the present peptide in aqueous solution were strongly concentration dependent. The calorimetric measurements showed that the enthalpy of transitions was 300-400 kJ/mol, i.e., 7-10 kJ/mol per VPGIG pentamer. The Tc of the (VPGIG)40 solution was highly affected by the addition of inert salts or SDS. The effects of salts were consistent with those observed in the lyotropic series or Hoffmeister series. The CD spectrum at low peptide concentrations indicated that the present peptide forms type II beta-turn-like structure(s) at higher temperatures, but the temperature dependence of random coil diminishment (195 nm) and beta-turn formation (210 nm) were not exactly coincident. A hypothetical mechanism of the (VPGIG)40 phase transition that could account for these observations was postulated. Observations suggest that the temperature-responsive properties of the elastin model peptides occur via a mechanism involving conformational change-association-aggregation and that the first two are strongly interactive.

The substrate binding domain of DnaK facilitates slow protein refolding.

We examined the effects of a fragment of the substrate binding domain of DnaK on protein refolding from chemically denatured states. The fragment DnaK384-638, containing a full-length substrate binding domain, tightly binds to the unfolded protein in solution. The effects of DnaK384-638 on the reactivation of beta-galactosidase and luciferase were examined at low substrate concentration and low temperature, conditions in which the folding is significantly slow (several days) but the reactivation yield is higher than those in ordinary refolding conditions. In the presence of DnaK384-638, the maximum yield of active beta-galactosidase was improved from 45% to 65% after a 48-h refolding reaction. Spectroscopic experiments showed that DnaK384-638 bound to partially structured monomers of beta-galactosidase and consequently suppressed aggregation. DnaK384-638 accelerated the refolding of luciferase to attain equilibrium in 8 h. On the other hand, DnaK386-561, which has no affinity for the substrate, had no chaperone activity for the reactivation of these proteins. These results indicate that the substrate binding of DnaK384-638 facilitates slow protein refolding.

Cold denaturation of proteins under high pressure.

The advantageous usage of the high pressure technique in studies of cold denaturation of proteins is reviewed, with a brief explanation of the theoretical background of this universal phenomenon. Various experimental results are presented and discussed, explaining the plausible image of the cold denatured state of proteins. In order to understand more clearly this phenomenon and protein structure transition in general, several studies on model polymer systems are also reviewed.

Effects of guanidine hydrochloride and high pressure on subsite flexibility of beta-amylase.

We investigated the effects of guanidine hydrochloride (GuHCl) and high pressure on the conformational flexibility of the active site of sweet potato beta-amylase by monitoring the sulfhydryl reaction and the enzymatic activity. The reactivity of Cys345 at the active site, one of six inert half cystine residues of this enzyme, was enhanced by GuHCl at concentrations below 0.5 M. A GuHCl-induced change of the active site was also observed through an intensity change in the near-UV circular dichroism (CD) spectrum. On the other hand, the native conformation of sweet potato beta-amylase observed through fluorescence polarization, far-UV CD spectrum and intrinsic fluorescence was not influenced by GuHCl at concentrations below 0.5 M. Therefore, Cys345 reaction caused by GuHCl was due to an alteration of the local conformation of the active site. GuHCl-induced reaction of Cys345, located in the vicinity of subsites 3 and 4, is attributed to enhanced subsite flexibility, which is responsible for substrate slipping in a single-chain attack mechanism. Due to the flexible conformation, the local region of the subsite is more susceptible to GuHCl perturbation than the molecule overall. The enzymatic activity of sweet potato beta-amylase was reversibly inhibited by GuHCl at concentrations below 0.5 M, and kinetic analysis of the enzymatic mechanism showed that GuHCl decreases the kcat value. High pressure below 400 MPa also inactivated sweet potato beta-amylase with an increase in Cys345 reactivity. These findings indicated that excessively enhanced subsite flexibility reduced the enzymatic activity of sweet potato beta-amylase.