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A physical entrapment technique has been developed for the surface engineering of preformed alginate fibers. Surface engineering was carried out at room temperature in aqueous solutions without additional solvent, a catalyst/initiator, a chemical cross-linking agent, or a temperature increase. Entrapment of surface-modifying molecules was achieved by exposing the alginate fibers to a Na(+)-rich NaCl/CaCl2 mixture solution, which caused the formation of a moderate dissociation layer into which the modifier could diffuse within a few seconds. The surface dissociation was then reversed by the addition of a large excess of multivalent cations, which resulted in collapse of the interface and immobilization of the modifying species. Rhodamine-tagged poly(ethylene glycol)s of different molecular weights were used as model molecules to investigate the effect of process parameters on the entrapment efficiency. It was found that the entrapment efficiency as well as the distribution of the modifier within the alginate fibers was determined by several factors, including the NaCl/CaCl2 ratio in the preswelling solution, exposure time, and concentration and molecular weight of the modifiers. The morphology of the fibers was not significantly changed in terms of shape and size after the entrapment process. By this technique, poly(L-lysine) (PLL) coupled with cell adhesion peptide sequence GRGDS (PLL-GRGDS) was entrapped within alginate fibers, and it was demonstrated that the modification promoted the attachment of mouse 3T3 fibroblasts.
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PMID:Novel surface entrapment process for the incorporation of bioactive molecules within preformed alginate fibers. 1576 37

Dehydrins constitute a class of intrinsically disordered proteins that are expressed under conditions of water-related stress. Characteristic of the dehydrins are some highly conserved stretches of seven to 17 residues that are repetitively scattered in their sequences, the K-, S-, Y-, and Lys-rich segments. In this study, we investigate the putative role of these segments in promoting structure. The analysis is based on comparative analysis of four full-length dehydrins from Arabidopsis (Arabidopsis thaliana; Cor47, Lti29, Lti30, and Rab18) and isolated peptide mimics of the K-, Y-, and Lys-rich segments. In physiological buffer, the circular dichroism spectra of the full-length dehydrins reveal overall disordered structures with a variable content of poly-Pro helices, a type of elongated secondary structure relying on bridging water molecules. Similar disordered structures are observed for the isolated peptides of the conserved segments. Interestingly, neither the full-length dehydrins nor their conserved segments are able to adopt specific structure in response to altered temperature, one of the factors that regulate their expression in vivo. There is also no structural response to the addition of metal ions, increased protein concentration, or the protein-stabilizing salt Na(2)SO(4). Taken together, these observations indicate that the dehydrins are not in equilibrium with high-energy folded structures. The result suggests that the dehydrins are highly evolved proteins, selected to maintain high configurational flexibility and to resist unspecific collapse and aggregation. The role of the conserved segments is thus not to promote tertiary structure, but to exert their biological function more locally upon interaction with specific biological targets, for example, by acting as beads on a string for specific recognition, interaction with membranes, or intermolecular scaffolding. In this perspective, it is notable that the Lys-rich segment in Cor47 and Lti29 shows sequence similarity with the animal chaperone HSP90.
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PMID:Structural investigation of disordered stress proteins. Comparison of full-length dehydrins with isolated peptides of their conserved segments. 1656 95

Ampholytic polymer gels based on N-isopropylacrylamide (NIPA) and natural amino acid L-lysine were prepared by free radical polymerization in aqueous solutions. To make amino acids attachable to the polymer chain, the acrylic group was added to the epsilon-amino group of lysine to obtain N-epsilon-acrylic-lysine (Z). Finally, a new temperature- and pH-sensitive (NIPA-Z) hydrogel was obtained. The presence of amino and carboxylic groups of amino acids gave us a possibility to control the amount and sign of the excessive charge on the polymeric network with respect to pH. The swelling behavior of the NIPA-Z hydrogels with respect to the amount of Z (0-4%), temperature, and pH was investigated. Temperature and pH were changed in the ranges of 20-50 degrees C and 2-12, respectively. To eliminate the influence of ionic strength on the swelling behavior, this parameter was kept constant in all experiments. It was found that the pH dependence of the degree of swelling for the NIPA-Z gels, measured at constant temperature, exhibits a minimum. Such a minimum was seen for the ampholyte networks with independent acidic and basic groups attached to the polymer chains. For the NIPA-Z gels, the minimum was wide, and the pH range over which it was spread corresponded well to the pH distribution of the zwitterions. The way the gel volume changed with an increase in temperature depends on the amino acid amount. It is initially discontinuous and turns to the continuous one for the higher percentage of amino acid. The temperature dependence of the swelling process obtained for different pH values clearly shows that for the pH region where the zwitterions dominate, the polymer networks collapse more efficiently.
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PMID:pH and temperature-sensitive N-isopropylacrylamide ampholytic networks incorporating L-lysine. 1692 72

We have used fluorescence correlation spectroscopy measurements to quantify the hydrodynamic sizes of monomeric polyglutamine as a function of chain length (N) by measuring the scaling of translational diffusion times (tau(D)) for the peptide series (Gly)-(Gln)(N)-Cys-Lys(2) in aqueous solution. We find that tau(D) scales with N as tau(o)N(nu) and therefore ln(tau(D)) = ln(tau(o)) + nuln(N). The values for nu and ln(tau(o)) are 0.32 +/- 0.02 and 3.04 +/- 0.08, respectively. Based on these observations, we conclude that water is a polymeric poor solvent for polyglutamine. Previous studies have shown that monomeric polyglutamine is intrinsically disordered. These observations combined with our fluorescence correlation spectroscopy data suggest that the ensemble for monomeric polyglutamine is made up of a heterogeneous collection of collapsed structures. This result is striking because the preference for collapsed structures arises despite the absence of residues deemed to be hydrophobic in the sequence constructs studied. Working under the assumption that the driving forces for collapse are similar to those for aggregation, we discuss the implications of our results for the thermodynamics and kinetics of polyglutamine aggregation, a process that has been implicated in the molecular mechanism of Huntington's disease.
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PMID:Fluorescence correlation spectroscopy shows that monomeric polyglutamine molecules form collapsed structures in aqueous solutions. 1707 61

Bacterial biofilms, i.e. surface-associated cells covered in hydrated extracellular polymeric substances (EPS), are often studied with high-resolution electron microscopy (EM). However, conventional desiccation and high vacuum EM protocols collapse EPS matrices which, in turn, deform biofilm appearances. Alternatively, wet-mode environmental scanning electron microscopy (ESEM) is performed under a moderate vacuum and without biofilm drying. If completely untreated, however, EPS is not electron dense and thus is not resolved well in ESEM. Therefore, this study was towards adapting several conventional SEM staining protocols for improved resolution of biofilms and EPS using ESEM. Three different biofilm types were used: 1) Pseudomonas aeruginosa unsaturated biofilms cultured on membranes, 2) P. aeruginosa cultured in moist sand, and 3) mixed community biofilms cultured on substrates in an estuary. Working with the first specimen type, a staining protocol using ruthenium red, glutaraldehyde, osmium tetroxide and lysine was optimized for best topographic resolution. A quantitative image analysis tool that maps relief, newly adopted here for studying biofilms, was used to compare micrographs. When the optimized staining and ESEM protocols were applied to moist sand cultures and aquatic biofilms, the smoothening effect that bacterial biofilms have on rough sand, and the roughening that aquatic biofilms impart on initially smooth coupons, were each quantifiable. This study thus provides transferable staining and ESEM imaging protocols suitable for a wide range of biofilms, plus a novel tool for quantifying biofilm image data.
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PMID:Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy. 1719 92

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.
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PMID:A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae. 1722 9

The volumetric response of polymer gels on cosolute addition depends on the interaction of the polymer with the cosolute and can be used as a simple and sensitive way of elucidating these interactions. Here we report on DNA networks, prepared by crosslinking double-stranded DNA with ethylene glycol diglycidyl ether (EGDE); these have been investigated with respect to their swelling in aqueous solution containing different additives, such as metal ions, polyamines, charged proteins, and surfactants. The deswelling on addition of metal ions occurs at lower concentrations with increasing valency of the counterion. The collapse of the gels in the presence of trivalent ions seems to follow the same kind of mechanism as the interaction in solution, but addition of these ions leads to DNA denaturation and formation of single-stranded DNA. Striking features were found in the deswelling of DNA gels by chitosan, spermine, spermidine, lysozyme, poly-l-lysine and poly-l-arginine. Chitosan is the most efficient cosolute of those investigated with respect to DNA gel collapse. The effect of the cationic surfactant tail length on the volume phase transition of DNA gels was studied as a function of surfactant concentration. Cationic surfactants effectively collapsed the gel from the critical aggregation concentration (cac), decreasing with increasing length of the hydrophobic tail. In several cases, the deswelling as a function of cosolute concentration shows a pronounced two-step behavior, which is interpreted in terms of a combination of DNA chain condensation and general osmotic deswelling. The studies included investigations on the state of the DNA chain after deswelling, on the reversibility of the deswelling as well as on the kinetics. With the exception for the trivalent lanthanide ions, it appears that the DNA chain always retains a double-helix conformation; with these metal ions, single-stranded DNA is found. The deswelling appears to be reversible as exemplified by addition of anionic surfactant subsequent to gel collapsed by cationic surfactant and addition of sodium bromide to gels collapsed by a polycation. An investigation of the kinetics shows that an increase in the surfactant tail length gives a pronouncedly slower deswelling kinetics.
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PMID:Effect of additives on swelling of covalent DNA gels. 1748 24

Acid dissociation constants of side-chain acidic groups of amino acid residues in peptides can be determined by 1H NMR, provided resonances can be resolved for carbon-bonded reporter protons located near the acidic group. We report here that the increased resolution of the band-selective homonuclear-decoupled (BASHD) TOCSY experiment greatly extends the range of application of the NMR method for determination of residue-specific, side-chain acid dissociation constants of peptides that contain multiple residues of the same amino acid. Chemical shift-pH titration curves are obtained from cross-peaks for reporter protons in BASHD-TOCSY spectra measured as a function of pH. The method is based on using sequence-dependent differences in the chemical shifts of resonances for the backbone CalphaH protons and the increased resolution in BASHD-TOCSY spectra from collapse of CalphaH multiplets to singlets in the F1 dimension to resolve resonances for the side-chain reporter protons. Application of the method is demonstrated by determination of residue-specific pKA values for each of the side-chain ammonium groups of the six lysine residues in the hexadecapeptide Ac-SRGKAKVKAKVKDQTK-NH2. Chemical shift-pH titration curves were obtained for the lysine side-chain CepsilonH2 reporter protons from their resolved CalphaH-CepsilonH2 TOCSY cross-peaks in BASHD-TOCSY spectra. Relative acidities of the six ammonium groups were also determined from the residue specific chemical shift-pH titration data by a pH-independent method, and calculation of fractional concentrations of protonation microspecies using the residue-specific pKAs is also described.
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PMID:Determination of residue-specific acid dissociation constants for peptides by band-selective homonuclear-decoupled (1)H NMR. 1767 82

Three distinct species of nucleocapsids of equine herpesvirus type-1, designated as either light (L), intermediate (I), or heavy (H) on the basis of their densities in Renografin-76 density gradients (Perdue et al. 1975), were characterized with respect to their amino acid content, DNA content, and role in the maturation process. Preparations of L and I nucleocapsids, shown previously to lack a densely staining core within the capsid, exhibited virtually identical amino acid compositions. Preparations of H nucleocapsids, which possess densely staining cores, contained significantly more lysine, glutamic acid, and serine than did L and I capsids and, in general, more closely resembled the enveloped virion in amino acid composition. The increased content of lysine and glutamic acid in H nucleocapsids indicates that polypeptide IVa (30,000 mol wt), which is present only in H nucleocapsids, is rich in these amino acids. These amino acids may be present as polylysine and polyglutamic acid which have been shown to collapse DNA and function in the DNA packaging event of bacterial viruses (Laemmli, 1975). DNA isolated from preparations of each of the three nucleocapsid species was characterized and shown to be identical to the DNA of enveloped virus in density (1.716 g/cm(3)) and size (50-55 S). The intranuclear production of the three nucleocapsid species was studied with respect to both the time and rate of synthesis of each and the relative amount of each species present during infection. All three species appeared at 6-8 hr postinfection and were produced in a logarithmic fashion until 15 hr postinfection. The ratio of L:I:H particles, based on the percentage that each species comprised of the total purified nucleocapsid population, remained constant and was approximately 45:45:10 at all times postinfection. Analysis of the fate of each of the three intranuclear nucleocapsid classes by pulse-chase experiments indicated that I and H species are removed from the nucleus by participation in the maturation process, but L capsids remain in the nucleus throughout infection and are defective by-products of virus assembly. Based on the evidence accumulated in this study and on previous data (Perdue et al. 1974, 1975), a model depicting equine herpesvirus maturation is proposed. This model proposes that the viral DNA is inserted into I nucleocapsids and that this process is accompanied by condensation of the internal protein resulting in the formation of a dense core and hence an H nucleocapsid.
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PMID:Biochemical studies of the maturation of herpesvirus nucleocapsid species. 1862 60

The PhoB/PhoR-dependent response to inorganic phosphate (Pi)-starvation in Vibrio cholerae O1 includes the expression of vc0719 for the response regulator PhoB, vca0033 for an alkaline phosphatase and vca1008 for an outer membrane protein (OMP). Sequences with high identity to these genes have been found in the genome of clinical and environmental strains, suggesting that the Pi-starvation response in V. cholerae is well conserved. VCA1008, an uncharacterized OMP involved in V. cholerae pathogenicity, presents sequence similarity to porins of Gram-negative bacteria such as phosphoporin PhoE from Escherichia coli. A three-dimensional model shows that VCA1008 is a 16-stranded pore-forming beta-barrel protein that shares three of the four conserved lysine residues responsible for PhoE anionic specificity with PhoE. VCA1008 beta-barrel apparently forms trimers that collapse into monomers by heating. Properties such as heat modifiability and resistance to denaturation by sodium dodecyl sulfate at lower temperatures permitted us to suggest that VCA1008 is a classical porin, more precisely, a phosphoporin due to its Pi starvation-induced PhoB-dependent expression, demonstrated by electrophoretic mobility shift assay and promoter fusion-lacZ assays.
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PMID:Molecular analysis of VCA1008: a putative phosphoporin of Vibrio cholerae. 1965 44


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