Chemoenzymatic synthesis of poly(L-alanine) in aqueous environment

L-alanine ethyl ester was polymerized into poly(L-alanine) (polyAla), one of the insoluble polypeptides, by papain in aqueous buffer at varying pH. At neutral pH, a maximum chain length of 11 repeats was observed. These polymers were dominated by random coiled structure and demonstrated a lack of patterned macromolecular assembly. Under alkaline conditions, longer polymer chain lengths were achieved, and the maximum chain length was 16 repeats. These longer chains showed distinct β-sheet formation and were capable of fibril assembly. The present study reports on chemoenzymatic synthesis of a hydrophobic homopolypeptide under aqueous conditions as well as demonstrates a chain length dependency of secondary structure formation and macromolecular assembly of chemoenzymatically synthesized polyAla, providing a new insight into material design of polypeptide.     

The interaction of histone H3 with histone H4 and with other histones studied by 19F nuclear magnetic resonance.

The behaviour, upon variations in ionic strength, pH and temperature of 19F nuclear nuclear magnetic resonance signals of the trifluoroacetonylated derivative of histone H3 is compared with those of the H3-H4 complex and of the Hv fraction (an equimolar mixture of H2A, H2B, H3 and h4). The line width of the 19F-labelled histone H3 signals increases with ionic strength or pH, an effect consistent with aggregation of the protein. In the case of H3-H4 complex or Hv the line width decreases at intermediate ionic strengths (0.1-0.25 M NaCl). This effect is interpreted as the consequence of the formation of a well defined structure with ionic strength. At high salt concentrations the line width increases as a consequence of the final rigid quaternary structure or of the formation of higher aggregates.     

Adsorption of polypeptides on the solid surface. II. Effect of secondary chain structure and helix–coil transition

The theory of adsorption of semistiff chains on a planar surface developed by the authors previously has been used to consider the helix–coil transition in single-stranded macromolecule interacting with an adsorbent plane. The cases of nonselective interaction when the adsorption energy is independent of the unit conformation (a) and selective interaction with only helical (b) or coiled (c) sequences active in adsorption were investigated. In case (b) the existence of secondary structure favors chain bonding to the surface. This leads to the increase in the stability of the helical state and complete polypeptide chain spiralization. The profile of the conformational helix–coil transition acquires an asymmetrical shape inherent to the second-order phase transition. In case (c) the bonding of a partially helical chain to the surface is similar to the adsorption of Gaussian coils and is accompanied by the destruction of secondary structure, this destruction being appreciable even if the helical state in space was favorable.     

Comparison of the conformation of RNA from phage MS2 and 16S rRNA. Interaction with dyes specific for the secondary structure of native RNA and RNA subjected to hydrolysis by nuclease S1

The interaction of ethidium bromide (EtBr) with double-stranded (ds), and acridine orange (AO) with single-stranded (ss) fragments of 16S rRNA Escherichia coli in a wide range of ionic strength, at various pH, Zn2+ ion concentrations and partial hydrolysis by nuclease S1 was investigated. It was shown that about 90% of the RNA molecule is accessible to both dyes, when the ionic strength is near of 0.01 (pH 7). Approximately half of the RNA becomes inaccessible to dyes, when the ionic strength was increased up to 0.08-0.24 (pH 4.7-7), independent on the presence of Zn2+ ions (10(-3) M). About a half of the ds-, and a quarter of the ss-segments of the RNA, deduced from the secondary structure model were protected from the interaction with EtBr and AO. The hydrolysis of about a half of ss-segments upon addition of the Zn2+ (10(-3) M) ions did not affect the RNA tertiary structure. The experimental data obtained confirm the idea of the existence of some "nucleus" (or "nuclei") within the 16S rRNA molecule. The "nucleus" seems to be inaccessible to the dyes and is very stable to heat denaturation. It was supposed that this structure is organized by means of interaction of some of the parallelly oriented ds-segments, as it was suggested earlier for the phage MS2 RNA structure.     

Cytochrome c impaled: investigation of the extended lipid anchorage of a soluble protein to mitochondrial membrane models

Cyt c (cytochrome c) has been traditionally envisioned as rapidly diffusing in two dimensions at the surface of the mitochondrial inner membrane when not engaged in redox reactions with physiological partners. However, the discovery of the extended lipid anchorage (insertion of an acyl chain of a bilayer phospholipid into the protein interior) suggests that this may not be exclusively the case. The physical and structural factors underlying the conformational changes that occur upon interaction of ferrous cyt c with phospholipid membrane models have been investigated by monitoring the extent of the spin state change that result from this interaction. Once transiently linked by electrostatic forces between basic side chains and phosphate groups, the acyl chain entry may occur between two parallel hydrophobic polypeptide stretches that are surrounded by positively charged residues. Alteration of these charges, as in the case of non-trimethylated (TML72K) yeast cyt c and Arg91Nle horse cyt c (where Nle is norleucine), led to a decline in the binding affinity for the phospholipid liposomes. The electrostatic association was sensitive to ionic strength, polyanions and pH, whereas the hydrophobic interactions were enhanced by conformational changes that contributed to the loosening of the tertiary structure of cyt c. In addition to proposing a mechanistic model for the extended lipid anchorage of cyt c, we consider what, if any, might be the physiological relevance of the phenomenon.     

Recombination of human erythrocyte apoprotein and lipid. I. Interaction of apoprotein and lipid at the air-water interface

The formation and stabilization of a complex between total erythrocyte apoprotein and monolayers of total erythrocyte lipid as measured by changes of surface pressure (Δπ) and rate of change of surface pressure (dπ/dt) was studied as a function of pH, ionic strength, and lipid surface pressure. Penetration of apoprotein into lipid monolayers was favored by conditions in which lipid and apoprotein were oppositely charged. Once the interaction was completed, the resultant surface complex was resistant to large changes in subphase pH and ionic strength as shown by the insensitivity of Δπ to these parameters. The dπ/dt, however, showed strong dependence on pH and ionic strength, but not on lipid surface pressure. A sharp decrease in dπ/dt around pH 3.5–4.5 is associated with the change in apoprotein charge from (+) to (?). Comparison of complex formation between apoprotein and bovine serum albumin, cytochrome c, and human hemoglobin suggests that erythrocyte apoprotein was specialized in its interaction with erythrocyte lipids. The data show that formation of an apoprotein-lipid complex at the air-water interface has both electrostatic and hydrophobic components. This contradicts results from other laboratories studying erythrocyte membrane recombination by bulk methods.     

Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro. A model of control of leucocytic lysosomal activity.

1. The activities of 30 different lysosomal enzymes were determined in vitro in the presence of the sulphated glycosaminoglycans, heparin and chondroitin sulphate, all the enzymes being measured on a density-gradient-purified lysosomal fraction. 2. Each enzyme was studied as a function of the pH of the incubation medium. In general the presence of sulphated glycosaminoglycans induced a strong pH-dependent inhibition of lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. However, in the particular case of lysozyme and phospholipase A2 the heparin-induced inhibition was maintained in the pH range 4.0-7.0. 3. For certain enzymes, such as acid beta-glycerophosphatase, alpha-galactosidase, acid lipase, lysozyme and phospholipase A2, the pH-dependent behaviour obtained in the presence of heparin was quite different to that obtained with chondroitin sulphate, suggesting the existence of physicochemical characteristic factors playing a role in the intermolecular interaction for each of the sulphated glycosaminoglycans studied. 4. Except in the particular case of peroxidase activity, in all other lysosomal enzymes measured the glycosaminoglycan-enzyme complex formation was a temperature-and time-independent phenomenon. 5. The effects of the ionic strength and pH on this intermolecular interaction reinforce the concept of an electrostatic reversible interaction between anionic groups of the glycosaminoglycans and cationic groups on the enzyme molecule. 6. As leucocytic primary lysosomes have a very acid intragranular pH and large amounts of chondroitin sulphate, we propose that this glycosaminoglycan might act as molecular regulator of leucocytic activity, by inhibiting lysosomal enzymes when the intragranular pH is below the pI of lysosomal enzymes. This fact, plus the intravacuolar pH changes described during the phagocytic process, might explain the unresponsiveness of lysosomal enzymes against each other existing in primary lysosomes as well as its full activation at pH values occurring in secondary lysosomes during the phagocytic process.     

Localization of energy domains in Bacillus intermedius 7P ribonuclease

Two independently melting regions (energetic domains) were localized in Bacillus intermedius 7P ribonuclease by methods of circular dichroism and high resolution X-ray analysis: the lov-temperature melting domain, containing C-terminal region of the molecule with five strands in antiparallel beta-structure and the high-temperature melting alpha-helical domain in the N-terminal region. The contact between these domains is stabilized mainly by ionic interaction Asp-22 - Lys+-48. At pH 2.4 and 30.5 0 C, when the low-temperature domain melts, half of the beta-structure content in binase is destroyed though the alpha-helical structure content is conserved. It has been shown that in pH interval 2.4-4.8 at 15 0 C no changes in secondary structure and local surrounding of aromatic amino acid residues could be identified. Thus, the changes in ionic interactions in the binase molecule due to protonation of Asp side chain groups does not effect the secondary or tertiary structure, though it changes the energetical state of the binase molecule, revealing a change of number and size of energetic domains.     

Chitosan–bovine serum albumin complex formation: A model to design an enzyme isolation method by polyelectrolyte precipitation

Interactions between a model protein (bovine serum albumin—BSA) and the cationic polyelectrolyte, chitosan (Chi), have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It has been found that the conformation of the BSA does not change significantly during the chain interaction between BSA and chitosan forming the non-covalently linked complex. The effects of pH, ionic strength and anions which modify the water structure around BSA were evaluated in the chitosan–BSA complex formation. A net coulombic interaction force between BSA and Chi was found as the insoluble complex formation decreased after the addition of NaCl. Around 80% of the BSA in solution precipitates with the Chi addition. A concentration of 0.05% (w/v) Chi was necessary to precipitate the protein, with a stoichiometry of 6.9 g BSA/g Chi. No modification of the tertiary and secondary structure of BSA was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan proved to be a useful framework to isolate proteins with a slightly acid isoelectrical pH by means of precipitation.     

Charge effects on folded and unfolded proteins

We develop a theory for the effects of charge on the stabilization of globular proteins. The folding process is modeled as occurring through a fictitious intermediate state along a two-part thermodynamic pathway in which the molecule (i) increases its density and then (ii) rearranges its ionic groups to the protein surface. The equilibrium for the binding of protons in salt solutions is assumed to be driven by the electrical potential due to the charge distribution, in addition to the intrinsic binding affinity and bulk proton concentration. The potential is calculated for inside and outside a porous sphere model of the protein using the Poisson-Boltzmann relation, wherein the interior dielectric constant is taken to be a linear function of the chain density. The model predicts the slope of the titration curves for native myoglobin in agreement with experiments by Breslow and Gurd (1962). From the similar experiments on the unfolded state, and from the experiments of Privalov et al. (1986) on the intrinsic viscosity of the unfolded molecules, the theory shows that the unfolded state has a much higher density than a chain in a theta solvent and that the density increases with ionic strength. In addition, from the free energy of proton binding to the protein, we also calculate the electrostatic contributions to protein stability, a major contribution deriving from changes in ionization. We consider the example of the stability of myoglobin as a function of pH, ionic strength, and ionic groups buried in the native protein structure. We show that although maximum stability of most proteins should occur at their isoelectric point, the burial of nontitratable groups should lead to maximum stabilities at pH values other than the isoelectric point.     

Modulation of the self-assembled structure of biomolecules: coarse grained molecular dynamics simulation

The mechanisms governing the self-assembled structure of biomolecules (single chain and bundle of chains) are studied with an AB copolymer model via the coarse grained molecular dynamics simulations. Non-local hydrophobic interaction is found to play a critical role in the pattern formation of the assembled structure of polymer chains. We show that the polymer structure could be controlled by adjusting the balance between local (short range) and non-local (long range) hydrophobic interaction which are influenced by various factors such as the sequences, chain length, stiffness, confinement, and the topology of polymers. In addition, the competition between the intrachain hydrophobic interaction and interchain hydrophobic interaction determines the structural transition of the chain bundles. This work may provide important insights into the fundamental physics in the structure control and the self-assembly of biomolecules for various practical applications.     

Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography.

1. Lipoamide dehydrogenase NADH: lipoamide oxidoreductase, (EC 1.6.4.3) from pig heart has been separated into two sets of isoenzymes by chromatography on lipoyl- and NAD+-derivatized Sepharose-4B matrices. The first fraction is eluted at 30 mM sodium phosphate buffer (pH 7.2), the other requires a higher ionic strength. The two groups originate from the alpha-ketoglutarate and the pyruvate dehydrogenase complex respectively. 2, Hydrophobic chromatography on a homologous series of alkyl-Sepharoses lead to similar results. The first fraction is eluted with 30 mM phosphate buffer in the case of propyl- and butyl-Sepharose but a high ionic strength is required in the case of an increased chain length (C5--C6). The second fraction is reversibly bound on Sepharose-NC3 and -NC4 but binding becomes irreversible at higher chain lengths. 3. Aminoalkyl-Sepharose behave qualitatively as the alkyl derivatives although elution, particularly in the case of the second fraction, can be realized at lower ionic strength. 4. Matrices which are negatively charged (Sepharose-NCnCOOH, n equal 3--7) have no affinity at pH 7.2. 5. The influence of a neutral polar substituent has been studied by comparing the following matrices: Sepharose-NC6OH, Sepharose-NC6NH2 and Sepharose NC6. Binding of the various isoenzymes is completely reversible in the case of a Sepharose-NC6OH matrix and the elution behaviour is identical to that on propyl- and butyl matrices.     

Modulation of the lipid binding properties of the N-terminal domain of human apolipoprotein E3.

Apolipoprotein E (apoE) plays a critical role in plasma lipid homeostasis through its function as a ligand for the low-density lipoprotein (LDL) receptor family. Receptor recognition is mediated by residues 130-150 in the independently folded, 22-kDa N-terminal (NT) domain. This elongated globular four-helix bundle undergoes a conformational change upon interaction with an appropriate lipid surface. Unlike other apolipoproteins, apoE3 NT failed to fully protect human LDL from aggregation induced by treatment with phospholipase C. Likewise, in dimyristoylglycerophosphocholine (Myr2Gro-PCho) vesicle transformation assays, 100 microg apoE3 NT induced only 15% reduction in vesicle (250 microg) light scattering intensity after 30 min. ApoE3 NT interaction with modified lipoprotein particles or Myr2Gro-PCho vesicles was concentration-dependent whereas the vesicle transformation reaction was unaffected by buffer ionic strength. In studies with the anionic phospholipid dimyristoylglycerophosphoglycerol, apoE3 NT-mediated vesicle transformation rates were enhanced > 10-fold compared with Myr2Gro-PCho and activity decreased with increasing buffer ionic strength. Solution pH had a dramatic effect on the kinetics of apoE3 NT-mediated Myr2Gro-PCho vesicle transformation with increased rates observed as a function of decreasing pH. Fluorescence studies with a single tryptophan containing apoE3 NT mutant (L155W) revealed increased solvent exposure of the protein interior at pH values below 4.0. Similarly, fluorescent dye binding experiments with 8-anilino-1-naphthalene sulfonate revealed increased exposure of apoE3 NT hydrophobic interior as a function of decreasing pH. These studies indicate that apoE3 NT lipid binding activity is modulated by lipid surface properties and protein tertiary structure.     

Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain.

The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60 degrees C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.     

Inhibition of hyaluronan hydrolysis catalysed by hyaluronidase at high substrate concentration and low ionic strength.

Hyaluronidase and high levels of hyaluronan are found together in tumours. It is highly likely that hyaluronidase activity controls the balance between high molecular mass hyaluronan and oligosaccharides, and thus plays an important role in cancer development. The hyaluronan hydrolysis catalysed by bovine testicular hyaluronidase was studied as a model. The kinetics was investigated at pH 5 and 37 degrees C using the colorimetric N-acetyl-d-glucosamine reducing end assay method. While the substrate dependence obtained in the presence of 0.15 mol L(-1) ionic strength exhibited a Michaelis-Menten behaviour, an atypical behaviour was observed under low ionic strength: for increasing hyaluronan concentrations, the initial reaction rate increased, reached a maximum and then decreased to a very low level, close to zero at high substrate concentrations. One of the various hypotheses examined to explain this atypical behaviour is the formation of non-specific complexes between hyaluronan and hyaluronidase based on electrostatic interactions. This hypothesis is the only one that can explain all the experimental results including the variation of the reaction medium turbidity as a function of time and the influence on the initial reaction rate of the hyaluronan concentration over hyaluronidase concentration. However, phenomena such as the high viscosity of highly concentrated hyaluronan solutions or the steric exclusion of hyaluronidase from hyaluronan solutions may contribute to the atypical behaviour. Finally, the biological implications of the non-linear and non-monotonous shape of the hyaluronan-hyaluronidase substrate dependence in the regulation of the hyaluronan chain molecular mass are discussed, in particular in the case of cancer development.     

Strong impact of ionic strength on the kinetics of fibrilar aggregation of bovine beta-lactoglobulin

We investigate the effect of ionic strength on the kinetics of heat-induced fibrilar aggregation of bovine beta-lactoglobulin at pH 2.0. Using in situ light scattering we find an apparent critical protein concentration below which there is no significant fibril formation for all ionic strengths studied. This is an independent confirmation of our previous observation of an apparent critical concentration for 13 mM ionic strength by proton NMR spectroscopy. It is also the first report of such a critical concentration for the higher ionic strengths. The critical concentration decreases with increasing ionic strength. Below the critical concentration mainly "dead-end" species that cannot aggregate anymore are formed. We prove that for the lowest ionic strength this species consists of irreversibly denatured protein. Atomic force microscopy studies of the morphology of the fibrils formed at different ionic strengths show shorter and curvier fibrils at higher ionic strength. The fibril length distribution changes non-monotonically with increasing ionic strength. At all ionic strengths studied, the fibrils had similar thicknesses of about 3.5 nm and a periodic structure with a period of about 25 nm.     

The insertion of D-beta-hydroxybutyrate apodehydrogenase into phospholipid monolayers and phospholipid vesicles

The strong interaction of D-beta-hydroxybutyrate dehydrogenase with phospholipid monomolecular films is demonstrated by the surface pressure increase of a film compressed up to 33 mN/m. Although the D-beta-hydroxybutyrate apodehydrogenase was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged monolayers such as those made from diphosphatidylglycerol. The weakest interaction was found with phosphatidylcholine, which is the reactivating phospholipid for the enzyme. These interactions were dependent on the phospholipid chain length, ionic strength, and pH. At basic pH the apoenzyme lost its specificity for negatively charged phospholipids, suggesting the deprotonation of a cationic amino acid residue of the enzyme polypeptide chain. The charge effects are in agreement with results obtained using phospholipid vesicles. Beside the electrostatic interactions, the influence of phospholipid chain length and the ionic strength indicate that D-beta-hydroxybutyrate apodehydrogenase penetrates into the hydrophobic part of the lipid interface.     

Ionic control of enzymic degradation of double-stranded RNA

The pattern of the degradation of various double-stranded polyribonucleotides by several ribonucleases (bovine RNAase A and its cross-linked dimer, bovine seminal RNAase, and pike-whale pancreatic RNAase) has been studied as a function of ionic strength and pH. It appears that (1) there is no direct correlation between the secondary structure of double-stranded RNA and its resistance against enzymatic breakdown, i.e., the stability of the secondary structure of double-helical RNA is not the main variable in the process. (2) The acstivity responses of the enzymes examined to changes of ionic strength and pH suggest that enzymic degradation of double-stranded RNA is mainly controlled by ion concentration, and that the process may fall within the phenomena interpreted by the theory of the ionic control of biochemical reactions advanced by Douzou and Maurel (Douzou, P. and Maurel, P. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1013--1015). (3) The activity curves of the enzyme studied show, at a given pH, a shift toward higher ionic strengths as a function of the basicity of the enzyme protein. This finding explains the already observed correlation between number and/or density of positive charges of a ribonuclease molecule and its ability to attack double-stranded RNA in 0.15 M sodium chloride/0.015 M sodium citrate (SSC). (4) A careful analysis of the influence of ionic strength and pH on the reaction appears to be necessary in order to characterize a ribonuclease which shows activity towards double-stranded RNAs, and to allow a meaningful comparison between different enzymes capable of attacking these substrates.     

Mechanistic and structural analyses of the roles of Arg409 and Asp402 in the reaction of the flavoprotein nitroalkane oxidase

The flavoprotein nitroalkane oxidase (NAO) catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones. The enzyme is a homologue of acyl-CoA dehydrogenase. Asp402 in NAO has been proposed to be the active site base responsible for removing the substrate proton in the first catalytic step; structurally it corresponds to the glutamate which acts as the base in medium chain acyl-CoA dehydrogenase. In the active site of NAO, the carboxylate of Asp402 forms an ionic interaction with the side chain of Arg409. The R409K enzyme has now been characterized kinetically and structurally. The mutation results in a decrease in the rate constant for proton abstraction of 100-fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-ray crystallography to a resolution of 2.65 A, shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively charged nitrogen in the side chain of the residue at position 409. The D402E mutation results in a smaller decrease in the rate constant for proton abstraction of 18-fold. The structure of the D402E enzyme, determined at 2.4 A resolution, shows that there is a smaller increase in the distance between Arg409 and the carboxylate at position 402, and the interaction of this residue with Ser276 is perturbed. These results establish the critical importance of the interaction between Asp402 and Arg409 for proton abstraction by nitroalkane oxidase.     

Formation of micelles and membrane action of the local anesthetic tetracaine hydrochloride

The formation of micelles of the local anesthetic tetracaine hydrochloride in aqueous phosphate buffer solution of pH 6.5 and ionic strength ($\text{I}$) 0.10 was examined at 22°C by surface tension and using the fluorescent indicators perylene (peri-dinaphthalene) and 8-anilino-1-naphthalene sulfonic acid, sodium salt (ANS). The critical micelle concentration was located at 0.069, 0.071 and 0.063 M by measurements of surface tension, perylene solubilization and enhancement of ANS fluorescence, respectively. In contrast to other cationic surfactants, the anesthetic monomer did not show evidence of forming a fluorescent molecular complex with ANS under the experimental conditions of this study.The formation of micelles by tetracaine-HCl showed a pronounced effect on lipid membranes by inducing an abrupt decrease in the scattered light of egg lecithin liposomes at an anesthetic concentration roughly similar to its critical micelle concentration. This optical behaviour is characteristic of liposome damage and can be interpreted to mean that the lipids become solubilized into tetracaine-HCl micelles.The ability of this local anesthetic to form micelles can be taken as a manifestation of the same hydrophobic forces that lead to partitioning of the drug into membranes.