Relationship between the molecular structure of aqueous solutions of polyethylene glycol and the compactness of double-stranded DNA molecules

The dependence of viscosity of the water solutions of poly(ethylene glycol) (PEG) on the molecular weight has been studied. It has been shown that there is a "transitional" region in PEG properties which accounts for the formation of fluctuation polymer network of the PEG molecules. It has been shown that the "transitional" region in properties of PEG which appears at a certain concentration of PEG (CtrPEG) is characteristic of the PEG preparations with molecular weights exceeding 600 and dependence of the value of CtrPEG on the molecular weight of PEG was obtained. Compactization of double-stranded DNA molecules in PEG-containing water-salt solutions has been studied and the dependence of the value of CcrPEG, . i.e. the concentration of PEG at which the compact particles of DNA appear in the solution, on the molecular weight of PEG was obtained. The correlation between these two dependences reflecting quite different physico-chemical processes shows that the double-stranded DNA molecules are constrained within the polymer network of the PEG molecules. The influence of ionic strength and ionic composition of the solution on the formation of a compact form was investigated. The transition of the DNA molecules from a linear to a compact state may occur only at a definite value of ionic strength of the solution. This transition may occur at the change of K+ for Na+ cations (at a constant value of CPEG). The extent of compactization of the DNA molecules in PEG-containing water-salt solutions is monitored by the molecular structure and by the ionic strength of the solvent. It is supposed that the peculiarities of compactization of the DNA molecules in PEG-containing water-salt solutions reflect some characteristics of conformational transitions of the DNA molecules which occur in vivo.     

Thermodynamic stability of the ordered conformations of carrageenan polyelectrolytes

Manning's counterion condensation theory has been applied to the temperature-induced conformational transition of κ- and ι-carrageenan in the solution and gel states. The formalism of the theory has been extended to transitions between conformations with charge densities below or across the counterion condensation threshold. Measurements of the dependence of the melting temperature on ionic strength, and of the enthalpy of melting, are interpreted with the theory as indicating that the conformational transition is intramolecular and that side-by-side dimerization of chains gives rise to the gel structure.     

Photoacoustic characterization of protein dynamics following CO photodetachment from fully reduced bovine cytochrome c oxidase

We report a protein conformational change following carbon monoxide photodetachment from fully reduced bovine cytochrome c oxidase that is hypothesized to be associated with changes in ligand mobility through a dioxygen access channel in the protein. Although not resolved by earlier photoacoustic or optical studies on this adduct, utilization of slightly lower temperatures revealed a process with a kinetic lifetime of about 70 ns at 10 degrees C. We measure an enthalpy change of about 8 kcal/mol in 0.050 M HEPES buffer that becomes less endothermic (DeltaH approximately 2 kcal/mol) at higher ionic strength. The volume contraction of about -0.7 mL/mol associated with the process almost doubles in higher ionic strength buffer systems. Measurements of samples in phosphate buffer systems are similar and appear to display the same subtle ionic strength dependence. Both the isolation of this photoacoustic signal component and the possible dependence on ionic strength of the thermodynamic parameters derived from its analysis appear analogous to and consistent with prior photoacoustic results monitoring CO photodetachment from the camphor complex of cytochrome P-450. Accordingly, we consider a similar model in which a conformational change results in movement of an exposed charged group or groups towards the interior of the protein, out of contact with solvent, as in the closing of a salt bridge.     

Effect of ionic strength and oxidation on the P-loop conformation of the protein tyrosine phosphatase-like phytase, PhyAsr

The protein tyrosine phosphatase (PTP)-like phytase, PhyAsr, from Selenomonas ruminantium is a novel member of the PTP superfamily, and the only described member that hydrolyzes myo-inositol-1,2,3,4,5,6-hexakisphosphate. In addition to the unique substrate specificity of PhyAsr, the phosphate-binding loop (P-loop) has been reported to undergo a conformational change from an open (inactive) to a closed (active) conformation upon ligand binding at low ionic strength. At high ionic strengths, the P-loop was observed in the closed, active conformation in both the presence and absence of ligand. To test whether the P-loop movement can be induced by changes in ionic strength, we examined the effect that ionic strength has on the catalytic efficiency of PhyAsr, and determined the structure of the enzyme at several ionic strengths. The catalytic efficiency of PhyAsr is highly sensitive to ionic strength, with a seven-fold increase in k(cat)/K(m) and a ninefold decrease in K(m) when the ionic strength is increased from 100 to 500 mm. Surprisingly, the P-loop is observed in the catalytically competent conformation at all ionic strengths, despite the absence of a ligand. Here we provide structural evidence that the ionic strength dependence of PhyAsr and the conformational change in the P-loop are not linked. Furthermore, we demonstrate that the previously reported P-loop conformational change is a result of irreversible oxidation of the active site thiolate. Finally, we rationalize the observed P-loop conformational changes observed in all oxidized PTP structures.     

Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase.

The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to be -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCl. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCl). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational prerequisites for formation of amyloid fibrils from histones

We demonstrate that bovine core histones are natively unfolded proteins in solutions with low ionic strength due to their high net positive charge at pH 7.5. Using a variety of biophysical techniques we characterized their conformation as a function of pH and ionic strength, as well as correlating the conformation with aggregation and amyloid fibril formation. Tertiary structure was absent under all conditions except at pH 7.5 and high ionic strength. The addition of trifluoroethanol or high ionic strength induced significant alpha-helical secondary structure at pH 7.5. At low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC indicate the histones are present as a hexadecamer of globular subunits. The secondary structure at low pH was independent of the ionic strength or presence of TFE, as judged by FTIR. The data indicate that histones are able to adopt five different relatively stable conformations; this conformational variability probably reflects, in part, their intrinsically disordered structure. Under most of the conditions studied the histones formed amyloid fibrils with typical morphology as seen by electron microscopy. In contrast to most aggregation/amyloidogenic systems, the kinetics of fibrillation showed an inverse dependence on histone concentration; we attribute this to partitioning to a faster pathway leading to non-fibrillar self-associated aggregates at higher protein concentrations. The rate of fibril formation was maximal at low pH, and decreased to zero by pH 10. The kinetics of fibrillation were very dependent on the ionic strength, increasing with increasing salt concentration, and showing marked dependence on the nature of the ions; interestingly Gdn.HCl increased the rate of fibrillation, although much less than NaCl. Different ions also differentially affected the rate of nucleation and the rate of fibril elongation.     

Structural changes in the contractile proteins of muscle fiber studied by polarization ultraviolet fluorescence microscopy. IX. The effect of the pH and ionic strength of the solution on the conformational restructurings of F-actin induced by the binding of heavy meromyosin

The dependence of F-actin conformational changes induced by the F-actin-HMM complex on pH and ionic strength was found by polarized ultraviolet fluorescence microscopy. It is discovered that pH affects sufficiently the cooperativity of F-actin structural changes, while the ionic strength affects their depth. The actomyosin complex was supposed to be at least in two structural states, differing in their orientation as well as in flexibility of F-actin monomers.     

Changes in chromatin folding in solution

The formation of higher order structures by nucleosome oligomers of graded sizes with increasing ionic strength has been studied in solution, by measuring sedimentation coefficients. Nucleosome monomers and dimers show no effect of ionic strength at the concentrations used, while trimers to pentamers show a linear dependence of the logarithm of sedimentation coefficient upon the logarithm of ionic strength between 5 and 25 mm, but no dependence above 25 mm. Between pentamer and hexamer a change occurs and the linear relationship is observed up to ionic strength 125 mm with hexamer and above.The simple power-law dependence of the sedimentation coefficient upon the ionic strength (s ∝ Iⁿ) is observed up to nucleosome 30mers, but by 60mer a jump in the sedimentation coefficient occurs between ionic strengths 45 and 55 mm, with the power-law applying both above and below the jump. Removal of histone H1 and non-histone proteins lowers the overall sedimentation rate and abolishes the jump.Cross-linking large oligomers at ionic strength 65 mm stabilizes the structure in the conformation found above the jump, leading to a simple power-law dependence throughout the range of ionic strength for cross-linked material. Cleavage of the cross-links restores the jump, presumably by allowing the conformational transition that causes it. Large oligomers are indistinguishable in sedimentation behaviour whether extracted from nuclei at low ionic strength or at 65 mm and maintained in the presence of salt.We interpret these results, together with the detailed electron microscopic studies reported by Thoma et al. (1979) under similar salt conditions, as showing the histone H1-dependent formation of superstructures of nucleosomes in solution induced by increasing ionic strength. The unit of higher order structure probably contains five or six nucleosomes, leading to the change in stability with hexamer. Although this size corresponds to the lower limit of size suggested for “superbeads” (Renz et al., 1977), we see no evidence that multiples of six nucleosomes have any special significance as might be predicted if superbeads had any structural importance. Rather, our results are compatible with a continuous pattern of condensation, such as a helix of nucleosomes (see e.g. Finch & Klug, 1976). The jump in sedimentation observed between ionic strengths 45 and 55 mm, together with the effect of cross-linking, suggests the co-operative stabilization of this structure at higher ionic strengths. A plausible hypothesis is that the turns of the solenoid are not tightly bonded in the axial direction below 45 mm, but come apart due to the hydrodynamic shearing forces in the larger particles leading to less compact structures with slower sedimentation rates. Above 55 mm the axial bonding is strong enough to give a stable structure of dimensions compatible with the 30 nm structures observed in the cell nucleus.     

Dynamic structure of metallothionein

Molecular sieve chromatography of rabbit liver metallothionein at different electrolyte concentrations revealed that this protein undergoes an increase in Stokes radius from 1.50 to 1.78 nm when the ionic strength is lowered from 0.5 to 0.015 indicating a change in molecular shape and/or hydration. The variation in ionic strength also affects the far-UV circular dichroism of metallothionein reflecting a conformational transition in the protein. The effects are attributed to changes in intramolecular repulsion between the strongly negatively charged metal-thiolate clusters of the protein. It is suggested that metallothionein exists in at least two interchangeable conformational states which differ in hydrodynamic properties and whose equilibrium concentrations are determined by the electrostatic free energy of the system.     

Modifications of single acetylcholine-activated channels in BC3H-1 cells. Effects of trimethyloxonium and pH

We have examined the effects of chemical modification with trimethyloxonium (TMO) and changes in external pH on the properties of acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by approximately 60%. G omega seal patch-clamp recordings of single ACh channel currents showed that the reduction in ACh sensitivity is due to alterations in both the current-carrying and the kinetic properties of the channels. Under all our experimental conditions, i.e., in external solutions of normal or low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh single-channel conductance to 70-90% of normal. The effects of TMO on channel kinetics were dependent on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by approximately 25%. A similar reduction in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics of modified channels. In low ionic strength solutions normal ACh channel open times were maximal at approximately pH 6.7 and decreased by three- to fourfold at both acid and alkaline pH. TMO modification removed the pH dependence of channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally titratable groups on the external surface of ACh channels that help to determine both the gating and permeability properties of ACh channels.     

'Smart' polymers and what they could do in biotechnology and medicine.

Stimulus-responsive or 'smart' polymers undergo strong conformational changes when only small changes in the environment (e. g. pH, temperature, ionic strength) occur. These changes result in phase separation from aqueous solution or order-of-magnitude changes in hydrogel size. Smart polymers are used in bioseparation and drug delivery, for the development of new biocatalysts, as biomimetic actuators, and as surfaces with switchable hydrophobic-hydrophilic properties.     

Binding of basic pancreatic trypsin inhibitor and related isoinhibitors to leukocytic elastase. Determination of thermodynamic parameters

The effect of temperature, ionic strength and solvation power of mono- and divalent cations on the interaction of BPTI-like inhibitors with human leukocytic elastase has been determined. The binding process is characterized by a non-linear dependence of the equilibrium association constant on 1/T indicating a thermal transition at temperature values ranging between 20 degrees C and 35 degrees C depending on the solvent. The marked dependence of the thermodynamic parameters (delta H degrees, delta S degrees, delta G degrees) and of the transition temperature on the concentration and nature of the cations present in solution seems to indicate that the transition, probably of conformational nature, is related to removal of water molecules upon enzyme/inhibitor complex formation.     

Inter-domain interaction and the structural flexibility of calmodulin in the connecting region of the terminal two domains

The calcium-dependent difference absorption spectrum of scallop calmodulin was measured in the presence of mastoparan. The difference spectrum at 286 nm (delta A286) showed biphasic response to Ca2+ concentration. The first change represents the conformational change around Tyr-138 and the second change may respond to an interaction between N- and C-domain of calmodulin which became apparent in the associated state with mastoparan. Calmodulin-mastoparan complex was eluted from a gel filtration column after free calmodulin in the presence of Ca2+, which indicates a more compact structure of calmodulin-mastoparan complex than of free calmodulin. The biphasic response of delta A286 was also observed with free calmodulin when the ionic strength was as low as 0.02 M NaCl. In the absence of NaCl, the Ca2+ dependence of delta A288 was monophasic, assuming identical affinity of Ca2+ to both domains. Increase in the sensitivity of calmodulin to trypsin was observed with decrease in ionic strength. These results suggest an ionic-strength-dependent decrease in ordered structure of the connecting region. Calmodulin may change shape depending upon the ionic strength by bending at the connecting region. We assumed from the observations that calmodulin in solution may fluctuate between the two extreme shapes of the bent and the dumbbell structure. Target proteins may select and fix the specific bent structure for their activation.     

The solution structure of small peptides: An IR CD study of aqueous solutions of (L-Ala)n [n = 3, 4, 5, 6] at different temperatures and ionic strengths

The solution conformation of a number of small, linear alanine oligomers was investigated via ir (or vibrational) CD (VCD). We find that these oligopeptides assume distinct solution conformations that depend primarily on chain lengths, and to a lesser degree on temperature, ionic strength, and pH. As expected, the longer chain oligomers exhibit more distinct VCD features and, presumably, more stable solution structures. At the level of the hexamer, however, aggregation of the peptide occurs. The fast time scale of VCD allows solution structures to be detected that may not be observable using slower techniques such as various forms of nmr spectroscopy. The VCD results reported here confirm that it is generally possible to obtain conformational information for small, linear homo- and heterooligopeptides via VCD spectroscopy. In this respect, the sensitivity of VCD is similar to that of electronic CD. Furthermore, the temperature dependence of the VCD results indicate that at elevated temperatures, the increasing number of conformational states results in a loss of discernible conformers, and consequently, a broadening and weakening of the VCD features. © 1998 John Wiley & Sons, Inc. Biopoly 46: 455–463, 1998     

Structural transformations of oligonucleosomes from pigeon erythrocyte chromatin

The methods of velocity sedimentation and circular dichroism have been used to investigate structural rearrangements of pigeon erythrocyte oligonucleosomes isolated after digestion with micrococcal nuclease (oligonucleosomes-M) or pancreatic DNase I (oligonucleosomes-D), in the wide range of ionic strength (mu from 0.005 to 0.5). The electrophoretic analysis of DNA isolated from the oligonucleosomes has revealed internal cuts in the DNA chain of oligonucleosomes-D. In spite of this fact the conformational parameters of DNA in both types of oligonucleosomes are practically indistinguishable, and their optical and hydrodynamic properties vary in a similar way with increasing ionic strength of the solution. The specificity of DNase I action results in the ability of oligonucleosomes-D to form homogeneous associates at mu = 0.065, which seems to be due to the existence of elongated intact ends of linker DNA in oligonucleosomes-D. It has been shown that the integrity of oligonucleosomes-D in a wide range of ionic strength is maintained by histones H1 and H5, because after their dissociation the sedimentation coefficient sharply decreases. The results obtained reveal the multifunctional role of lysine-rich histones and intact linker in the processes of compaction and association of oligonucleosomes.     

Probing the electrostatic shielding of DNA with capillary electrophoresis

The free solution mobility of a 20-bp double-stranded DNA oligomer has been measured in diethylmalonate (DM) and Tris-acetate buffers, with and without added NaCl or TrisCl. DM buffers have the advantage that the buffering ion is anionic, so the cation composition in the solution can be varied at will. The results indicate that the free solution mobility of DNA decreases linearly with the logarithm of ionic strength when the ionic strength is increased by increasing the buffer concentration. The mobility also decreases linearly with the logarithm of ionic strength when NaCl is added to NaDM buffer or TrisCl is added to TrisDM buffer. Nonlinear effects are observed if the counterion in the added salt differs from the counterion in the buffer. The dependence of the mobility on ionic strength cannot be predicted using the Henry, Debye-Hückel-Onsager, or Pitts equations for electrophoresis. However, the mobilities observed in all buffer and buffer/salt solutions can be predicted within approximately 20% by the Manning equation for electrophoresis, using no adjustable parameters. The results suggest that the electrostatic shielding of DNA is determined not only by the relative concentrations of the various ions in the solution, but also by their equivalent conductivities.     

Partially unfolded states of beta(2)-microglobulin and amyloid formation in vitro

Dialysis-related amyloidosis (DRA) involves the aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils. Using Congo red and thioflavin-T binding, electron microscopy, and X-ray fiber diffraction, we have determined conditions under which recombinant monomeric beta(2)m spontaneously associates to form fibrils in vitro. Fibrillogenesis is critically dependent on the pH and the ionic strength of the solution, with low pH and high ionic strength favoring fibril formation. The morphology of the fibrils formed varies with the growth conditions. At pH 4 in 0.4 M NaCl the fibrils are approximately 10 nm wide, relatively short (50-200 nm), and curvilinear. By contrast, at pH 1.6 the fibrils formed have the same width and morphology as those formed at pH 4 but extend to more than 600 nm in length. The dependence of fibril growth on ionic strength has allowed the conformational properties of monomeric beta(2)m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pK(a) of 4.7 destabilizes native beta(2)m and generates a partially unfolded species. On average, these molecules retain significant secondary structure and have residual, non-native tertiary structure. They also bind the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), show line broadening in one-dimensional (1)H NMR spectra, and are weakly protected from hydrogen exchange. Further acidification destabilizes this species, generating a second, more highly denatured state that is less fibrillogenic. These data are consistent with a model for beta(2)m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies.     

Pharmacology and Surface Electrostatics of the K Channel Outer Pore Vestibule

In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.1 channels was more sensitive to solution ionic strength than was TEA block, a result consistent with a contribution from an electrostatic potential near the blocking site. In contrast, TEA block of another type of K channel (Kv2.1) was insensitive to solution ionic strength and these channels were resistant to block by gallamine. Neutralizing either of two lysine residues in the outer vestibule of these Kv2.1 channels conferred ionic strength sensitivity to TEA block. Kv2.1 channels with both lysines neutralized were sensitive to block by gallamine, and the ionic strength dependence of this block was greater than that for TEA. These results demonstrate that Kv3.1 (like Shaker) channels contain negatively charged residues in the outer vestibule of the pore that influence quaternary ammonium pharmacology. The presence of specific lysine residues in wild-type Kv2.1 channels produces an outer vestibule with little or no net charge, with important consequences for quaternary ammonium block. Neutralizing these key lysines results in a negatively charged vestibule with pharmacological properties approaching those of other types of K channels.     

Thermodynamic and conformational studies on an immunoglobulin light chain which reversibly precipitates at low temperatures.

A lambda light chain, isolated from an immunoglobulin G molecule, was found to reversibly precipitate at low temperatures. This cryoprecipitation was a function of pH, ionic strength, protein concentration, and time as well as temperature. The lambda chain underwent a cooperative conformational change as the temperature was lowered from 26 to 0 degrees C as judged by ultraviolet difference spectroscopy and circular dichroism. Normal lambda chains showed no conformational change. By difference spectroscopy it was possible to calculate the equilibrium constant governing the conformational change. The change was strongly exothermic (delta H approximately -80 kcal mol-1) and accompanied by a large decrease in entropy (delta S approximately -280 eu). The midpoint of the transition was dependent on the initial protein concentration, suggesting that only the noncovalent dimer of the lambda chain exhibited the conformational change. The existence of a monomer-dimer eqiulibrium (KA approximately 4 X 10(5) M-1) was confirmed by sedimentation velocity. No conformational change was observed by circular dichroism at concentrations where greater than 95% of lambda chain was in the form of a monomer. Although high ionic strength inhibited cryoprecipitation, it had no effect on the conformational change. Stabilization of the dimer by forming an interchain disulfide bond between two monomers abolished both the conformational change and cryoprecipitation. A fragment corresponding to the constant region was isolated from both peptic and tryptic digests of the lambda chain. This fragment neither cryoprecipitated nor showed temperature dependence conformational changes. It proved impossible to isolate a fragment corresponding to the variable region. Both qualitative and quantitative models are presented to account for the behavior of the lambda chain at low temperatures.