Slow relaxation process in DNA at different levels of hydration

There are many speculations about the dynamic transition observed in hydrated bio-polymers at temperatures T 200 – 230 K being an important factor for enabling of their functions. The transition shows up as a sharp increase of atomic mean-squared displacements above this temperature. The nature of the dynamic transition is not yet clear. Using inelastic neutron scattering we show in this Note that the transition in DNA is related to the appearance of a slow relaxation process. Decrease in the hydration level suppresses the process and the dynamic transition. It is found that, in terms of dynamics, the decrease in water content is similar in effect to a decrease in temperature. The obtained results support the idea that the dynamic transition is mediated by the water of hydration since bulk water has a dynamic transition around the same temperature.     

Influence of hydration on the dynamics of lysozyme

Quasielastic neutron and light-scattering techniques along with molecular dynamics simulations were employed to study the influence of hydration on the internal dynamics of lysozyme. We identified three major relaxation processes that contribute to the observed dynamics in the picosecond to nanosecond time range: 1), fluctuations of methyl groups; 2), fast picosecond relaxation; and 3), a slow relaxation process. A low-temperature onset of anharmonicity at T approximately 100 K is ascribed to methyl-group dynamics that is not sensitive to hydration level. The increase of hydration level seems to first increase the fast relaxation process and then activate the slow relaxation process at h approximately 0.2. The quasielastic scattering intensity associated with the slow process increases sharply with an increase of hydration to above h approximately 0.2. Activation of the slow process is responsible for the dynamical transition at T approximately 200 K. The dependence of the slow process on hydration correlates with the hydration dependence of the enzymatic activity of lysozyme, whereas the dependence of the fast process seems to correlate with the hydration dependence of hydrogen exchange of lysozyme.     

Separating the contributions to 15N transverse relaxation in a fibronectin type III domain

In proteins, dynamic mobility is an important feature of structure, stability, and biomolecular recognition. Uniquely sensitive to motion throughout the milli- to picosecond range, rates of transverse relaxation, R2, are commonly obtained for the characterization of chemical exchange, and the construction of motional models that attempt to separate overall and internal mobility. We have performed an in-depth study of transverse relaxation rates of backbone 15N nuclei in TNfn31–90, the third fibronectin type III domain from human tenascin. By combining the results of spin-echo (CPMG) and off-resonance T1 experiments, we present R2 rates at effective field strengths of 2 to 40 krad/s, obtaining a full spectrum of 16 independent R2 data points for most residues. Collecting such a large number of replicate measurements provides insight into intrinsic uncertainties. The median standard deviation in R2 for non-exchanging residues is 0.31, indicating that isolated measurements may not be sufficiently accurate for a precise interpretation of motional models. Chemical exchange events on a timescale of 570 s were observed in a cluster of residues at the C terminus. Rates of exchange for five other residues were faster than the sampled range of frequencies and could not be determined. Averaged 'exchange free' transverse relaxation rates, R20, were used to calculate the diffusion tensor for rotational motion. Despite a highly asymmetric moment of inertia, the narrow angular dispersion of N-H vectors within the sandwich proves insufficient to define deviations from isotropic rotation. Loop residues provide exclusive evidence for axially symmetric diffusion (Dpar/Dper=1.55).     

Effect of CPT on the calf thymus Topoisomerase I-mediated DNA breakage-reunion reaction: Optimal conditions for the formation and reversal of the CPT trapped Topoisomerase I cleavable complex

The effects of CPT on the calf thymus Topoisomerase I-mediated DNA breakage-reunion reaction were studied at an enzyme concentration range proper for evidencing, at the same time, both DNA relaxation and DNA cleavage/religation. Some of the requirements and the optimal conditions for the formation and reversal of the CPT-trapped Topoisomerase I-DNA cleavable complex are also characterized. We conclude that:

1. Calf thymus (100 kDa) Topoisomerase I requires, for maximal DNA cleavage activity, specific and characteristic reaction conditions.
2. CPT does not affect these optimal conditions, but only stabilizes the normal enzyme-DNA intermediate. In this way, the drug lowers the religation process, becoming responsible for the relaxation inhibition.
3. The optimum of monovalent salt concentration for cleavable complex formation is found between 30 and 70 mM. These values are lower than those required for the relaxation activity optimum (75–125 mM NaCl).
4. The addition of 0.5 M monovalent salt causes reversal of the reaction, and shifts the equlibrium distribution between cleavable intermediate and closed relaxed DNA in the direction of DNA resealing. Therefore, it is suggested that salt affects the cleavage but not the religation reaction.

     

Dimyristoylphosphatidic acid/cholesterol bilayers

Lipid bilayers and monolayers composed of dimyristoylphosphatidic acid (DMPA) and cholesterol were characterized by differential scanning calorimetry and film balance measurements. Increasing cholesterol content decreases the bilayer phase transition temperature and enthalpy in a manner similar to that observed before for other lipid/cholesterol systems. In monomolecular films at the air-water interface cholesterol exhibits the well known condensing effect in the liquid-expanded phase, while the liquid-condensed phase is less affected. As with the bilayer phase transition, the transition temperature and change in area at the liquid-condensed to liquid-expanded phase transition, as measured from isobars at 25 dynes/cm, decreases with increasing cholesterol content. The kinetics of the phase transition of DMPA/cholesterol bilayers were measured using the pressure jump relaxation technique with optical detection. Three relaxation times were observed. The relaxation times and amplitudes pass through maximum values at the transition midpoint. With increasing cholesterol content the maximum values of the relaxation times decrease but not in a linear fashion. The time constants display an intermediate maximum at ca. 10% to 12 mol% cholesterol. This observation is discussed in terms of a possible change in the nature of the phase transition from first-order with phase separation to a continuous second-order transition. The dependence of the relaxation amplitudes on cholesterol content gave evidence for nucleation being the rate limiting step for the transition in this particular system.Abbreviations DMPA dimyristoylphosphatidic acid - DMPC dimyristoylphosphatidylcholine - DMPE dimyristoylphosphatidylethanolamine - DPPC dipalmitoylphosphatidylcholine - DSC differential scanning calorimetry Part of this research has been presented at the VIII. Discussion Group Meeting Fast Reactions in Solution of the Royal Society of Chemistry and the Max-Planck-Gesellschaft, Berlin, 26th–29th August 1984     

The reaction of aromatic peptides with double helical DNA. Quantitative characterisation of a two step reaction scheme

The binding of LysTrpLys and LysTyrLys to calf thymus DNA has been investigated by the field jump method using fluorescence detection. Two separate relaxation processes, clearly distinguished on the time scale and by opposite ampli- tudes, are observed for the binding of LysTrpLys to DNA with ~ 30000 base pairs. The concentration dependence of the relaxation time constants demonstrates a mechanism with a bimolecular step followed by a slow intramolecular transition with a forward rate of 6.4 X 103 s^?1 and an equilibrium constant of 11. Measurements at various degrees of peptide binding demonstrate that the binding mechanism associated with low binding rates is restricted to a rather low number of binding sites (roughly one site in 15 base pairs). The binding of LysTyrLys to the same DNA is not associated with relaxation pro- cesses of opposite amplitudes; nevertheless two processes could be identified and assigned to a two step mechanism corre- sponding to that observed in the case of LysTrpLys. In the presence of sonicated DNA both peptides show a single relaxa- tion process with characteristics similar to those observed for the slow process in the binding to high molecular DNA. The data indicate that the intramolecular step is faster for low than for high molecular DNA. These results suggest an assignment of the intramolecular step to an insertion of the aromatic residues into the DNA associated with bending of the helix. The increase in the rate of the intramolecular step with decreasing chain length of the DNA may then be explained by a higher flexibility of the double helix at lower chain lengths.     

Sodium ion and solvent nuclear relaxation results in aqueous solutions of DNA

The field-dependent ²³Na nuclear relaxation in aqueous DNA solutions has been obtained for a range of temperatures, including the DNA melting region. At least two correlation times are needed to characterize the spectral density function for the ²³Na relaxation. For the slow process (with the largest correlation time), the temperature dependence of the coupling constant and the correlation time were determined, and important premelting effects were observed. Possible origins of the slow process are discussed. The last process is shown to be correlated with the properties of the hydration water of DNA as reflected by the ¹⁷O relaxation rates in these solutions. The influence of the polyelectrolyte and NaCl concentrations on the ²³Na relaxation rate is compared with previous results from solutions of linear flexible polyelectrolytes.     

Identification of a splice-site mutation in the low density lipoprotein receptor gene by denaturing gradient gel electrophoresis

We have applied the denaturing gradient gel electrophoresis (DGGE) technique to detect sequence variations in exon 9 of the low density lipoprotein receptor (LDLR) gene in individuals with heterozygous familial hypercholesterolemia (FH). A fragment containing exon 9 and 25 base pairs (bp) of the intron boundary sequence at either side was amplified. To this fragment a 40-bp GC-clamp was attached by the polymerase chain reaction (PCR). We have analyzed a total of 165 DNA samples of FH patients and have detected a mutation in three cases. Two patients were found to have the previously described South African G to A transition in codon 408. In a third patient, we observed a different banding pattern of the DNA fragments on DGGE indicating a different mutation. The mutant homoduplex band of this sample was purified from the gel, cloned in an AT-vector and sequenced. Sequence analysis demonstrated a G to A transition of the consensus G-nucleotide at the intron 9 splice donor site. Cosegregation between this mutation and elevated plasma cholesterol levels was observed in family members of this FH patient. This mutation probably prevents normal splicing of the mRNA and represents the first identified splice-site mutation in the LDLR gene. We conclude that the use of DGGE of GC-clamped PCR-amplified exon sequences offers a general strategy for the detection of disease-producing mutations in the LDLR gene.     

Dynamic fluorescence measurements on the main phase transition of dipalmytoylphosphatidylcholine vesicles

The kinetics of the main phase transition in dipalmytoylphosphatidylcholine (DPPC) vesicles have been investigated using our iodine laser-Tjump technique with fluorescence detection. A set of three fluorescent probes has been used to sense different parts of the bilayer hydrocarbon chain region. The well established membrane probes DPH and TMADPH as well as DPHPC, a labelled DPPC molecule. We report three relaxation signals in the s and ms time range, which are detected with all three probes. This result supports our model of the main phase transition in DPPC vesicles.Abbreviations DMPC Dimyristoylphosphatidylcholine - DPPC Dipalmytoylphosphatidylcholine - DPH 1,6-Diphenylhexa-1,3,5-triene - TMADPH 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene - DPHPC Diphenylhexatriene-phosphatidylcholine - T_m Temperature of the main phase transition     

Influence of Bacteriophage PBS1 and φW-14 Deoxyribonucleic Acids on Homologous Deoxyribonucleic Acid Uptake and Transformation in Competent Bacillus subtilis

Both bacteriophage PBS1 deoxyribonucleic acid (DNA) (in which all the thymine residues are replaced by uracil) and phage W-14 DNA [in which half the thymine residues are replaced by 5-(aminobutylaminomethyl)uracil or 5-putrescinylthymine] exhibit comparable competing abilities for uptake of homologous DNA in a Bacillus subtilis competent system. But, whereas PBS1 DNA leads to a decrease in transformation frequencies compatible with its competing ability for DNA uptake, W-14 DNA decreases transformation frequencies by a factor up to eightfold higher. The effect of W-14 DNA on transformation frequencies is visible even at a concentration level that does not decrease transforming DNA uptake. No such effect was observed with heterologous DNA containing presumably ionically bound putrescine. Low concentrations of W-14 DNA decreased the number of double (nonlinked) transformants more than single transformants. The influence on transformation was abolished when W-14 DNA was added 20 min after addition of transforming DNA, i.e., when the recombination process was terminated. The putrescine-containing DNA also decreased retention of trichloroacetic acid-precipitable radioactivity of homologous DNA taken up. We conclude that W-14 DNA inhibits some intracellular process(es) at the level of recombination. In addition, there is evidence that W-14 DNA, but not heterologous DNA with ionically bound putrescine, binds also to site(s) on the cell surface other than receptors for homologous DNA.     

Broadband dielectric spectroscopy and calorimetric investigations of d-lyxose

Using broadband dielectric spectroscopy, we have studied different types of relaxation processes, namely, primary (α), secondary (β), and another sub-T_g process called γ-process, in the supercooled state of d-lyxose, over a wide frequency (10^-2–) and temperature range (120–340 K). In addition, the same sample was analyzed by differential scanning calorimeter. The temperature dependence of the relaxation times as well as the dielectric strength of different processes has been critically examined. It has been observed that the slower secondary relaxation (designated as β-) process shifts to lower frequencies with increasing applied pressure, but not the faster one. This pressure dependence indicates that the observed slower secondary relaxation (β-) is Johari–Goldstein relaxation process and faster one (γ-process) is probably the rotation of hydroxymethyl (–CH₂OH) side group attached to the sugar ring, that is, of intramolecular origin.     

Quasielastic light scattering: Effect of ionic strength on the internal dynamics of DNA

Quasielastic light scattering is used to study the effect of ionic strength on the dynamic behaviour of DNA. In a first approach the spectrum of scattered light is analyzed in terms of a single relaxation process. The large difference between the observed behaviour and that expected according to a pure diffusional process reflects the contribution associated with internal modes, which increases with decreasing ionic strength. Such behaviour is better analyzed in terms of a double relaxation process by using two relaxation times, the reciprocals of which are equal to DK² and DK² + τ_i^?1 (K), respectively, where τ_i (K) is an average value describing the set of modes observed at a given K value. Relative intensity and relaxation times, which are the more accurate parameters, were used to interpret the results. The observed increase of the relative contribution of internal modes with decreasing ionic strength is actually a relative decrease of the diffusional contribution induced by a corresponding increase of the radius of gyration R_G. On the other hand, the reciprocal τ_i^?1 (K) of the relaxation time is a linear function of K² in the analyzed KR_G range and is insensitive to ionic strength between 10^?2M and 1M. These results, when discussed according to Rouse's model, lead to define for each value of τ_i^?1 (K) a corresponding mean-squared equilibrium length 〈μ〉 which is found to be a linear function of K^?2.     

Dielectric relaxation studies of ionic processes in lysolecithin-packaged gramicidin channels

Summary Dielectric permittivities have been determined for suspensions of lysolecithin packaged malonyl gramicidin channels over the frequency range of 5kHz to 900 MHz and under conditions of approximately equimolar concentrations (10mM) of channels and salts. The salts were lithium chloride, sodium chloride and thallium acetate. A relaxation process unique to the thallium acetate-channel system was observed which on analysis gave rise to a relaxation time at 25⁰ of 120 nsec. The permittivity data, as well as a comparison of binding constants, indicate that the relaxation process results from Tl⁺ being bound within the channel and more specifically from an intrachannel ion translocation with a rate constant of approximately 4×10⁶ sec^–1 and with an energy of activation of less than 6.7 kcal/mole. These data compare favorably with data from conductance studies on planar bilayers and with ion and carbon-13 nuclear magnetic studies on the lysolecithin packaged malonyl gramicidin channels which combine to indicate that the relaxation process is due to the jump of the thallium ion across a central barrier.     

Ionic strength effects on macroion diffusion and excess light-scattering intensities of short DNA rods

Quasielastic and static light-scattering measurements were made on DNA isolated from chicken erythrocyte mononucleosomes as a function of ionic strength between 6 × 10^?4 and 1.0M. A transition from single-exponential autocorrelation functions to markedly non-single-exponential decays was observed around 10^?2M ionic strength and was accompanied by a large decrease in the excess light-scattering intensity. Autocorrelation functions recorded below 10^?2M salt were well fit by the sum of two exponential relaxation which differed by as much as 100-fold in time constants. Apparent diffusion coefficients for the fast and slow processes plateaued around 10^?3M with numerical values approximately 10-fold and 1/10, respectively, of the translational diffusion coefficient for mononucleosome DNA at high ionic strength. This behavior is similar to that observed with poly(L -lysine), for which the slow decay has been associated with a transition to an extraordinary phase. The strong and complex salt dependence observed here illustrates potential difficulties in deriving structural information from scattering by polyions at low ionic strength.     

Observing the Temperature Dependent Transition of the GP2 Peptide Using Terahertz Spectroscopy

The GP2 peptide is derived from the Human Epidermal growth factor Receptor 2 (HER2/nue), a marker protein for breast cancer present in saliva. In this paper we study the temperature dependent behavior of hydrated GP2 at terahertz frequencies and find that the peptide undergoes a dynamic transition between 200 and 220 K. By fitting suitable molecular models to the frequency response we determine the molecular processes involved above and below the transition temperature (T _D). In particular, we show that below T _D the dynamic transition is dominated by a simple harmonic vibration with a slow and temperature dependent relaxation time constant and that above T _D, the dynamic behavior is governed by two oscillators, one of which has a fast and temperature independent relaxation time constant and the other of which is a heavily damped oscillator with a slow and temperature dependent time constant. Furthermore a red shifting of the characteristic frequency of the damped oscillator was observed, confirming the presence of a non-harmonic vibration potential. Our measurements and modeling of GP2 highlight the unique capabilities of THz spectroscopy for protein characterization.     

Activation of human topoisomerase I by protein kinase CK2

The enzymatic studies were performed to reveal a mode of activation of human topoisomerase I by a direct interaction with protein kinase CK2. In the absence of ATP CK2 kinase activated DNA relaxation about twofold. CK2 subunit was identified as solely responsible for the stimulation of relaxing activity by CK2 kinase. CK2 activated the relaxation only at the excess of the substrate over topoisomerase I. At the equimolar ratio of the substrate DNA and topoisomerase I the activation was not observed. There was also no effect of CK2 on camptothecin-induced cleavage of DNA by htopo I. These results identify an accelerated movement of topoisomerase I between substrate molecules as a cause of the activation of DNA relaxation by CK2 kinase.     

Brownian motion of highly charged poly(L-lysine). Effects of salt and polyion concentration

The Brownian motion of a single sample of high-molecular-weight poly(L -lysine) [(Lys)_n, n = 955] has been studied by dynamic light scattering over a wide range of NaBr concentrations and at three different polyion concentrations. A substantial decrease in scattered intensity is associated with the transition from the ordinary phase to the low-salt extraordinary phase. At the salt concentration where the transition takes place the relaxations are non-exponential and appear to exhibit at all angles a rapid relaxation (τ ? 10 μsec) that is presumed to be a manifestation of the kinetics of the transition process. The K² dependence of the slow relaxation rates in the extraordinary phase has been confirmed within the experimental error. The extrapolated infinite-dilution values of the diffusion coefficients in the ordinary phase are observed to decline precipitously below 10^?2M salt to astonishingly small values, indicating a dramatic rise in the friction factors of the isolated polyions. An extensive discussion of these findings in relation to the theory employed here and to existing data in the literature is also given.     

DNA condensation by cobalt hexaammine(III) in alcohol–water mixtures: Dielectric constant and other solvent effects

DNA molecules condense into compact structures in the presence of a critical concentration of multivalent cations. To probe the contribution ofelectrostatic forces to condensation, we used mixtures of water with methanol (MeOH), ethanol (EtOH), and isopropanol (iPrOH) to vary the dielectric constant ? from 80 to 50. The condensation of pUC18 plasmids by hexaammine cobalt (III), Co(NH₃), was monitored by total intensity and dynamic light scattering, electron microscopy, andCD. The total scattering intensity increased as ? went from 80 to 70, and then decreased as ? decreased further. Ultraviolet spectrophotometry confirmed that the loss of intensity at low ? was not due to the particles' settling out of solution. The rate as well as the extent of condensation increased as? was lowered from 80 to 70, and also depended on the species of alcohol (MeOH < EtOH < iPrOH). The hydrodynamic radii R_H of the particles, however, remained roughly the same at 300–350 A and was independent of the species of alcohol. R_H increased below ? = 70. The critical concentration of Co(NH₃) required to induce DNA condensation decreased from 21 μM to about 16 μM as the dielectric constant decreased from 80 to 70, and decreased moderately with the nonpolarity of the alcohol. The fraction of DNA charge neutralized at the onset of DNA condensation was calculated by a modification of Manning's two-variable counterion condensation theory to be 0.90 ± 0.01, independent of ?. By electron microscopy we observed that the condensed particles changed from about 93% toroids at ? = 80 to 89% rods at ? = 70 and 98% rods at ? = 65. At epsi; lower than 65, DNA collapsed into a network of multistranded fibers. The morphology of condensed DNA particles, whether toroids, rods, or fibers, was independent of the alcohol species. CD spectra in ethanol–water mixtures indicated that both closed circular and linearized plasmids were in the B conformation when condensed with Co(NH₃)³⁺₆ at ?≥ 70, although the closed circular molecules exhibited a weak Ψ-DNA spectrum. A transition from the B to A formtook place between ? = 70 and 60, well above the normal dielectric constant of ? = 40 for this transition, indicating that ethanol and Co(NH₃) synergistically promote the B–A transition. We interpret these results to mean that alcohols have both electrostatic and structural effects on DNA, leading to three regimes of condensation. At the lowest alcohol concentrations the B conformation is stableand condensation is relatively slow, allowing time for the packing adjustments necessary to form toroids. At intermediate alcohol concentrations condensation is faster, and the combined effects of solvent and Co(NH₃) locally destabilize the double helix, permitting DNA foldbacks that lead to rodlike condensates. Rods become shorter as wellas more numerous as ? decreases from 80 to 65–60, indicatingincreasing destabilization as alcohol increases. At the lowest dielectric constants, alcohol and Co(NH₃) produce A-DNA, which strongly self-adheres and rapidly aggregates intofibrous networks, not allowing time for more compact condensates to form. © 1995 John Wiley & Sons, Inc.     

Spectral analysis of current noise generated by carrier-mediated ion transport

Summary It is shown that valinomycin-mediated alkali ion transport is associated with a characteristic type of current noise. The spectral intensity of the noise which is measured under equilibrium conditions, i.e., at zero net current, is frequency independent (white) both at high and at low frequencies. The transition between the low- and the high-frequency limit occurs in a frequency range which is related to the characteristic relaxation time constants of the transport system. This behavior is predicted from the carrier transport model on the basis of Nyquist's theorem.     

Phase transition kinetics of lipid bilayer membranes studied by time-resolved pressure perturbation calorimetry

The relaxation kinetics of aqueous lipid dispersions after a pressure jump (p-jump) was investigated using time-resolved pressure perturbation calorimetry (PPC). Analysis of the calorimetric response curves by deconvolution with the instrumental response function gives information about slow processes connected with the lipid phase transition. The lipid transition from the gel to the liquid-crystalline state was found to be a multi-step process with relaxation constants in the seconds range resolvable by time-resolved PPC and faster processes with relaxation times shorter than ca. 5 s that could not be resolved by the instrument. The faster processes comprise ca. 50% of the total heat uptake at the transition midpoint. This is the first calorimetric measurement showing the multi-step nature of the transition. The results are in good agreement with data obtained with other detection methods and with molecular modelling experiments describing the transition as a multi-step process with nucleation and growth steps.