Exchange Lifetime Research Articles

Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme.

In common with other self-cleaving RNAs, the lead-dependent ribozyme (leadzyme) undergoes dynamic fluctuations to a chemically activated conformation. We explored the connection between conformational dynamics and self-cleavage function in the leadzyme using a combination of NMR spin-relaxation analysis of ribose groups and conformational restriction via chemical modification. The functional studies were performed with a North-methanocarbacytidine modification that prevents fluctuations to C2′-endo conformations while maintaining an intact 2′-hydroxyl nucleophile. Spin-relaxation data demonstrate that the active-site Cyt-6 undergoes conformational exchange attributed to sampling of a minor C2′-endo state with an exchange lifetime on the order of microseconds to tens of microseconds. A conformationally restricted species in which the fluctuations to the minor species are interrupted shows a drastic decrease in self-cleavage activity. Taken together, these data indicate that dynamic sampling of a minor species at the active site of this ribozyme, and likely of related naturally occurring motifs, is strongly coupled to catalytic function. The combination of NMR dynamics analysis with functional probing via conformational restriction is a general methodology for dissecting dynamics-function relationships in RNA.

Photochemistry of cis,trans-[Pt(en)(I)2(OH)2] complex in aqueous solutions

Diiodido Pt(IV) complexes (earlier tested as prodrugs in photodynamic therapy) could be considered as model mixed-ligand platinum complexes. In this work, photochemistry of cis,trans‐[PtIV(en)(I)2(OH)2] complex (1) in aqueous solutions was examined in time interval from hundreds of femtoseconds to hours. UV and NMR spectroscopy, nanosecond laser flash photolysis and ultrafast spectroscopy were applied to study the primary photochemical processes of 1. As it was shown earlier, photolysis of the complex is multistage. On the first stage, mer-[PtIV(en)(I)(OH)3] complex is formed with the quantum yield of 0.06 (irradiation at 282nm). Further ligand exchange and photoreduction occur with much lower quantum yield. The characteristic lifetime of ligand exchange is about 90ps. The spectrum of the key intermediate (KI) was recorded in the picosecond time domain. Depending on the attribution of the KI, two tentative schemes of the primary photophysical and photochemical processes are proposed.

Developing High Field MRI Contrast Agents by Tuning the Rotational Dynamics: Bisaqua GdAAZTA‐based Dendrimers

AbstractMonomeric and dimeric AAZTA‐based bifunctional chelators (AAZTA=6‐amino‐6‐methylperhydro‐1,4‐diazepine tetraacetic acid) were attached to different generations (G0, G1 and G2) of ethylenediamine‐cored PAMAM dendrimers (PAMAM=polyamidoamine) to obtain a series of six dendrimeric systems with 4 to 32 chelates at the periphery. These GdIII‐loaded dendrimers have molecular weight ranging from 3.5 to 25 kDa, thus allowing a systematic investigation on the changes in relaxivity (r1) with the variation of the rotational dynamics following the increase in molecular size. Variable‐temperature 17O NMR (on the dimeric building block Gd2L2) and 1H Nuclear Magnetic Relaxation Dispersion measurements at different temperatures indicate that the water exchange lifetime (τM∼90 ns) of the two inner sphere water molecules does not represent a limiting factor to the relaxivity of the systems. The r1 values at 1.5 T (60 MHz) and 298 K increases from 10.2 mM−1 s−1 for the monomer GdL1 to 31.4 mM−1 s−1 for the dendrimer Gd32G2‐32 (+308 %). However, the relaxivity (per Gd) does not show a linear dependence on the molecular mass, but rather the enhancement tends to attenuate markedly for larger systems. This effect has been attributed to the growing decrease in correlation between local rotational motions and global molecular tumbling.

Models of Isotopic Water Diffusion in Spherical Aerosol Particles.

Isotopic exchange experiments that utilize D2O and H2O have received attention as a method for studying water diffusion in high viscosity aerosol particles. However, the mathematical models used to retrieve diffusion coefficients from these measurements have yet to be critically examined. Here, two models for the isotopic exchange of D2O and H2O in spherical particles are analyzed and compared. The primary difference between the two models is the choice of boundary condition at the surface of the spherical particle. In one model, it is assumed that the concentration of D2O at the surface is fixed, while in the other model, it is assumed that, at the particle surface, the concentration of D2O in the condensed phase is in equilibrium with D2O vapor. Closed-form expressions for the two boundary value problems that describe these physical models are found and discussed. Then, specific examples of aqueous droplets containing either sucrose, citric acid, and shikimic acid are examined with both models. It is found that at low relative humidities the choice of boundary condition has a negligible effect on the predicted lifetime of isotopic exchange, while at high relative humidities predicted lifetimes can differ by orders of magnitude. The implication of this result is that the choice of model can greatly affect diffusion coefficients retrieved from experimental measurements under certain conditions. Finally, discrepancies between diffusion coefficients measured using isotopic exchange and water sorption and desorption experiments are discussed.

On allocating one active redundancy to coherent systems with dependent and heterogeneous components' lifetimes

This article studies coherent systems of heterogenous and statistically dependent components' lifetimes. We present a sufficient and necessary condition for a stochastically longer system lifetime resulted by allocating a single active redundancy. For exchangeable components' lifetimes, allocating the redundancy to the component with more minimal path sets is proved to produce a more reliable system, and for systems with stochastic arrangement increasing components' lifetimes and symmetric structure with respect to two components, allocating the redundancy to the weaker one brings forth a larger reliability. Several numerical examples are presented to illustrate the theoretical results as well. © 2016 Wiley Periodicals, Inc. Naval Research Logistics 63: 335–345, 2016

Global Dynamics and Exchange Kinetics of a Protein on the Surface of Nanoparticles Revealed by Relaxation-Based Solution NMR Spectroscopy.

The global motions and exchange kinetics of a model protein, ubiquitin, bound to the surface of negatively charged lipid-based nanoparticles (liposomes) are derived from combined analysis of exchange lifetime broadening arising from binding to nanoparticles of differing size. The relative contributions of residence time and rotational tumbling to the total effective correlation time of the bound protein are modulated by nanoparticle size, thereby permitting the various motional and exchange parameters to be determined. The residence time of ubiquitin bound to the surface of both large and small unilamellar liposomes is ∼20 μs. Bound ubiquitin undergoes internal rotation about an axis approximately perpendicular to the lipid surface on a low microsecond time scale (∼2 μs), while simultaneously wobbling in a cone of semiangle 30-55° centered about the internal rotation axis on the nanosecond time scale. The binding interface of ubiquitin with liposomes is mapped by intermolecular paramagnetic relaxation enhancement using Gd(3+)-tagged vesicles, to a predominantly positively charged surface orthogonal to the internal rotation axis.

Re-evaluation of the water exchange lifetime value across red blood cell membrane

The water exchange lifetime (τi) through red blood cell (RBC) membranes can be measured by analyzing the water protons bi-exponential T1 and T2 curves when RBCs are suspended in a medium supplemented with paramagnetic species. Since the seminal papers published in the early '70s of the previous century, paramagnetic Mn2+ ions were used for doping the extracellular compartment in the RBCs suspension. The obtained τi values fall in the range of 9.8–14ms. Conversely, other physic-chemical measurements afforded longer τi values.Herein, it is shown that the replacement of Mn2+ with the highly stable, hydrophilic Gd(III) complexes used as paramagnetic magnetic resonance imaging (MRI) contrast agents led to measure τi values of 19.1±0.65ms at 25°C. The observed difference is ascribed to the occurrence of enhanced permeability of RBC membrane in the presence of Mn2+ ions. This view finds support from the observation that an analogous behavior was shown in the presence of other divalent cations, such Ca2+ and Zn2+ ions. A possible role of scramblase has been hypothesized.Finally, τi has been measured in presence of alcohols to show that the herein proposed method can detect minor changes in RBC membranes' stiffness upon the incorporation of aliphatic alcohols.

Dynamically achieved active site precision in enzyme catalysis.

ConspectusThe grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes’ enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme–substrate interactions and beyond.This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C–H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed.

Computing subsignatures of systems with exchangeable component lifetimes

The subsignatures of a system with continuous and exchangeable component lifetimes form a class of indexes ranging from the Samaniego signature to the Barlow–Proschan importance index. These indexes can be computed through explicit linear expressions involving the values of the structure function of the system. We show how the subsignatures can be computed more efficiently from the reliability function of the system via identifications of variables, differentiations, and integrations.

Gd(III)-labeled peptide nanofibers for reporting on biomaterial localization in vivo.

Bioactive supramolecular nanostructures are of great importance in regenerative medicine and the development of novel targeted therapies. In order to use supramolecular chemistry to design such nanostructures, it is extremely important to track their fate in vivo through the use of molecular imaging strategies. Peptide amphiphiles (PAs) are known to generate a wide array of supramolecular nanostructures, and there is extensive literature on their use in areas such as tissue regeneration and therapies for disease. We report here on a series of PA molecules based on the well-established β-sheet amino acid sequence V3A3 conjugated to macrocyclic Gd(III) labels for magnetic resonance imaging (MRI). These conjugates were shown to form cylindrical supramolecular assemblies using cryogenic transmission electron microscopy and small-angle X-ray scattering. Using nuclear magnetic relaxation dispersion analysis, we observed that thermal annealing of the nanostructures led to a decrease in water exchange lifetime (τm) of hundreds of nanoseconds only for molecules that self-assemble into nanofibers of high aspect ratio. We interpret this decrease to indicate more solvent exposure to the paramagnetic moiety on annealing, resulting in faster water exchange within angstroms of the macrocycle. We hypothesize that faster water exchange in the nanofiber-forming PAs arises from the dehydration and increase in packing density on annealing. Two of the self-assembling conjugates were selected for imaging PAs after intramuscular injections of the PA C16V3A3E3-NH2 in the tibialis anterior muscle of a murine model. Needle tracts were clearly discernible with MRI at 4 days postinjection. This work establishes Gd(III) macrocycle-conjugated peptide amphiphiles as effective tracking agents for peptide amphiphile materials in vivo over the timescale of days.

Dependence of exchangeable residual lifetimes subject to failure

We provide exact formulas about the survival copula of an exchangeable random vector of residual lifetimes when some of the components have failed. Specific computations are carried out in the case of Archimedean copulas and the evolution of dependence after successive failures of some components is shown numerically via measures of association.

Subsignatures of systems

We introduce the concept of subsignature for semicoherent systems as a class of indexes that range from the system signature to the Barlow–Proschan importance index. Specifically, given a nonempty subset M of the set of components of a system, we define the M-signature of the system as the |M|-tuple whose kth coordinate is the probability that the kth failure among the components in M causes the system to fail. We give various explicit linear expressions for this probability in terms of the structure function and the distribution of the component lifetimes. We also examine the case of exchangeable lifetimes and the special case when M is a modular set.

Amide Proton Solvent Protection in Amylin Fibrils Probed by Quenched Hydrogen Exchange NMR

Amylin is an endocrine hormone that accumulates in amyloid plaques in patients with advanced type 2 diabetes. The amyloid plaques have been implicated in the destruction of pancreatic β-cells, which synthesize amylin and insulin. To better characterize the secondary structure of amylin in amyloid fibrils we assigned the NMR spectrum of the unfolded state in 95% DMSO and used a quenched hydrogen-deuterium exchange technique to look at amide proton solvent protection in the fibrils. In this technique, partially exchanged fibrils are dissolved in 95% DMSO and information about amide proton occupancy in the fibrils is determined from DMSO-denatured monomers. Hydrogen exchange lifetimes at pH 7.6 and 37°C vary between ∼5 h for the unstructured N-terminus to 600 h for amide protons in the two β-strands that form inter-molecular hydrogen bonds between amylin monomers along the length of the fibril. Based on the protection data we conclude that residues A8-H18 and I26-Y37 comprise the two β-strands in amylin fibrils. There is variation in protection within the β-strands, particularly for strand β1 where only residues F15-H18 are strongly protected. Differences in protection appear to be due to restrictions on backbone dynamics imposed by the packing of two-layers of C2-symmetry-related β-hairpins in the protofilament structure, with strand β1 positioned on the surface and β2 in the interior.

Utilizing a Water-Soluble Cryptophane with Fast Xenon Exchange Rates for Picomolar Sensitivity NMR Measurements

Hyperpolarized (129)Xe chemical exchange saturation transfer ((129)Xe Hyper-CEST) NMR is a powerful technique for the ultrasensitive, indirect detection of Xe host molecules (e.g., cryptophane-A). Irradiation at the appropriate Xe-cryptophane resonant radio frequency results in relaxation of the bound hyperpolarized (129)Xe and rapid accumulation of depolarized (129)Xe in bulk solution. The cryptophane effectively "catalyzes" this process by providing a unique molecular environment for spin depolarization to occur, while allowing xenon exchange with the bulk solution during the hyperpolarized lifetime (T(1) ≈ 1 min). Following this scheme, a triacetic acid cryptophane-A derivative (TAAC) was indirectly detected at 1.4 picomolar concentration at 320 K in aqueous solution, which is the record for a single-unit xenon host. To investigate this sensitivity enhancement, the xenon binding kinetics of TAAC in water was studied by NMR exchange lifetime measurement. At 297 K, k(on) ≈ 1.5 × 10(6) M(-1) s(-1) and k(off) = 45 s(-1), which represent the fastest Xe association and dissociation rates measured for a high-affinity, water-soluble xenon host molecule near rt. NMR line width measurements provided similar exchange rates at rt, which we assign to solvent-Xe exchange in TAAC. At 320 K, k(off) was estimated to be 1.1 × 10(3) s(-1). In Hyper-CEST NMR experiments, the rate of (129)Xe depolarization achieved by 14 pM TAAC in the presence of radio frequency (RF) pulses was calculated to be 0.17 μM·s(-1). On a per cryptophane basis, this equates to 1.2 × 10(4)(129)Xe atoms s(-1) (or 4.6 × 10(4) Xe atoms s(-1), all Xe isotopes), which is more than an order of magnitude faster than k(off), the directly measurable Xe-TAAC exchange rate. This compels us to consider multiple Xe exchange processes for cryptophane-mediated bulk (129)Xe depolarization, which provide at least 10(7)-fold sensitivity enhancements over directly detected hyperpolarized (129)Xe NMR signals.

AGING FUNCTIONS AND MULTIVARIATE NOTIONS OF NBU AND IFR

For d≥2, let X=(X1, …, Xd) be a vector of exchangeable continuous lifetimes with joint survival function $\overline{F}$. For such models, we study some properties of multivariate aging of $\overline{F}$ that are described by means of the multivariate aging function $B_{\overline{F}}$, which is a useful tool for describing the level curves of $\overline{F}$. Specifically, the attention is devoted to notions that generalize the univariate concepts of New Better than Used and Increasing Failure Rate. These multivariate notions are satisfied by random vectors whose components are conditionally independent and identically distributed having univariate conditional survival function that is New Better than Used (respectively, Increasing Failure Rate). Furthermore, they also have an interpretation in terms of comparisons among conditional survival functions of residual lifetimes, given a same history of observed survivals.

Systems with Failure-Dependent Lifetimes of Components

A model for describing the lifetimes of coherent systems, where the failures of components may have an impact on the lifetimes of the remaining components, is proposed. The model is motivated by the definition of sequential order statistics (cf. Kamps (1995)). Sequential order statistics describe the successive failure times in a sequential k-out-of-n system, where the distribution of the remaining components' lifetimes is allowed to change after every failure of a component. In the present paper, general component lifetimes which can be influenced by failures are considered. The ordered failure times of these components can be used to extend the concept of sequential order statistics. In particular, a definition of sequential order statistics based on exchangeable components is proposed. By utilizing the system signature (cf. Samaniego (2007)), the distribution of the lifetime of a coherent system with failure-dependent exchangeable component lifetimes is shown to be given by a mixture of the distributions of sequential order statistics. Furthermore, some results on the joint distribution of sequential order statistics based on exchangeable components are given.

Systems with Failure-Dependent Lifetimes of Components

A model for describing the lifetimes of coherent systems, where the failures of components may have an impact on the lifetimes of the remaining components, is proposed. The model is motivated by the definition of sequential order statistics (cf. Kamps (1995)). Sequential order statistics describe the successive failure times in a sequentialk-out-of-nsystem, where the distribution of the remaining components' lifetimes is allowed to change after every failure of a component. In the present paper, general component lifetimes which can be influenced by failures are considered. The ordered failure times of these components can be used to extend the concept of sequential order statistics. In particular, a definition of sequential order statistics based on exchangeable components is proposed. By utilizing the system signature (cf. Samaniego (2007)), the distribution of the lifetime of a coherent system with failure-dependent exchangeable component lifetimes is shown to be given by a mixture of the distributions of sequential order statistics. Furthermore, some results on the joint distribution of sequential order statistics based on exchangeable components are given.

Supramolecular Protein Cage Composite MR Contrast Agents with Extremely Efficient Relaxivity Properties

A DTPA-Gd containing polymer was grown in the interior of a heat shock protein cage resulting in T(1) particle relaxivities of 4200 mM(-1) sec(-1) for the 12 nm particle. Relaxivity parameters were determined, and this analysis suggests that the rotational correlation time has been optimized while the water exchange lifetime is longer than optimal. This synthetic approach holds much promise for the development of next generation contrast agents and this report will aid in their design.

Tracking the relative in vivo pharmacokinetics of nanoparticles with PARACEST MRI.

A noninvasive assay that tracks the relative in vivo pharmacokinetics of two nanoparticles may accelerate the development of nanoparticles for biomedical applications, and may provide a method to select personalized nanomedicines for individual patients. To develop an in vivo competitive assay, two MRI contrast agents that could be selectively detected through paramagnetic chemical exchange saturation transfer (PARACEST) were conjugated to a second generation and fifth generation polyamidoamine (PAMAM) dendrimer. The CEST effects of each agent was calibrated relative to concentration. The effects of T(1) relaxivities of these dendritic PARACEST magnetic resonance imaging (MRI) contrast agents were found to be negligible relative to their CEST effects with respect to changes in image contrast, which facilitated the measurement of the ratios of their chemical exchange lifetimes. Injection of both contrast agents into a mouse model of mammary carcinoma resulted in a temporal increase in the CEST effect from each agent in the flank tumor. Although the in vivo CEST effects could not be used to determine the absolute concentrations of each agent within the tumor, the ratio of the in vivo CEST effects was used to measure the ratio of the concentrations of the agents. This result demonstrated that the relative in vivo pharmacokinetics of two nanoparticles may be evaluated using PARACEST MRI.

Housing Status and HIV Risk Behaviors Among Homeless and Housed Persons With HIV

To compare drug, alcohol, and sexual HIV transmission risk behaviors of homeless and housed people living with HIV/AIDS. Data were from 8075 respondents in a cross-sectional, multisite behavioral survey of adults recently reported to have HIV infection. At interview, 310 respondents (4%) were homeless. Compared with homeless respondents, housed respondents were more likely to be sexually active (past 12 months). However, sexually active homeless respondents had more sex partners (lifetime and past 12 months), greater sex exchange for money or drugs (lifetime and past 12 months), and greater unprotected vaginal or anal sex with an unknown serostatus partner. Homeless respondents were more likely to have possible alcohol abuse (lifetime), used drugs (last 12 months), and injected drugs (lifetime and past 12 months). After controlling for potential confounding variables, housing status remained a significant predictor of number of sex partners (past 12 months), sex exchange (lifetime and past 12 months), unprotected sex with unknown status partner, and all drug and alcohol use variables. Homeless people living with HIV/AIDS are more likely to have ever or recently engaged in substance use and HIV transmission risk behaviors. Findings underscore the need to provide HIV prevention services to homeless persons and address their housing needs.