Kinetics Of Escape Research Articles

Anomalous reaction-diffusion as a model of nonexponential DNA escape kinetics

We show that data from recent experiments carried out on the kinetics of DNA escape from alpha-hemolysin nanopores [M. Wiggin, C. Tropini, C. T. Cossa, N. N. Jetha, and A. Marziali, Biophys. J. 95, 5317 (2008)] may be rationalized by a model of chain dynamics based on the anomalous diffusion of a particle moving in a harmonic well in the presence of a delta function sink. The experiments of Wiggin et al. found, among other things, that the occasional occurrence of unusually long escape times in the distribution of chain trapping events led to nonexponential decays in the survival probability, S(t), of the DNA molecules within the nanopore. Wiggin et al. ascribed this nonexponentiality to the existence of a distribution of trapping potentials, which they suggested was the result of stochastic interactions between the bases of the DNA and the amino acids located on the surface of the nanopore. Based on this idea, they showed that the experimentally determined S(t) could be well fit in both the short and long time regimes by a function of the form (1+t/tau)(-alpha) (the so called Becquerel function). In our model, S(t) is found to be given by a Mittag-Leffler function at short times and by a generalized Mittag-Leffler function at long times. By suitable choice of certain parameter values, these functions are found to fit the experimental S(t) even better than the Becquerel function. Anomalous diffusion of DNA within the trap prior to escape over a barrier of fixed height may therefore provide a second, plausible explanation of the data, and may offer fresh perspectives on similar trapping and escape problems.

Adenovirus Virion Stability and the Viral Genome: Size Matters

Ongoing investigations into the fundamental aspects of virus biology continue to provide crucial insights for the development of more effective virus-based therapeutics. Within the field of gene therapy, these discoveries have translated into virus vectors with improved tissue tropism, prolonged transgene expression, and enhanced safety. However, radical modifications to the viral genome or capsid constituents can have marked effects on virion stability. In particular, recent insights into the role of viral genomes in contributing to the physical stability of the virion highlight the need to reconsider this heretofore underappreciated aspect of vector design. Here we discuss the impact of the genome on virion stability, with particular emphasis on the development of adenovirus (Ad)-based vectors.

Differential patterns of immune escape at Tat-specific cytotoxic T cell epitopes in pigtail macaques

Cytotoxic T lymphocyte responses to conserved proteins such as Gag within HIV- or SIV-infected hosts can facilitate partial control of viremia. However, the utility of targeting variable viral proteins by CTL responses is unclear. We studied CTL responses to regulatory and accessory proteins of SIV in pigtail macaques. The regulatory and accessory proteins were the most commonly targeted proteins by CTL responses from pigtail macaques. We identified 2 novel Tat-specific CTL responses that were both restricted by the Mane-A10 allele. Viral escape at one of the Tat epitopes, KSA10, was slower in comparison to another Tat epitope KVA10. The kinetics of escape of the KSA10 Tat epitope were more similar to an immunodominant KP9 Gag epitope also restricted by Mane-A10. Our results suggest that some regulatory or accessory CTL epitopes may be useful targets for vaccination against HIV.

High Content Screening Applications for RNA Therapeutics

RNA interference (RNAi) is a post‐transcriptional gene silencing process mediated by short, interfering RNA molecules (siRNAs) targeting complementary mRNA. siRNAs prevent protein expression and create an opportunity to use RNAi for drug target identification, validation, and therapeutics for treating human diseases. Successful application requires efficient delivery approaches, a hurdle which can be overcome through packaging siRNA inside lipid nanoparticles (LNPs). To evaluate biological activities of LNPs, we developed innovative assays and microscopy techniques to monitor tissue biodistribution, cell internalization, endosomal escape, and gene silencing. In combination with fluorescent markers, we visualized Cy5‐labeled siRNA encapsulated in LNPs and quantified kinetics of cell uptake and endosomal escape in various cells. Some LNPs displayed defects in cell uptake whereas other internalized into endosomes and distributed rapidly to the cytosol. Use of endocytosis inhibitors reveal clathrin and non‐clathrin coated pits participate, in part, in internalization in hepatocytes. Multiplexed, image‐based assays were employed to evaluate potential cellular toxicities and bioinformatics tools were developed to visualize multiparameter HCS data and mine phenotypic signatures.

Force spectroscopy of a single artificial biomolecule bond: The Kramers’ high-barrier limit holds close to the critical force

We use a minimal system with a single micron-size bead trapped with optical tweezers to investigate the kinetics of escape under force. Surprisingly, the exponential decay of the off rate with the barrier energy is still valid close to the critical force. Hence, the high viscosity approximation derived by Kramers in the case of a high energy barrier holds even for an energy barrier close to the thermal energy. Several recent models describe a single biomolecule bond by a smooth single-barrier energy profile. When this approach is accurate enough, our result justifies the use of Kramers' approximation in the high-force regime, close to the critical force of the system, as done in recent single biomolecule bond studies.

Nonexponential Kinetics of DNA Escape from α-Hemolysin Nanopores

Throughput and resolution of DNA sequence detection technologies employing nanometer scale pores hinge on accurate kinetic descriptions of DNA motion in nanopores. We present the first detailed experimental study of DNA escape kinetics from α-hemolysin nanopores and show that anomalously long escape times for some events result in nonexponential kinetics. From the distribution of first-passage times, we determine that the energy barrier to escape follows a Poisson-like distribution, most likely due to stochastic weak binding events between the DNA and amino acid residues in the pore.

Cage Escape Competes with Geminate Recombination during Alkane Hydroxylation by the Diiron Oxygenase AlkB

AlkB stops the radical clock: Three structurally analogous radical-clock substrates with a large span in their rearrangement rates are hydroxylated by AlkB to afford similar amounts of rearranged (2) and unrearranged products (1). Such a result is in accord with radical rebound competing with cage escape of the geminate substrate radical. The results show that radical clocks can measure both the radical lifetime and the kinetics of cage escape.

Kinetics of promoter escape varies as a function of KCl concentration

The efficiency of promoter escape in E. coli transcription initiation is dependent on two factors: 1) the rate of escape, and 2) the extent of RNA polymerase (RNAP) partitioning into escape‐competent open complexes. Both parameters can be obtained from a single cycle assay performed under RNAP‐limiting conditions. For T5 N25 and N25anti promoters, these two factors are governed by the initial transcribed sequence (ITS). N25 and N25anti promoters differ only in +3 to +20 region of the ITS, but exhibit half‐lives of ∼3 min and ∼42 min, respectively, for full‐length RNA synthesis. Interestingly, the slower escaping N25anti promoter forms a higher fraction of productive open complexes and yields higher levels of productive RNA than N25; this result was unexpected. The above studies were performed in 200 mM KCl solution. We next examined the effect of KCl concentration and found that lowering [KCl] from 200 – 10 mM increased the rate of escape for N25 while also raising its productive fraction. For N25anti, the escape rate remained the same while lower [KCl] brought about higher productive synthesis. We conclude that KCl concentration affects the open complex formation and partitioning similarly in both promoters, but influences their open complex collapse and escape differently, confirming that the rate of promoter escape is ITS‐sequence dependent. Supported by an NSF grant (RUI‐0418316) to L. M. Hsu.

Vaccination and Timing Influence SIV Immune Escape Viral Dynamics In Vivo

CD8+ cytotoxic T lymphocytes (CTL) can be effective at controlling HIV-1 in humans and SIV in macaques, but their utility is partly offset by mutational escape. The kinetics of CTL escape and reversion of escape mutant viruses upon transmission to MHC-mismatched hosts can help us understand CTL-mediated viral control and the fitness cost extracted by immune escape mutation. Traditional methods for following CTL escape and reversion are, however, insensitive to minor viral quasispecies. We developed sensitive quantitative real-time PCR assays to track the viral load of SIV Gag164–172 KP9 wild-type (WT) and escape mutant (EM) variants in pigtail macaques. Rapid outgrowth of EM virus occurs during the first few weeks of infection. However, the rate of escape plateaued soon after, revealing a prolonged persistence of WT viremia not detectable by standard cloning and sequencing methods. The rate of escape of KP9 correlated with levels of vaccine-primed KP9-specific CD8+ T cells present at that time. Similarly, when non-KP9 responder (lacking the restricting Mane-A*10 allele) macaques were infected with SHIVmn229 stock containing a mixture of EM and WT virus, rapid reversion to WT was observed over the first 2 weeks following infection. However, the rate of reversion to WT slowed dramatically over the first month of infection. The serial quantitation of escape mutant viruses evolving during SIV infection shows that rapid dynamics of immune escape and reversion can be observed in early infection, particularly when CD8 T cells are primed by vaccination. However, these early rapid rates of escape and reversion are transient and followed by a significant slowing in these rates later during infection, highlighting that the rate of escape is significantly influenced by the timing of its occurrence.

In Vivo Fitness Costs of Different Gag CD8 T-Cell Escape Mutant Simian-Human Immunodeficiency Viruses for Macaques

The kinetics of immune escape and reversion depend upon the efficiency of CD8 cytotoxic T lymphocytes (CTL) and the fitness cost of escape mutations. Escape kinetics of three simian immunodeficiency virus Gag CTL epitopes in pigtail macaques were variable; those of KP9 and AF9 were faster than those of KW9. Kinetics of reversion of escape mutant virus to wild type upon passage to naïve major histocompatibility complex-mismatched macaques also varied. Rapid reversion occurred at KP9, gradual biphasic reversion occurred at AF9, and escape mutant KW9 virus failed to revert. The fitness impact of these mutations is KP9 > AF9 > KW9. These data provide insights into the differential utility of CTL in controlling viremia.

On Stochastic Models of Dynamic Disorder

In this paper we investigate some general aspects of stochastic models of dynamic disorder. First, we reexamine the Zwanzig model for the kinetics of escape through a fluctuating hole. We show that this model is trivially connected to the canonical model of the broadening of the zero-phonon line (ZPL) in crystals. This provides a new perspective of the Wang-Wolynes expression for the rate of escape from a geometric bottleneck with non-Markovian Gaussian fluctuations. Motivated by recent single-molecule experiments, we examine more general examples of fluctuation processes from the perspective of cumulant expansions. Finally, we discuss recent single-molecule experiments probing enzyme turnover performed by Xie and co-workers.

Modeling the Endosomal Escape of Cell-Penetrating Peptides: Transmembrane pH Gradient Driven Translocation across Phospholipid Bilayers

Cell-penetrating peptides (CPPs) are able to mediate the efficient cellular uptake of a wide range of cargoes. Internalization of a number of CPPs requires uptake by endocytosis, initiated by binding to anionic cell surface heparan sulfate (HS), followed by escape from endosomes. To elucidate the endosomal escape mechanism, we have modeled the process for two CPPs: penetratin (pAntp) and the N-terminal signal peptide of the unprocessed bovine prion protein (bPrPp). Large unilamellar phospholipid vesicles (LUVs) were produced encapsulating either peptide, and an ionophore, nigericin, was used to create a transmembrane pH gradient (DeltapH(mem), inside acidic) similar to the one arising in endosomes in vivo. In the absence of DeltapH(mem), no pAntp escape from the LUVs is observed, while a fraction of bPrPp escapes. In the presence of DeltapH(mem), a significant amount of pAntp escapes and an even higher degree of bPrPp escape takes place. These results, together with the differences in kinetics of escape, indicate different escape mechanisms for the two peptides. A minimum threshold peptide concentration exists for the escape of both peptides. Coupling of the peptides to a cargo reduces the fraction escaping, while complexation with HS significantly hinders the escape. Fluorescence correlation spectroscopy results show that during the escape process the LUVs are intact. Taken together, these results suggest a model for endosomal escape of CPPs: DeltapH(mem)-mediated mechanism, following dissociation from HS of the peptides, above a minimum threshold peptide concentration, in a process that does not involve lysis of the vesicles.

Determinants of Human Immunodeficiency Virus Type 1 Escape from the Primary CD8+ Cytotoxic T Lymphocyte Response

CD8+ cytotoxic T lymphocytes (CTLs) play an important role in containment of virus replication in primary human immunodeficiency virus (HIV) infection. HIV's ability to mutate to escape from CTL pressure is increasingly recognized; but comprehensive studies of escape from the CD8 T cell response in primary HIV infection are currently lacking. Here, we have fully characterized the primary CTL response to autologous virus Env, Gag, and Tat proteins in three patients, and investigated the extent, kinetics, and mechanisms of viral escape from epitope-specific components of the response. In all three individuals, we observed variation beginning within weeks of infection at epitope-containing sites in the viral quasispecies, which conferred escape by mechanisms including altered peptide presentation/recognition and altered antigen processing. The number of epitope-containing regions exhibiting evidence of early CTL escape ranged from 1 out of 21 in a subject who controlled viral replication effectively to 5 out of 7 in a subject who did not. Evaluation of the extent and kinetics of HIV-1 escape from >40 different epitope-specific CD8 T cell responses enabled analysis of factors determining escape and suggested that escape is restricted by costs to intrinsic viral fitness and by broad, codominant distribution of CTL-mediated pressure on viral replication.

Diffusive escape and reentry through a channel in the cavity wall

We show that the kinetics of diffusive escape from a cavity through a narrow channel in the cavity wall and successive reentry can be described by a formal kinetic scheme for reversible dissociation of a spherical binding site with appropriately defined effective association and dissociation rate constants. Initially the population of the cavity decays exponentially, with the rate constant determined by the cavity volume and the channel length and radius. A crossover to the universal inverse power law behavior, which does not depend on the channel geometry, occurs at long times. These simple predictions are in excellent agreement with the results of Brownian dynamics simulations.

Parameters affecting cellular invasion and escape from the parasitophorous vacuole by different infective forms of Trypanosoma cruzi.

In this study we have examined certain aspects of the process of cell invasion and parasitophorous vacuole escape by metacyclic trypomastigotes and extracellular amastigote forms of Trypanosoma cruzi (G strain). Using Vero (and HeLa) cells as targets, we detected differences in the kinetics of vacuole escape by the two forms. Alcalinization of intercellular pH influenced both invasion as well as the escape from the parasitophorous vacuole by metacyclic trypomastigotes, but not the escape kinetics of extracellular amastigotes. We used sialic acid mutants as target cells and observed that the deficiency of this molecule facilitated the escape of both infective forms. Hemolysin activity was only detected in extracellular amastigotes and neither form presented detectable transialidase activity. Invasion of extracellular amastigotes and trypomastigotes in Vero cells was affected in different ways by drugs that interfere with host cell Ca2+ mobilization. These results are in line with previous results that indicate that metacyclic trypomastigotes and extracellular amastigote forms utilize mechanisms with particular features to invade host cells and to escape from their parasitophorous vacuoles.

Escape and reentry of a Brownian particle through a hole in a cavity

We show that the kinetics of diffusive escape from a cavity through a small hole can be described, to a sufficient degree of accuracy, by a formal kinetic scheme for reversible dissociation of a spherical binding site with appropriately defined effective association and dissociation rate constants. At short times, the population of the cavity decays exponentially, while at long times a crossover to the inverse power law behavior is observed. These simple predictions are in excellent agreement with the results of Brownian dynamics simulations.

Kinetics of escape through a small hole

We study the time dependence of the survival probability of a Brownian particle that escapes from a cavity through a round hole. When the hole is small the escape is controlled by an entropy barrier and the survival probability decays as a single exponential. We argue that the rate constant is given by k=4Da/V, where a and V are the hole radius and the cavity volume and D is the diffusion constant of the particle. Brownian dynamics simulations for spherical and cubic cavities confirmed both the exponential decay of the survival probability and the expression for the rate constant for sufficiently small values of a.

Water penetration and escape in proteins

The kinetics of water penetration and escape in cytochrome c (cyt c) is studied by molecular dynamics (MD) simulations at various temperatures. Water molecules that penetrate the protein interior during the course of an MD simulation are identified by monitoring the number of water molecules in the first coordination shell (within 3.5 A) of each water molecule in the system. Water molecules in the interior of cyt c have 0-3 water molecules in their first hydration shell and this coordination number persists for extended periods of time. At T = 300 K we identify over 200 events in which water molecules penetrate the protein and reside inside for at least 5 picoseconds (ps) within a 1.5 nanoseconds (ns) time period. Twenty-seven (27) water molecules reside for at least 300 ps, 17 water molecules reside in the protein interior for times longer than 500 ps, and two interior water molecules do not escape; at T = 360 K one water molecule does not escape; at 430 K all water molecules exchange. Some of the internal water molecules show mean square displacements (MSD) of 1 A2 characteristic of structural waters. Others show MSD as large as 12 A2, suggesting that some of these water molecules occupy transient cavities and diffuse extensively within the protein. Motions of protein-bound water molecules are rotationally hindred, but show large librations. Analysis of the kinetics of water escape in terms of a survival time correlation function shows a power law behavior in time that can be interpreted in terms of a broad distribution of energy barriers, relative to kappa BT, for water exchange. At T = 300 K estimates of the roughness of the activation energy distribution is 4-10 kJ/mol (2-4 kappa BT). Activation enthalpies for water escape are 6-23 kJ/mol. The difference in activation entropies between fast exchanging (0.01 ns) and slow exchanging (0.1-1 ns) water molecules is -27 J/K/mol. Dunitz (Science 1997;264:670.) has estimated the maximum entropy loss of a water molecule due to binding to be 28 J/K/mol. Therefore, our results suggest that the entropy of interior water molecules is similar to entropy of bulk water.

Water penetration and escape in proteins

The kinetics of water penetration and escape in cytochrome c (cyt c) is studied by molecular dynamics (MD) simulations at various temperatures. Water molecules that penetrate the protein interior during the course of an MD simulation are identified by monitoring the number of water molecules in the first coordination shell (within 3.5 Å) of each water molecule in the system. Water molecules in the interior of cyt c have 0–3 water molecules in their first hydration shell and this coordination number persists for extended periods of time. At T = 300 K we identify over 200 events in which water molecules penetrate the protein and reside inside for at least 5 picoseconds (ps) within a 1.5 nanoseconds (ns) time period. Twenty-seven (27) water molecules reside for at least 300 ps, 17 water molecules reside in the protein interior for times longer than 500 ps, and two interior water molecules do not escape; at T = 360 K one water molecule does not escape; at 430 K all water molecules exchange. Some of the internal water molecules show mean square displacements (MSD) of 1 Å2 characteristic of structural waters. Others show MSD as large as 12 Å2, suggesting that some of these water molecules occupy transient cavities and diffuse extensively within the protein. Motions of protein-bound water molecules are rotationally hindered, but show large librations. Analysis of the kinetics of water escape in terms of a survival time correlation function shows a power law behavior in time that can be interpreted in terms of a broad distribution of energy barriers, relative to κBT, for water exchange. At T = 300 K estimates of the roughness of the activation energy distribution is 4–10 kJ/mol (2–4 κBT). Activation enthalpies for water escape are 6–23 kJ/mol. The difference in activation entropies between fast exchanging (0.01 ns) and slow exchanging (0.1–1 ns) water molecules is −27 J/K/mol. Dunitz (Science 1997;264:670.) has estimated the maximum entropy loss of a water molecule due to binding to be 28 J/K/mol. Therefore, our results suggest that the entropy of interior water molecules is similar to entropy of bulk water. Proteins 2000;38:261–272. Published 2000 Wiley-Liss, Inc.

Reaction dynamics on a thermally fluctuating potential

This paper analyzes the kinetics of escape of a particle over a barrier fluctuating between two states, the fluctuations being produced by thermal noise. By this we mean that the jump rates for transitions between the two states are position-dependent, satisfying detailed balance at any point along the reaction coordinate. The fast-fluctuation limit can be analyzed in terms of the potential of mean force, and for high barriers the survival probability is found to be a single exponential. In the slow-fluctuation regime the survival probability is a linear combination of two exponentials. In the case of a linear potential the slow-fluctuation solution describes the kinetics, as obtained from simulations, quite well over the entire range of the jump rates between the two states. Our analysis suggests that this is true for more general forms of the potential. Further, for a thermally fluctuating potential the mean lifetime is shown to decrease monotonically as the jump rate increases. This is in contrast to the turnover behavior, or resonant activation, which can occur when fluctuations are produced by nonthermal noise. An extension of our approach to systems with thermal fluctuations between more than two states is discussed.