Bernard S. Gerstman

Laser induced bubble formation in the retina

The immediate thermodynamic effects of absorption of a laser pulse in the retina are theoretically studied to understand underlying physical damage mechanisms at threshold fluences. Damage is most likely to occur at threshold levels in the retinal pigment epithelium due to the strong absorption by the melanosomes.

Melanin granule model for laser-induced thermal damage in the retina

An analytical model for thermal damage of retinal tissue due to absorption of laser energy by finite-sized melanin granules is developed. Since melanin is the primary absorber of visible and near-IR light in the skin and in the retina, bulk heating of tissue can be determined by superposition of individual melanin granule effects. Granules are modeled as absorbing spheres surrounded by an infinite medium of water. Analytical solutions to the heat equation result in computations that are quick and accurate. Moreover, the model does not rely on symmetric beam profiles, and so arbitrary images can be studied. The important contribution of this model is to provide a more accurate biological description of sub-millisecond pulse exposures than previous retinal models, while achieving agreement for longer pulses. This model can also be naturally extended into the sub-microsecond domain by including vaporization as a damage mechanism. It therefore represents the beginning of a model which can be applied across the entire pulse duration domain.

Self-organization in protein folding and the hydrophobic interaction.

Self-organization is a critical aspect of living systems. During the folding of protein molecules, the hydrophobic interaction plays an important role in the collapse of the peptide chain to a compact shape. As the hydrophobic core tightens and excludes water, not only does the number of hydrophobic side chain contacts increase, but stabilization is further enhanced by an increase in strength of each hydrophobic interaction between side chains in the core. Thus, the self-organization of the protein folding process augments itself by enhancing the stability of the core against large-scale motions that would unfold the protein. Through calculations and computer simulations on a model four-helix bundle protein, we show how the strengthening of the hydrophobic interaction is crucial for stabilizing the core long enough for completion of the folding process and quantitatively manifests self-organizing dynamical behavior.

Stress confinement, shock wave formation, and laser-induced damage

The concept of confinement is that if energy deposition into a system occurs during durations shorter than a confinement time, the response of the system depends only on the total energy deposited and not on the deposition time. For stress confinement, the relevant response is the pressure that is produced. We have shown previously that for laser absorption by a spherical absorber, stress confinement is not valid at the core of the absorber and the tensile stresses continue to grow as the pulse duration shrinks well below any characteristic response time of the system. We have now calculated the pressure response in the cellular medium outside the absorber. We find that for a variety of energies, stress confinement is valid. We find that the characteristic confinement time agrees well with that expected for pressure transmission across the absorber. We show that even though the peak pressure that is produced varies slowly as a function of pulse duration, there is a sudden onset of shock wave production when the pulse duration is shortened below the confinement time. Since damage results from pressure gradients, the sudden onset of shock waves implies a sharp increase in the potential for damage.

Exploring the diffusion of molecular oxygen in the red fluorescent protein mCherry using explicit oxygen molecular dynamics simulations.

The development of fluorescent proteins (FPs) has revolutionized cell biology research. The monomeric variants of red fluorescent proteins (RFPs), known as mFruits, have been especially valuable for tagging and tracking cellular processes in vivo. Determining oxygen diffusion pathways in FPs can be important for improving photostability and for understanding maturation of the chromophore. We use molecular dynamics (MD) calculations to investigate the diffusion of molecular oxygen in one of the most useful monomeric RFPs, mCherry. We describe a pathway that allows oxygen molecules to enter from the solvent and travel through the protein barrel to the chromophore. We calculate the free-energy of an oxygen molecule at points along the path. The pathway contains several oxygen hosting pockets, which are identified by the amino acid residues that form the pocket. We also investigate an RFP variant known to be significantly less photostable than mCherry and find much easier oxygen access in this variant. The results provide a better understanding of the mechanism of molecular oxygen access into the fully folded mCherry protein barrel and provide insight into the photobleaching process in these proteins.

Biophysical effects of pulsed lasers in the retina and other tissues containing strongly absorbing particles: shockwave and explosive bubble generation

Damage by pulsed lasers to the retina or other tissues containing strongly absorbing particles may occur through biophysical mechanisms other than simple heating. Shockwaves and bubbles have been observed experimentally, and depending on pulse duration, may be the cause of retinal damage at threshold fluence levels. We perform detailed calculations on the shockwave and bubble generation expected from pulsed lasers. For a variety of different laser pulse durations and fluences, we tabulate the expected strength of the shockwave and size of the bubble that will be generated. We also explain how these results will change for absorbing particles with different physical properties such as absorption coefficient, bulk modulus, or thermal expansion coefficient. This enables the assessment of biological danger, and possible medical benefits, for lasers of a wide range of pulse durations and energies, incident on tissues with absorbing particles with a variety of thermomechanical characteristics.

Hydrogen Bond Flexibility Correlates with Stokes Shift in mPlum Variants

Fluorescent proteins have revolutionized molecular biology research and provide a means of tracking subcellular processes with extraordinary spatial and temporal precision. Species with emission beyond 650 nm offer the potential for deeper tissue penetration and lengthened imaging times; however, the origin of their extended Stokes shift is not fully understood. We employed spectrally resolved transient grating spectroscopy and molecular dynamics simulations to investigate the relationship between the flexibility of the chromophore environment and Stokes shift in mPlum. We examined excited state solvation dynamics in a panel of strategic point mutants of residues E16 and I65 proposed to participate in a hydrogen-bonding interaction thought responsible for its red-shifted emission. We observed two characteristic relaxation constants of a few picoseconds and tens of picoseconds that were assigned to survival times of direct and water-mediated hydrogen bonds at the 16-65 position. Moreover, variants of the largest Stokes shift (mPlum, I65V) exhibited significant decay on both time scales, indicating the bathochromic shift correlates with a facile switching between a direct and water-mediated hydrogen bond. This dynamic model underscores the role of environmental flexibility in the mechanism of excited state solvation and provides a template for engineering next-generation red fluorescent proteins.

Structural information content and Lyapunov exponent calculation in protein unfolding

A lattice model with Monte Carlo dynamics is used to carry out computer simulations of protein dynamics on a four α-helix bundle. The interaction energies in the model can be set so that either the helix bundle structure remains relatively stable or changed so that it unfolds. The computer model produces output that simulates experimental measurements relating to the structure. We show how this output can be used with analytical techniques of nonlinear dynamics to obtain important information about the complex underlying protein dynamics. Time-delay reconstruction plots of structural parameters of unfolding bundles resemble strange attractors in a space of dimension 3–4. We calculate Lyapunov exponents for these unfolding runs and find positive Lyapunov exponents implying chaotic dynamics. For stable runs the Lyapunov exponents are close to zero. We use these Lyapunov exponents to calculate the rate of loss of structural information during the unfolding process and show how the approach may be useful for investigating the folding dynamics of proteins.

The Ebola virus protein VP40 hexamer enhances the clustering of PI(4,5)P2 lipids in the plasma membrane

The Ebola virus is a lipid-enveloped virus that obtains its lipid coat from the plasma membrane of the host cell it infects during the budding process. The Ebola virus protein VP40 localizes to the inner leaflet of the plasma membrane and forms the viral matrix, which provides the major structure for the Ebola virus particles. VP40 is initially a dimer that rearranges to a hexameric structure that mediates budding. VP40 hexamers and larger filaments have been shown to be stabilized by PI(4,5)P2 in the plasma membrane inner leaflet. Reduction in the plasma membrane levels of PI(4,5)P2 significantly reduce formation of VP40 oligomers and virus-like particles. We investigated the lipid-protein interactions in VP40 hexamers at the plasma membrane. We quantified lipid-lipid self-clustering by calculating the fractional interaction matrix and found that the VP40 hexamer significantly enhances the PI(4,5)P2 clustering. The radial pair distribution functions suggest a strong interaction between PI(4,5)P2 and the VP40 hexamer. The cationic Lys side chains are found to mediate the PIP2 clustering around the protein, with cholesterol filling the space between the interacting PIP2 molecules. These computational studies support recent experimental data and provide new insights into the mechanisms by which VP40 assembles at the plasma membrane inner leaflet, alters membrane curvature, and forms new virus-like particles.

Activated Rate Processes and a Specific Biochemical Mechanism for Explaining Delayed Laser Induced Thermal Damage to the Retina

Laser induced thermal damage to the retina is investigated. The one step Arrhenius type thermal damage integral of Henriques is analyzed for its strengths and weaknesses. The zero-order activated rate process is shown to well represent the data for pulse durations greater than 10 μs. A zero-order biochemical damage mechanism involving free radical formation and thermal disruption of the melanosomes protein coat is proposed as the initial molecular process that leads to cellular damage which appears after a delay. Data are presented that show the photoactivation of melanin granule oxidative reactivity. This in vitro data is evidence for an important step in our hypothesized damage pathway.