Charles M. Knobler

Osmotic pressure inhibition of DNA ejection from phage

Bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes. We demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure. In the particular case of bacteriophage λ, the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria; it is completely suppressed by a pressure of 20 atmospheres. Furthermore, our experiments monitor directly a dramatic decrease of the stress inside the unopened phage capsid upon addition of polyvalent cations to the host solution, in agreement with many recent theories of DNA interactions.

Seeing Phenomena in Flatland: Studies of Monolayers by Fluorescence Microscopy

Monolayers formed at the interface between air and water can be seen with fluorescence microscopy. This allows the phase behavior of these monolayers to be determined by direct observation and opens up the possibility of following the kinetics of phase transformations in two-dimensional systems. Some unexpected morphologies have been discovered that provide information about the nature of monolayer phases and have connections to pattern formation in other systems.

Light scattering studies of phase separation in isobutyric acid + water mixtures

Measurements are reported of light scattering by isobutyric acid + water mixtures that have been quenched into the two‐phase region by a pressure‐jump technique. The conditions of the jump method are discussed in detail. Examples are given of the behavior of the scattered intensity I (k,t) as a function of the time t and scattering vector k. Power‐law expressions of the form km =A′t−a′ are found to represent the time dependence of the angle of maximum scattering. The intensity at the maximum can be represented by power laws as well: I (km) =A″ta″. For mixtures of the critical composition both a′ and a″ depend on quench depth; for other mixtures a′?0.3 and a″?1.1. Two measurements made at the cloud point show an initial t2 growth in I (km) followed by a slower growth ∝t1/2.

Self-Assembly of Viral Capsid Protein and RNA Molecules of Different Sizes: Requirement for a Specific High Protein/RNA Mass Ratio

ABSTRACT Virus-like particles can be formed by self-assembly of capsid protein (CP) with RNA molecules of increasing length. If the protein “insisted” on a single radius of curvature, the capsids would be identical in size, independent of RNA length. However, there would be a limit to length of the RNA, and one would not expect RNA much shorter than native viral RNA to be packaged unless multiple copies were packaged. On the other hand, if the protein did not favor predetermined capsid size, one would expect the capsid diameter to increase with increase in RNA length. Here we examine the self-assembly of CP from cowpea chlorotic mottle virus with RNA molecules ranging in length from 140 to 12,000 nucleotides (nt). Each of these RNAs is completely packaged if and only if the protein/RNA mass ratio is sufficiently high; this critical value is the same for all of the RNAs and corresponds to equal RNA and N-terminal-protein charges in the assembly mix. For RNAs much shorter in length than the 3,000 nt of the viral RNA, two or more molecules are assembled into 24- and 26-nm-diameter capsids, whereas for much longer RNAs (>4,500 nt), a single RNA molecule is shared/packaged by two or more capsids with diameters as large as 30 nm. For intermediate lengths, a single RNA is assembled into 26-nm-diameter capsids, the size associated with T=3 wild-type virus. The significance of these assembly results is discussed in relation to likely factors that maintain T=3 symmetry in vivo.

Frustration‐limited clusters in liquids

We present a continuum theory of frustration‐limited clusters in one‐component glass‐forming liquids that accounts, in part, for the recently reported [Fischer et al., J. Non‐Cryst. Solids, 131–133, 134 (1991)], and quite unexpected, presence in simple glass‐forming liquids of stable clusters at low temperatures (T) and the even less expected persistence for very long times of these clusters at higher T’s. The model is based on the idea that there is a local structure that is energetically preferred over simple crystalline packing, which is strained (frustrated) over large distances; although in a curved space the preferred packing could lead to ‘‘ideal’’ crystallization at temperatures that are usually above the actual freezing temperature, in ‘‘flat’’ space this transition is narrowly avoided. We are led to a new ansatz for the T dependence of the viscosity, which permits us to collapse data for many liquids onto a universal curve.

Predicting the sizes of large RNA molecules

We present a theory of the dependence on sequence of the three-dimensional size of large single-stranded (ss) RNA molecules. The work is motivated by the fact that the genomes of many viruses are large ssRNA molecules—often several thousand nucleotides long—and that these RNAs are spontaneously packaged into small rigid protein shells. We argue that there has been evolutionary pressure for the genome to have overall spatial properties—including an appropriate radius of gyration, Rg—that facilitate this assembly process. For an arbitrary RNA sequence, we introduce the (thermal) average maximum ladder distance (〈MLD〉) and use it as a measure of the “extendedness” of the RNA secondary structure. The 〈MLD〉 values of viral ssRNAs that package into capsids of fixed size are shown to be consistently smaller than those for randomly permuted sequences of the same length and base composition, and also smaller than those of natural ssRNAs that are not under evolutionary pressure to have a compact native form. By mapping these secondary structures onto a linear polymer model and by using 〈MLD〉 as a measure of effective contour length, we predict the Rg values of viral ssRNAs are smaller than those of nonviral sequences. More generally, we predict the average 〈MLD〉 values of large nonviral ssRNAs scale as N0.67±0.01, where N is the number of nucleotides, and that their Rg values vary as 〈MLD〉0.5 in an ideal solvent, and hence as N0.34. An alternative analysis, which explicitly includes all branches, is introduced and shown to yield consistent results.

Textures and phase transitions in Langmuir monolayers of fatty acids. A comparative Brewster angle microscope and polarized fluorescence microscope study

Brewster angle microscopy (BAM) and polarized fluorescence microscopy (PFM) are used to observe the distinctive textures of and the transitions between condensed phases in Langmuir monolayers of n‐alkanoic acids. BAM is sensitive to film anisotropy even when the molecules are not tilted as long as the unit cell is anisotropic. Every transition is visible with one or both of the techniques, either as a dramatic change in the degree of contrast or as a sudden alteration of the mosaic domain texture. The two techniques are generally consistent, although the presence of the fluorescent probe impurity (for PFM) causes a subtle difference in the appearance of one transition and small shifts in transition surface pressures.

A chiral recognition polymer based on polyaniline

Abstract When polyaniline is doped with either R- or S-camphorsulfonic acid (CSA) it adopts a chiral structure. Removing the chiral acid dopant leads to a new form of chiral polyaniline. Such chiral dedoped polyanilines have the ability to discriminate among enantiomers of amino acids such as DL-phenylalanine. For example, the dedoped form of R-CSA polyaniline preferentially complexes with L-phenylalanine as indicated by a “doping” type absorption observed at ~450 nm in circular dichroism spectra. Since the dedoped form of R-CSA polyaniline does trot complex with d -phenylaniline, this chiral polymer can be used to separate racemic mixtures of DL-phenylalanine. Moreover, the amino acid enantiomer bound by the chiral polyaniline can be released by an organic solvent. The chiral separation mechanism is further explored by examining the interactions of phenylalanine derivatives with the chiral dedoped polyanilines.

How does dew form

Abstract Dew is a phenomenon that is experienced daily. It is formed of water droplets that imperfectly wet the substrate on which they condense. The formation of dew is connected to rich and numerous physical phenomena: heterogeneous nucleation, phase transitions, heat transfer, wetting. Although the growth of an isolated droplet still remains imperfectly understood, growth of an assembly of drops is better comprehended: the fact that drops coalesce during their growth, and that the substrate (plane, line, fractal …) is of lower dimensionality than that of the drops is at the origin of surprising properties (self-similarity, constant and universal value of the surface coverage, correlation of position). Experiments and numerical simulations of condensation of water vapor on planar or unidimensional solid substrates are presented. Condensation on a liquid substrate (oil) modifies the interactions between drops because the substrate is locally curved; the drops can then self-organize in two-dimensional (he...

Physical Chemistry of DNA Viruses

The relative simplicity of viruses makes it possible to apply generic physical approaches to the understanding of their structure and function. We focus here on viruses that have double-stranded (ds)DNA genomes that are enclosed in a protein container called the capsid. Their structures are now known in precise detail from cryo-electron microscopy. dsDNA is a stiff, highly charged polymer, and typical viral DNAs have contour lengths 1000 times longer than the radius of the capsid into which they are introduced in the assembly process, which is driven by a biological motor. As a result, the confined DNA is highly stressed. The energy stored in the dsDNA, which is compressed to crystalline densities, drives the ejection of the genome into the host at the start of an infection. Experiments have examined the packaging and ejection of the genomes, which have also been the subject of analytic theories and simulations.