Session U34: Focus Session: Non-equilibrium Fluctuations in Biomolecules

Fluctuations in Proteins

Proteins are the machines of life. In order to perform their functions, they must move continuously. The motions correspond to equilibrium fluctuations and to non-equilibrium relaxations. At least three different fluctuation processes occur: $\alpha$- and $\beta$-fluctuations and processes that occur even below one Kelvin. The $\alpha$-fluctuations can be approximated by the Vogel-Tammann-Fulcher relation, while the $\beta$-fluctuations appear to follow a conventional Arrhenius law (but may in some cases be better characterized by a Ferry law). Both are usually nonexponential in time. These phenomena are similar in proteins and glasses, but there is a fundamental difference between fluctuations in glasses and proteins: In glasses, they are independent of the environment, in proteins the $\alpha$-fluctuations are slaved to the $\alpha$-fluctuations in the solvent surrounding the protein; they follow their rate coefficients but they are entropically slowed. The studies of the protein motions are actually still in their infancy, but we can expect that future work will not only help understanding protein functions, but will also feed back to the physics of glasses.   

A Tunable Chemical Pattern Filter Constructed by Networks of Reaction Compartments and Tubes

We study numerically the filtering capabilities of nanoscale networks built up of containers and tubes hosting chemical reactions. Spatio-temporal patterns of substrate molecules are injected into the network. The substrate propagates by diffusion and reacts with enzymes distributed in the network prior to the injections. The dimensions of the network are tailored in a way that the transport and reaction rates are comparable in size, a situation in which the overall behavior is highly influenced by the geometry and topology of the network. This property is crucial for the functionality of the pattern filter developed in here. It is demonstrated that input patterns can be classified in a crude way using a simple setup (two micrometer-sized containers joined together by a nanotube) and that the classification can be tuned by changing the geometry of the network (the length of the tube connecting the two containers). The filter device we investigate can also be viewed as a primitive chemistry-based computational element since the information encoded in the input patterns is processed using chemical reactions. In particular it is argued that the filter can be used as a frequency sensor.   

Effect of Orientation in Translocation of Polymers through Nanopores

The motion of a polymer with inhomogeneous structure through a nanopore is discussed theoretically. Specifically, we consider the translocation of polymer consisting of one double-stranded and one single-stranded blocks. Since only the single-stranded chain can pass through the nanopore, the double-stranded segment has to unzip before translocating. Utilizing a simple analytical model, translocation times are calculated explicitly for the different polymer orientations - when the single-stranded block enters the pore first and when the double-stranded one enters first. Their dependence on external fields, energy of interaction in the double-stranded segment, total size of the polymer, and the fraction of double-stranded to single-stranded blocks lengths is analyzed. It is found that the order of entrance into the pore has a significant effect on the translocation dynamics.   

Assisted DNA hairpin retraction from nanopores

We present results from recent experimental and theoretical investigations of DNA hairpin retraction from an $\alpha$-hemolysin nanopore in the presence of an assisting voltage. By mapping the translocation process to that of biased diffusion of a Brownian particle we compute the probability of the polymer to stay in the pore as a function of time. Using this model we back out the diffusion constant and the drift velocity of the polymer as a function of the assisting voltage. While the drift-diffusion model gives good agreement with experiments at low voltages it fails for high assisting voltages. We discuss possible reasons for this along with the implications of our work.   

Pharmaceuticals in nanopores - A strategy to manipulate the phase behavior

The manipulation of the crystalline state of substances existing in different polymorphic forms is an important issue in many fields of application. In case of pharmaceuticals the stabilization of unstable forms is interesting since solubility and bioavailability are improved. We will show in this presentation that it is possible to manipulate the crystallization behavior of pharmaceuticals and to stabilize unstable crystalline forms by confining the substance in pores with diameters in the range 20-400 nanometers. \footnote{G.T. Rengarajan et al. $J. Am. Chem. Soc.$, to be published.} The crystallization behavior of a pharmaceutical model system in two different types of nanostructured inorganic host systems is studied by DSC and x-ray scattering. The results clearly show that the most unstable crystalline form of this pharmaceutical melts and is stable for long times under confinement which was never observed for bulk samples. This allows to extract the thermodynamic parameters of this crystalline form which have not been reported so far and shows that this is an interesting field of application for nanostructured host-guest systems. The influences of pore geometry and surface interaction are studied and possible explanations for the differences between the crystallization behavior in the bulk and under confinement are discussed.   

Thermal fluctuation spectroscopy in histone and nucleosomes during denaturation

Thermal stability of biomolecules is an important issue. We have studied thermal denaturation of histone and nucleosome using precision thermal fluctuation spectroscopy (TFS) . - a problem that we believe has not been studied experimentally before. TFS uses a very sensitive noise calorimeter which can detect thermal fluctuations of micro Kelvin at around room temperature. We find that the thermal denaturation of histones (in particular H1) as well as that of the nucleosome are associated with large fluctuations, which are few orders higher than those away from the denaturation temperature. It involves large energy exchange which can be few tens of kBT0 (T0=300K). It appears that the denaturation occurs in three distinct steps – 1. breaking of bonds leading to the cooling jumps, 2. the change in its secondary, tertiary structure leading to slow dynamics and 3. formation of bonds as it is unfolding and in the newly folded high temperature phase which accounts for the heating jumps.   

Development of High-Resolution Magnetic Tweezers for Single-Molecule Measurements

Magnetic tweezers can sense single-molecule DNA-protein interactions through optical tracking of the motion of a colloidal particle. This is typically done by relating changes in the colloid's diffraction pattern to its position. While diffraction-tracking is relatively simple to implement, it is intrinsically limited in its resolution. To improve this, we have developed a tracking technique based on Reflection Interference Contrast Microscopy (RICM). RICM relies on interference between light reflected from the colloid and a glass surface. To optimize the interference pattern, the reflecting surfaces of the colloid and the glass substrate were coated with gold and dielectric thin-films, respectively. To maintain the focal position of objective against the defocusing due to a thermal drift, the objective was automatically focused on the glass/water interface using feedback control with a piezo-driven actuator. We evaluated the system's performance by measuring fundamental physical properties of the DNA.   

Single-biomolecule circuits with carbon nanotube wiring

Because of their size and chemistry, carbon nanotubes offer a unique opportunity to couple solid-state electronics with individual proteins or other biomolecules. This talk will describe our success covalently attaching single proteins to functioning, nanotube-based electronic devices. Because the nanotubes are sensitive, one-dimensional conductors, their electrical properties are greatly altered by this attachment, even when only one or two proteins are bound. The single-molecule circuits which result allow the dynamics of molecules to be directly observed without ensemble averaging. This work is partly supported by NSF grant EF-0404057.   

Probing DNA-Protein Interactions on Surfaces Using Spectral Self-interference Fluorescence Microscopy

We are probing the interactions between double-stranded DNA and integration host factor (IHF) proteins [1] on surfaces using Spectral Self-interference Fluorescence Microscopy (SSFM) [2].The probing technique utilizes the spectral fringes produced by interference of direct and reflected emission from fluorescent molecules. The modified spectrum provides a unique signature of the axial position of the fluorophores. Using the SSFM technique, we probe the average location of the fluorescent markers attached to the DNA molecules to study the conformational changes in double-stranded DNA tethered to SiO$_{2}$ surfaces. In the presence of IHF, a DNA bending protein, we observe reduction in the vertical position of fluorescent molecules suggesting the formation of IHF-DNA complex and IHF-induced DNA bending. We also discuss the results with different IHF strains and different binding conditions. [1] Q. Bao et. al., Gene, Vol.343 pp.99-106 (2004) [2] L.A. Moiseev et. al., Journal of Applied Physics, Vol.96, pp. 5311-5315 (2004)   

Dynamics of Single Actin Filaments and Bundles in Flow

Actin filaments, aside from their biological renown as providing the `skeleton' of cells, also proffer an ideal platform from which to study -- more generally -- the properties of semi-flexible polymers. Microfluidic devices made using soft-lithography are easily adapted in dimension and geometry to create well-defined flow environments. Actin filaments are visualized in continuous flow in a microfluidic channel by stroboscopic laser light illumination. A detailed analysis of filament orientation, center-of-mass distribution, and thermal fluctuations as a function of flow rate and channel geometry is reported. In addition, the non-equilibrium bundling behavior of actin in the presence of actin-binding proteins or multivalent ions is studied in microchannel devices using FRET microscopy.   

Segregation of molecules in self-spreading lipid bilayer at ultra-small metal nano-gaps arrayed on solid surface

Diffusion of target molecules incorporated in the self-spreading lipid bilayer was controlled by the introduction of periodic array of metallic architecture on solid surface. Retardation of the progress of target molecules became significant when the size of gap between small metal architectures was less than a few hundred nm. The self-spreading dynamics of the lipid bilayer depending on the size of the small gap were analyzed semi-quantitatively. Estimated change in the driving force of the spreading layer suggests that highly localized compression of the spreading layer causes selective segregation of molecules. Surface-modified metal nano-architectures were also used to tune the selectivity of the molecules.   

Calibration of Micromachined Force Sensors by Gravitational Force on Precision Microspheres

To complement the existing tools for applying and measuring piconewton-level forces on biomolecules (e.g. optical tweezers, magnetic tweezers, AFM), we are developing a compliant micromachined spring for simple and direct measurements in an aqueous environment. Accurate calibration of the spring constant is crucial and we will present a gravitational method that uses NIST-traceable size standard microspheres. The method is applicable to calibration of other soft cantilevers of both in-plane and out-of-plane varieties. We affixed two microspheres to the force sensor and measured a deflection per bead of 196 nm $\pm $ 6{\%}. Using a weight of 150 pN $\pm $ 4.8{\%} per microsphere, we obtained a spring constant of 0.76 pN / nm $\pm $ 8{\%}. The method proved simpler and more reliable when compared to two other methods: high resolution SEM and thermal equipartition. The versatility of surface micromachining should enable use of the spring in new platforms for biophysical force measurement, for example on-chip load cells for dynamic DNA stretching.   

Multimode Analysis of SHG Signal from Complex Biological Systems: Parameterization of Features Using Nearest-Neighbor Analysis and Wavelet Transforms

We have developed a novel computational approach for quantifying structural disorder in biomolecular lattices with nonlinear susceptibility based on analysis of polarization-modulated second harmonic signal. Transient, regional disorder at the level of molecular organization is identified using a novel signal processing algorithm sufficiently compact for near real-time analysis with a desktop computer. Global disorder within the biostructure is assessed using a two-dimensional wavelet transform of the magnitude and phase of the second harmonic signal. Selection of coefficients and the specific wavelet family~is based on topological considerations. Experimental results suggest our signal processing method represents a robust, scaleable tool that allows us to detect both regional and global alterations in signal characteristics of biostructures with a high degree of discrimination.