Session C2: Symposium on Soft Matter: Biophysics II

Vibrating virus capsids and interactions with short light pulses -- picking up good vibrations

Viruses are the simplest ``life'' form. They reproduce by borrowing the machinery of their host cell. Viruses consist of an outer coat (capsid) that protects its genomic material inside. They are pathogenic to plants, bacteria, animals, and of course humans. Experimental studies at ASU by Tsen et al. have discovered that ultra-short laser pulses are capable of ``inactivating'' viruses. One potential mechanism is the coupling of light to the soft dynamical modes of the capsid. We describe theoretical modeling of this effect.   

Broadband Liquid Dielectric Spectrometer

A dielectric spectrometer was built to measure the dielectric relaxation of proteins in solution. The dielectric cell consisted of two parallel stainless-steel electrodes (separation of 8.5 mm) embedded in PTFE. To provide temperature stability, thermally regulated water flowed through both electrodes. The cell was connected to a 4294A Precision Impedance Analyzer, providing impedance measurements from 40Hz to 110 MHz. Due to electrode polarization and high frequency parasitics, useful measurements were obtained for frequencies ranging from 10 kHz to 10 MHz. Calibration was performed using air, iso-propanol and deionized water. Experiments were also conducted on buffers and salt solutions. The dielectric relaxation of the protein beta-lactoglobulin was measured at mg/ml concentrations.   

Image Processing Diagnostics: Emphysema

Currently the computerized tomography (CT) scan can detect emphysema sooner than traditional x-rays, but other tests are required to measure more accurately the amount of affected lung. CT scan images show clearly if a patient has emphysema, but is unable by visual scan alone, to quantify the degree of the disease, as it appears merely as subtle, barely distinct, dark spots on the lung. Our goal is to create a software plug-in to interface with existing open source medical imaging software, to automate the process of accurately diagnosing and determining emphysema severity levels in patients. This will be accomplished by performing a number of statistical calculations using data taken from CT scan images of several patients representing a wide range of severity of the disease. These analyses include an examination of the deviation from a normal distribution curve to determine skewness, a commonly used statistical parameter. Our preliminary results show that this method of assessment appears to be more accurate and robust than currently utilized methods which involve looking at percentages of radiodensities in air passages of the lung.   

Three-Pulse Photon Echo Peak Shift (3PEPS) as a probe of conformational distribution in heme protein folding

Line broadening of electronic spectra of cofactor consists of two contributions: homogeneous broadening and inhomogeneous broadening. Inhomogeneous broadening reflects conformational diversity of the cofactor and its surrounding environment and is a key to quantifying the disorder of soft condensed matter system. To clarify the relation between contributions to the lineshapes and protein conformation, we investigated the equilibrium unfolding sample of Zn-cytochrome c (Zn-cyt c) in guanidine hydrochloride (GuHCl). Soret band UV-vis spectra were first measured as a function of GuHCl, and fitted with both two- and three- state models of protein folding. Then, we measured 3PEPS signal of the folded, midpoint and unfolded samples. By tuning the laser wavelength over Soret band on midpoint sample and comparing experimental asymptotic peak shift with simulated from two- and three-state models, we attributed folding of Zn-cyt c to a two-state model. This is a novel approach in understanding protein folding and should be applicable to investigate of other proteins.   

Thrombin flux and wall shear rate regulate fibrin fiber deposition state during polymerization under flow

Thrombin is released as a soluble enzyme from the platelet surface to trigger fibrin polymerization during thrombosis under flow conditions. While isotropic fibrin polymerization under static conditions involves protofibril extension and lateral aggregation leading to a gel, factors regulating fiber diameter and orientation are poorly quantified under hemodynamic flow due to the difficulty of setting thrombin fluxes. A membrane microfluidic device allowed combined control of both thrombin wall flux (10$^{-13}$ to 10$^{-11}$ nmol/$\mu$ m$^2$ s) and the wall shear rate (10 to 100 s$^{-1}$) of a flowing fibrinogen solution. At the thrombin flux of 10$^{-12}$ nmol/$\mu$ m$^2$ s, both fibrin deposition and fiber thickness decreased as the wall shear rate increased from 10 to 100 s$^{-1}$. Direct measurement and transport-reaction simulations at 12 different thrombin flux-wall shear rate conditions demonstrated that two dimensionless numbers, the Peclet number (Pe) and the Damkohler number (Da),defined a phase diagram to predict fibrin morphology. For Da$<$10,we only observed thin films at all Pe. For 10$<$Da$<$100, we observed either mats of surface fibers or gels depending on the Pe. For Da$>$900 and Pe$<$100, we observed three-dimensional gels. These results indicate that increase wall shear rate first quenches lateral aggregation and then protofibril extension.   

Characterization of Manufactured Binding Sites in BPTI Using Laser Polarized $^{129}$Xe

We measure the NMR chemical shift of laser polarized $^{129}$Xe in wild type, Y35G, Y23A, and F45S BPTI (Bovine Pancreatic Trypsin Inhibitor) solutions of varying concentration. Our technique uses $^{129}$Xe in unprecedented low concentrations as a biosensor. The results provide structural information concerning the aforementioned proteins [2,3]. We use a flow-through polarizer that outputs $^{129}$Xe hyperpolarized to $\sim $10{\%}. Hyperpolarized gas coupled with a high resolution NMR spectrometer, enables us to measure sub-ppm chemical shifts at very low Xe and protein concentrations. In accordance with the fast exchange regime, we observe a single resonance that is chemically shifted as a function of protein concentration. Consistent with a rigid lattice and a manufactured binding site, Y23A and F45S demonstrate strong binding relative to wild type and Y35G. Wild type is believed not to have a specific binding site. Other experiments on Y35G have demonstrated a hydrophobic cavity and extensive solution-phase motion [1]. Weak binding supports the notion that a small fraction of solution-phase Y35G is in a conformation such that the cavity is accessible to Xe. [1] S. A. Beeser, \textit{J. Mol. Biol.}, \textbf{269}, 154-164v [2] W. M. Hanson \textit{et al.}, \textit{J. Mol. Biol.} 2007, \textbf{366}, 230-243 [3] A. T. Danishefsky \textit{et al.}, \textit{Protein Sci.} 1993, \textbf{2}, 577-587