Ewa Banachowicz

Solution Structure of Biopolymers: A New Method of Constructing a Bead Model

We propose a new, automated method of converting crystallographic data into a bead model used for the calculations of hydrodynamic properties of rigid macromolecules. Two types of molecules are considered: nucleic acids and small proteins. A bead model of short DNA fragments has been constructed in which each nucleotide is represented by two identical, partially overlapping spheres: one for the base and one for the sugar and phosphate group. The optimum radius sigma = 5.0 A was chosen on the basis of a comparison of the calculated translational diffusion coefficients (D(T)) and the rotational relaxation times (tau(R)) with the corresponding experimental data for B-DNA fragments of 8, 12, and 20 basepairs. This value was assumed for the calculation D(T) and tau(R) of tRNA(Phe). Better agreement with the experimental data was achieved for slightly larger sigma = 5.7 A. A similar procedure was applied to small proteins. Bead models were constructed such that each amino acid was represented by a single sphere or a pair of identical, partially overlapping spheres, depending on the amino acids size. Experimental data of D(T) of small proteins were used to establish the optimum value of sigma = 4.5 A for amino acids. The lack of experimental data on tau(R) for proteins restricted the tests to the translational diffusion properties.

Successful FCS experiment in nonstandard conditions.

Fluorescence correlation spectroscopy (FCS) is frequently used to measure the self-diffusion coefficient of fluorescently labeled probes in solutions, complex media, and living cells. In a standard experiment water immersion objectives and window thickness in the range of 0.13-0.19 mm are used. We show that successful FCS measurements can be performed using samples of different refractive index placed in cells having windows of different thickness, even much thicker than nominally allowed. Different water, oil, and silicon oil immersion as well as long working distance dry objectives, equipped with the correction collar, were tested and compared. We demonstrate that the requirements for FCS experiments are less stringent than those for high resolution confocal imaging and reliable relative FCS measurements can be performed even beyond the compensation range of the objectives. All these features open new possibilities for construction of custom-made high temperature and high pressure cells for FCS.

Three dimensional model of severe acute respiratory syndrome coronavirus helicase ATPase catalytic domain and molecular design of severe acute respiratory syndrome coronavirus helicase inhibitors

The modeling of the severe acute respiratory syndrome coronavirus helicase ATPase catalytic domain was performed using the protein structure prediction Meta Server and the 3D Jury method for model selection, which resulted in the identification of 1JPR, 1UAA and 1W36 PDB structures as suitable templates for creating a full atom 3D model. This model was further utilized to design small molecules that are expected to block an ATPase catalytic pocket thus inhibit the enzymatic activity. Binding sites for various functional groups were identified in a series of molecular dynamics calculation. Their positions in the catalytic pocket were used as constraints in the Cambridge structural database search for molecules having the pharmacophores that interacted most strongly with the enzyme in a desired position. The subsequent MD simulations followed by calculations of binding energies of the designed molecules were compared to ATP identifying the most successful candidates, for likely inhibitors—molecules possessing two phosphonic acid moieties at distal ends of the molecule.

Structural similarity of E. coli 5S rRNA in solution and within the ribosome.

The article presents translational and rotational diffusion coefficients of 5S rRNA determined experimentally by the method of dynamic light scattering (DLS) and its comparison with the values predicted for different models of this molecule. The tertiary structure of free 5S rRNA was proposed on the basis of the atomic structures of the 5S rRNA from E. coli and H. marismortui extracted from the ribosome. A comparison of the values of DT, τR, and Rg predicted for different models with experimental results for the free molecule in solution suggests that free 5S rRNA is less compact than that in the complex with ribosomal proteins. In general, the molecules of 5S rRNA consist of three domains: a short one and two longer ones. As follows from a comparison of the results of our simulations with experimental values, in the molecule in solution the two closest helical fragments of the longer domains remain collinear, whereas the short domain takes a position significantly deviated from them.

High-pressure small-angle neutron scattering studies of glucose isomerase conformation in solution

Small-angle neutron scattering (SANS) of solutions of glucose/xylose isomerase from Streptomyces rubiginosus was measured as a function of pressure. It is shown that the structure of the enzyme in solution as seen by SANS is practically the same as that in the crystal and does not change with pressure up to 150 MPa. This reflects the unusually high structural stability of this material, which makes it extremely interesting to use as a secondary standard for pressure-dependent SANS experiments. This lack of pressure dependence of the SANS data also indicates that any possible change in hydration of the protein induced by pressure is not visible in the SANS curves. An appropriate correction procedure must be used for the SANS data in order to account for the distortion of the intensity curve due to hard-sphere and electrostatic interactions. After this correction, the isomerase can be readily used as a secondary standard for SANS measurements.

Temperature dependent FCS studies using a long working distance objective: Viscosities of supercooled liquids and particle size

In this work, we describe new experimental setups for Fluorescence Correlation Spectroscopy (FCS) where a long working distance objective is used. Using these setups, FCS measurements in a broad temperature range for a small sample volume of about 50 μl can be performed. The use of specially designed cells and a dry long working distance objective was essential for avoiding temperature gradients in the sample. The performance of the new setups and a traditional FCS setup with immersion objectives is compared. The FCS data in combination with the Stokes-Einstein (SE) relation were used to obtain the values of the nanoviscosity of a fluid. We show for selected molecular van der Waals supercooled liquids that despite the fact that in these systems, a characteristic length scale can be defined, the nanoviscosity obtained from FCS is in a very good agreement with the macroscopic (rheometric) viscosity of the sample in a broad temperature range. This result corroborates the applicability of the SE relation to supercooled liquids at temperatures above 1.2 Tg. We also show that the temperature dependent size of thermoresponsive microgel particles can be determined by FCS using the designed cells and a long working distance objective in a broader size range without a need to use the correction procedure since the size correction is proportional to the square of the ratio of the hydrodynamic radius to the confocal volume size.

Structural properties of the intrinsically disordered, multiple calcium ion-binding otolith matrix macromolecule-64 (OMM-64)

Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed.

Expression and characterization of a barley phosphatidylinositol transfer protein structurally homologous to the yeast Sec14p protein.

Phosphatidylinositol transfer proteins (PITPs) include a large group of proteins implicated in the non-vesicular traffic of phosphatidylinositol (PI) between membranes. In yeast, the structure and function of the PITP Sec14-p protein have been well characterized. In contrast, the knowledge on plant PITP proteins is very scarce. In this work, we characterized a novel type of PITP protein in barley named HvSec14p and related to the yeast Sec14-p protein. Our data reveal that HvSec14p consists of only the Sec14p-domain structurally homologous to the yeast phosphoinositide binding domain. We show that HvSec14p expression is up-regulated at both transcript and protein levels at specific stages of development during seed formation and germination, and in leaves of a drought-tolerant barley genotype under osmotic constraints. Modeling analyses of the protein three-dimensional structure revealed its capacity to dock the phosphoinositides, PtdIns(3)P, PtdIns(4)P, PtdIns(5)P and PtdIns(3,5)P2. Consistently, the recombinant HvSec14p protein is able to bind in vitro most PIP types, the highest affinity being observed with PtdIns(3,5)P2. Based on the high gene expression at specific developmental stages and in drought-tolerant barley genotypes, we propose that HvSec14p plays essential roles in the biogenesis of membranes in expanding cells and in their preservation under osmotic stress conditions.

Dynamic Light Scattering and NMR Studies of Napin

We analyze the structure of napin (BngNAP1), a storage protein (m.w. 14.5 kDa) from Brassica napus. On the basis of the results of 1H NMR spectroscopy and dynamic light scattering (DLS) studies, the overall shape and secondary structure of the molecule are estimated.

Nano-viscosity of supercooled liquid measured by fluorescence correlation spectroscopy: Pressure and temperature dependence and the density scaling

The Stokes-Einstein relation allows us to calculate apparent viscosity experienced by tracers in complex media on the basis of measured self-diffusion coefficients. Such defined nano-viscosity values can be obtained through single particle techniques, like fluorescence correlation spectroscopy (FCS) and particle tracking (PT). In order to perform such measurements, as functions of pressure and temperature, a new sample cell was designed and is described in this work. We show that this cell in combination with a long working distance objective of the confocal microscope can be used for successful FCS, PT, and confocal imaging experiments in broad pressure (0.1-100 MPa) and temperature ranges. The temperature and pressure dependent nano-viscosity of a van der Waals liquid obtained from the translational diffusion coefficient measured in this cell by means of FCS obeys the same scaling as the rotational relaxation and macro-viscosity of the system.