10 research outputs found
Volume of Hsp90 ligand binding and the unfolding phase diagram as a function of pressure and temperature
No full textDetermination of the volume changes induced by ligand binding to heat shock protein 90 using high-pressure denaturation
No full textReal-time CUDA-based stereo matching using Cyclops2 algorithm
No full textAbstract This paper presents a novel stereo matching algorithm Cyclops2. The algorithm produces a disparity image, provided two rectified grayscale images. The matching is based on the concept of minimising a weight function calculated using the absolute difference of pixel intensities. We present three simple and easily parallelizable weight functions. Each presented function gives a different trade-off between algorithm processing time and reconstructed depth image accuracy. Detailed description of the algorithm implementation in CUDA is provided. The implementation was specifically optimised for embedded NVIDIA Jetson platform. NVIDIA Jetson TK1 and TX1 boards have been used to evaluate the algorithms. We evaluated seven algorithm variations with different parameter values. Each variation results in a different speed accuracy trade-off, demonstrating that our algorithm can be used in various situations. The presented algorithm achieves up to 70 FPS processing time on lower resolution images (750 × 500 pixels) and up to 23 FPS on high-resolution images (1500 × 1000 pixels). The use of optional post-processing stage (median filter) has also been investigated. We conclude that despite its limitations, our algorithm is relevant in the field of real-time obstacle avoidance
Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay – Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII
No full textThe analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90aN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 ºC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90aN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding
A Quantitative Model of Thermal Stabilization and Destabilization of Proteins by Ligands
No full textAbstractEquilibrium binding ligands usually increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. High-throughput screening for the discovery of drug-like compounds uses an assay based on thermal stabilization. The mathematical description of this stabilization is well developed, and the method is widely applicable to the characterization of ligand-protein binding equilibrium. However, numerous cases have been experimentally observed where equilibrium binding ligands destabilize proteins, i.e., diminish protein melting temperature by an amount proportional to the concentration and affinity of the ligand. Here, we present a thermodynamic model that describes ligand binding to the native and unfolded (denatured) protein states explaining the combined stabilization and destabilization effects. The model also explains nonsaturation and saturation effects on the protein melting temperature when the ligand concentration significantly exceeds the protein concentration. Several examples of the applicability of the model are presented, including specific sulfonamide binding to recombinant hCAII, peptide and ANS binding to the Polo-box domain of Plk1, and zinc ion binding to the recombinant porcine growth hormone. The same ligands may stabilize and destabilize different proteins, and the same proteins may be stabilized and destabilized by different ligands
High pressure spectrofluorimetry – a tool to determine protein-ligand binding volume
No full textThe change in protein volume observed upon protein-ligand interaction (termed as the binding volume) is an important but largely neglected thermodynamic parameter from the perspective of both fundamental science and potential applications in the development of specific protein ligands. The binding volume is the pressure derivative of the Gibbs energy, thus elevated pressure is required to determine the volumetric properties of proteins. Here we describe the use of high-pressure spectrofluorimetry to determine both unfolding and ligand binding- induced volume changes of a protein. The degree of protein unfolding at elevated pressures was monitored by an intrinsic tryptophan fluorescence. Different approaches of experimental fluorescence spectra analysis are described and the impact on the quality of thermodynamic parameters is discussed
