ABSTRACT
High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of -32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 µM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.
Subject(s)
Anilino Naphthalenesulfonates/metabolism , Darunavir/metabolism , HIV Protease/chemistry , HIV Protease/metabolism , Protein Folding , Protein Stability/drug effects , Atmospheric Pressure , Dimerization , HIV Protease Inhibitors/metabolism , Humans , Kinetics , Models, Molecular , Protein Conformation , Protein Multimerization , Spectrometry, Fluorescence , Thermodynamics , Tryptophan/metabolismABSTRACT
The behavior of microparticles exposed to gravitational and lift forces and to the velocity gradient in flow velocity profile formed in microfluidic conduits is studied from the viewpoint of the transient period (the relaxation) between the moment at which a particle starts to be transported by the hydrodynamic flow and the time at which it reaches an equilibrium position, characterized by a balance of all active forces. The theoretical model allowing the calculation of the relaxation time is proposed. The numerical calculus based on the proposed model is compared with the experimental data obtained under different experimental conditions, namely, for different lengths of microfluidic channels, different average linear velocities of the carrier liquid, and different sizes and densities of the particles used in the study. The results are important for the optimization of microfluidic separation units such as microthermal field-flow fractionation channels in which the separation or manipulation of the microparticles of various origin, synthetic, natural, biological, etc., is performed under similar experimental conditions but by applying an additional thermodynamic force.
Subject(s)
Microfluidic Analytical Techniques , Hydrodynamics , Particle SizeABSTRACT
The steady-state movement of the spherical and non-spherical particles, such as prolate or oblate rotational ellipsoids, cylinders, or parallelepipeds, suspended in a liquid and exposed to a unidirectional temperature gradient, is analyzed theoretically. The differences in the ratios of the rotational to translational diffusion coefficients of the non-spherical to spherical particles, the heterogeneity of thermal conductivity of the particle body, and the heterogeneity in surface chemical nature make possible to separate the particles according to differences in shape. Preliminary experimental separations of Gram-positive and Gram-negative, nearly spherical and rod-shaped bacteria performed by Microthermal Field-Flow Fractionation confirmed that the fractionation of the cells according to differences in shape is possible.
Subject(s)
Bacteria/chemistry , Fractionation, Field Flow/methods , Bacteria/isolation & purification , Particle SizeABSTRACT
The retention of Staphylococcus epidermidis bacteria cells, achieved with the use of micro-thermal field-flow fractionation and described in this paper, represents the first experimental proof that the separation and characterization of the bio-macromolecules and biological particles is possible by exploiting Ludwig-Soret effect of thermal diffusion. The experiments were carried out under gentle experimental conditions preventing the denaturation of the bacteria. Lift forces, appearing at high linear velocities of the carrier liquid, generated the focusing mechanism of the retention which resulted in high-speed and high-performance separation performed in less than 10 min.
Subject(s)
Fractionation, Field Flow/methods , Staphylococcus epidermidis/isolation & purification , Humans , Skin/microbiologyABSTRACT
The separation of Staphylococcus epidermidis and Rhodococcus erythropolis bacteria was achieved with the use of Micro-Thermal Focusing Field-Flow Fractionation. This is the first performance of separation exploiting the Ludwig-Soret effect (thermal diffusion) of living biological cells, combined with lift forces and resulting in the focusing mechanism of separation. The experiments were carried out under carefully chosen experimental conditions preventing the denaturation of the bacteria.
