RESUMEN
Chemotaxis Y (CheY), upon metal binding, displays a drastic alteration in its structure and stability. This premise prompted us to study the effect of crowding on the two conformationally distinct states of the same test protein. A comparative analysis on the structure and thermal stability in the presence and absence of the macromolecular crowder, ficoll, and its monomeric unit, sucrose, revealed a contrasting effect of ficoll on the apo and holo forms. In the presence of ficoll while the thermal stability (Tm) of the apo form is enhanced, the thermal stability of the holo form is reduced. The selective lowering of Tm for the holo form in the combined presence of ficoll and sucrose and not in sucrose alone suggests that the contrasting effect is due to the macromolecular nature of ficoll. Since metal-protein interaction remains unperturbed in the presence of ficoll and Mg2+ sequestration is ruled out in a systematic manner the alternative possibility for the exclusive reduction in the thermal stability of the holo form is the ficoll-induced modulation of the relative population of apo and holo forms of CheY.
Asunto(s)
Proteínas Bacterianas , Quimiotaxis , Ficoll , Desnaturalización Proteica , Sustancias MacromolecularesRESUMEN
To study the effect of non-specific interactions arising from proteins being in a crowded environment on physiological processes, the self-interaction of concentrated Dextran T70 and Ficoll 70 and the interactions between a dilute protein and these polymeric macromolecules were quantified using non-ideal tracer sedimentation equilibrium. Sedimentation equilibria of each polymer were measured between 5 and 37 °C, and sedimentation equilibria of 2 mg cm-3 superoxide dismutase (SOD) in 0-0.1 g cm-3 of each polymer was also measured. Results were analyzed using a model-free thermodynamic virial expression of activity coefficients in terms of the concentration of polymer and a structural model using a statistical thermodynamics approximation. The equilibrium gradients of each of the polymers suggest repulsive interaction, which is independent of temperature. However, the net repulsive interaction between superoxide dismutase (SOD) species and the polymers is dependent on temperature. The ratio of the solvation energy of SOD in Dextran T70 to that in Ficoll 70, lnγSOD(Dex)/lnγSOD(Fic) at the same w/v concentration was about 1.8 at 37 °C, 1.6 at the intermediate temperature, and ranges from 1.2 to 1.6 at 5 °C over the entire concentration range. The enthalpy and entropy of interaction of SOD with dilute Dextran T70 are - 14 kJ mol-1 and - 5.6 J K-1 mol-1, respectively. For SOD in dilute Ficoll 70, the enthalpy and entropy are - 8.1 kJ mol-1 and 12.9 J K-1 mol-1, respectively. Thus, Dextran T70 contributes more to the attractive protein-polymer interaction and to self-association of protein than Ficoll 70 and reasons for this are discussed.
Asunto(s)
Dextranos/metabolismo , Ficoll/metabolismo , Superóxido Dismutasa/metabolismo , Temperatura , Cinética , Unión Proteica , SolucionesRESUMEN
The effect molecular crowding, defined as the volume exclusion exerted by one soluble inert molecule upon another soluble molecule, has on the structure and self-interaction of lipid-free apoA-I were explored. The influence of molecular crowding on lipid-free apoA-I oligomerization and internal dynamics has been analyzed using electron paramagnetic resonance (EPR) spectroscopy measurements of nitroxide spin label at selected positions throughout the protein sequence and at varying concentrations of the crowding agent Ficoll-70. The targeted positions include sites previously shown to be sensitive for detecting intermolecular interaction via spin-spin coupling. Circular dichroism was used to study secondary structural changes in lipid-free apoA-I imposed by increasing concentrations of the crowding agent. Crosslinking and SDS-PAGE gel analysis was employed to further characterize the role molecular crowding plays in inducing apoA-I oligomerization. It was concluded that the dynamic apoA-I structure and oligomeric state was altered in the presence of the crowding agent. It was also found that the C-terminal was slightly more sensitive to molecular crowding. Finally, the data described the region around residue 217 in the C-terminal domain of apoA-I as the most sensitive reporter of the crowding-induced self-association of apoA-I. The implications of this behavior to in vivo functionality are discussed. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 683-692, 2016.
Asunto(s)
Apolipoproteína A-I/química , Ficoll/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Humanos , Dominios ProteicosRESUMEN
How the crowded environment inside cells affects folding, stability and structures of proteins is a vital question, since most proteins are made and function inside cells. Here we describe how crowded conditions can be created in vitro and in silico and how we have used this to probe effects on protein properties. We have found that folded forms of proteins become more compact in the presence of macromolecular crowding agents; if the protein is aspherical, the shape also changes (extent dictated by native-state stability and chemical conditions). It was also discovered that the shape of the macromolecular crowding agent modulates the folding mechanism of a protein; in addition, the extent of asphericity of the protein itself is an important factor in defining its folding speed.