Abstract
Interactions between proteins and ligands, which are fundamental to many biochemical processes essential to life, are mostly studied at dilute buffer conditions. The effects of the highly crowded nature of biological cells and the effects of liquid-liquid phase separation inducing biomolecular droplet formation as a means of membrane-less compartmentalization have been largely neglected in protein binding studies. We investigated the binding of a small ligand (ANS) to one of the most multifunctional proteins, bovine serum albumin (BSA) in an aqueous two-phase system (ATPS) composed of PEG and Dextran. Also, aiming to shed more light on differences in binding mode compared to the neat buffer data, we examined the effect of high hydrostatic pressure (HHP) on the binding process. We observe a marked effect of the ATPS on the binding characteristics of BSA. Not only the binding constants change in the ATPS system, but also the integrity of binding sites is partially lost, which is most likely due to soft enthalpic interactions of the BSA with components in the dense droplet phase of the ATPS. Using pressure modulation, differences in binding sites could be unravelled by their different volumetric and hydration properties. Regarding the vital biological relevance of the study, we notice that extreme biological environments, such as HHP, can markedly affect the binding characteristics of proteins. Hence, organisms experiencing high-pressure stress in the deep sea need to finely adjust the volume changes of their biochemical reactions in cellulo.
Highlights
The exact concentration of the solution was determined by measuring the absorbance at 280 nm by means of UV/Vis spectroscopy (UV-1800 spectrometer from Shimadzu Corporation) and using a molar extinction coefficient of 43600 M−1 cm−1
The interaction between BSA and ANS was followed by means of steady-state fluorescence spectroscopy using a K2 fluorometer from ISS, Inc. (Champaign, IL, USA) at the temperature of 25 °C
The titrations were performed by recording the ANS emission spectra by exciting the solutions at 350 nm and recording the emission intensities in the range 400–550 nm
Summary
A stock solution of bovine serum albumin (BSA, MW of 66 kDa, 583 residues) was prepared by dissolving the protein in Tris buffer. The exact concentration of the solution was determined by measuring the absorbance at 280 nm by means of UV/Vis spectroscopy (UV-1800 spectrometer from Shimadzu Corporation) and using a molar extinction coefficient of 43600 M−1 cm−1. Subsequent dilution in buffer was performed to determine the exact concentration.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
