Structure, Activity, and Dynamics of Human Serum Albumin in a Crowded Pluronic F127 Hydrogel

Abstract

Structure, activity, and dynamics of a plasma protein, human serum albumin (HSA), inside a crowded environment of F127 gel are studied by circular dichroism (CD), fluorescence correlation spectroscopy (FCS), and picosecond time-resolved fluorescence spectroscopy. For this purpose, the protein is covalently labeled by a maleimide dye, 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methyl-coumarin (CPM). The circular dichroism (CD) spectra suggest that the protein is more structured in the gel reflecting about the biological activities of the protein. FCS results demonstrate that compared to that in bulk water (buffer solution), translational diffusion is about 59 times slower inside the F127 gel. This indicates higher translational friction (viscosity) sensed by the probe (CPM). On the contrary, rotational relaxation (and hence, rotational friction) is more or less similar in F127 gel and in bulk water. FCS results further indicate that the time scales of conformational relaxation of the protein are substantially slow inside the crowded environment of F127 gel. The fast component of conformational relaxation is retarded by ∼55 times, and the slow component by ∼20 times. Fluorescence maximum of CPM bound to HSA show a ∼5 nm red shift, implying that the microenvironment of the probe, CPM, is more polar inside the gel. Solvation dynamics of CPM-labeled HSA inside the gel (⟨τ s ⟩ ∼ 300 ps) is faster compared to that for the protein in bulk water (⟨τ s ⟩ ∼ 600 ps).

Time Evolution of Local pH Around a Photo‐Acid in Water and a Polymer Hydrogel: Time Resolved Fluorescence Spectroscopy of Pyranine

“…It was already reported that in a F127 polymer hydrogel, the local viscosity rapidly varies from ∼20 cP in the corona region to ∼300 cP in the core . Thus, measurement of local viscosity is a useful probe to ascertain the location of the probe HPTS molecule.…”

“…All the measurements were carried out maintaining this temperature. Method of preparation of F127 gel is discussed earlier …”

“…We have chosen a complex hydrogel medium consisting of a pluoronic co‐polymer F127 [(PEO) 100 –(PPO) 70 –(PEO) 100 )]. The pluoronic copolymer F127 forms a hydrogel in aqueous medium at high concentration (∼30 wt %) . According to small angle neutron scattering (SANS) studies, this hydrogel consists of nearly spherical immobilized micelles ∼5 nm (core) surrounded by a hydrophilic shell having thickness of 5–6 nm (Scheme ) whose motions are arrested .…”

“…Such close packing of the spherical micelles leaves nearly 50 % volume as void occupied by free bulk‐like water molecules. Local viscosity measurement in such complex systems has implications in intra‐cellular viscosity . We apply fluorescence correlation spectroscopy (FCS) to estimate the local translational viscosity and fluorescence anisotropy decay for rotational friction (viscosity).…”

“…The pluoronic copolymer F127 forms a hydrogel in aqueous medium at high concentration (~30 wt %). [34][35][36][37][38][39][40] According to small angle neutron scattering (SANS) studies, this hydrogel consists of nearly spherical immobilized micelles~5 nm (core) surrounded by a hydrophilic shell having thickness of 5-6 nm (Scheme 2) whose motions are arrested. [34][35][36][37] Such close packing of the spherical micelles leaves nearly 50 % volume as void occupied by free bulk-like water molecules.…”