Direct Imaging of Protein Stability and Folding Kinetics in Hydrogels

Abstract

We apply fast relaxation imaging (FReI) as a novel technique for investigating the folding stability and dynamics of proteins within polyacrylamide hydrogels, which have diverse and widespread uses in biotechnology. FReI detects protein unfolding in situ by imaging changes in fluorescence resonance energy transfer (FRET) after temperature jump perturbations. Unlike bulk measurements, diffraction-limited epifluorescence imaging combined with fast temperature perturbations reveals the impact of local environment effects on protein-biomaterial compatibility. Our experiments investigated a crowding sensor protein (CrH2) and phosphoglycerate kinase (PGK), which undergoes cooperative unfolding. The crowding sensor quantifies the confinement effect of the cross-linked hydrogel: the 4% polyacrylamide hydrogel is similar to aqueous solution (no confinement), while the 10% hydrogel is strongly confining. FRAP measurements and protein concentration gradients in the 4% and 10% hydrogels further support this observation. PGK reveals that noncovalent interactions of the protein with the polymer surface are more important than confinement for determining protein properties in the gel: the mere presence of hydrogel increases protein stability, speeds up folding relaxation, and promotes irreversible binding to the polymer even at the solution-gel interface, whereas the difference between the 4% and the 10% hydrogels is negligible despite their large difference in confinement. The imaging capabilities of FReI, demonstrated to be diffraction limited, further revealed spatially homogeneous protein unfolding across the hydrogels at 500 nm length scales and revealed differences in protein properties at the gel-solution boundary.

An in vitro mimic of in‐cell solvation for protein folding studies

“…The FReI apparatus has been described previously . Briefly, a computer‐controlled, continuous‐wave 2 μm laser (AdValue Photonics, Tucson, Arizona) is used to rapidly apply a step‐function shaped temperature perturbation to the sample.…”

“…The FReI apparatus has been described previously. 36 Briefly, a computer-controlled, continuous-wave 2 μm laser (AdValue Photonics, Tucson, Arizona) is used to rapidly apply a step-function shaped temperature perturbation to the sample. As long as the perturbation is applied at a time scale faster than the underlying conformational dynamics (T-jump), it will elicit a timedependent response in the protein's FRET signal that can be used to probe the reaction.…”

Comparative screening of recombinant antigen thermostability for improved leptospirosis vaccine design

“…Diagnostic and binding studies that incorporate Lig proteins could also be impacted by thermostability issues. Depending on the surface chemistry in assays, the native fold of affixed proteins can be altered from proteins that are free in solution (Kisley et al, ; Weltz, Schwartz, & Kaar, ). In future Lig protein studies, the lack of comprehensive domain thermostability under relevant conditions needs to be carefully considered during experimental design to ensure proper comparability and interpretation of outcomes.…”

Heat shock-induced chaperoning by Hsp70 is enabled in-cell

“…Protein expression was carried out according to previously established protocol [30]. Fusion protein sequences were cloned into pDream 2.1/MCS vector by Genscript Corp. and used for dual expression in E. coli and mammalian cells unless stated otherwise.…”

“…Cells were imaged in Opti-MEM (Fisher) supplemented with 15% FBS. mEGFP in the cells was excited by a white LED by passing the light through a Chroma ET470/40x bandpass filter and mCherry was excited through a ET580/25x bandpass filter [30]. Both mEGFP and mCherry emission was monitored on a Lt225 camera equipped with a CMOS sensor (Lumenera).…”