Direct Imaging of Protein Stability and Folding Kinetics in Hydrogels

• DOI: 10.1021/acsami.7b01371
• Publication Date: 2017-05-29T12:14:06Z
• Authors: Lydia Kisley; Kali A. Miller; Drishti Guin; Xinyu Kong; Martin Gruebele; Deborah E. Leckband
• Keywords: Phosphoglycerate kinase; Polyacrylamide; Macromolecular Crowding; Protein Engineering

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