Protein Folding Stability and Kinetics in Alginate Hydrogels

Area of Science:

• Biophysics
• Materials Science
• Biochemistry

Background:

• Proteins are frequently encapsulated in alginate gels for applications in drug delivery and tissue engineering.
• Limited understanding exists regarding how this encapsulation affects intrinsic protein properties, including folding stability and unfolding kinetics.

Purpose of the Study:

• To quantitatively assess the impact of alginate hydrogel concentration on the stability and folding dynamics of encapsulated proteins.
• To investigate the in situ protein unfolding and folding kinetics within alginate hydrogels.

Main Methods:

• Utilized fast relaxation imaging (FReI) to monitor protein unfolding in situ within alginate hydrogels.
• Employed Förster resonance energy transfer (FRET)-labeled phosphoglycerate kinase (PGK) to measure changes in protein folding.
• Applied temperature jumps to induce unfolding and analyzed FRET response across varying alginate concentrations.

Main Results:

• Alginate gels significantly stabilize PGK, increasing its melting temperature by up to 18.4 °C, with stabilization showing a non-monotonic dependence on alginate density.
• Encapsulation in denser gels caused PGK to deviate more from two-state folding behavior.
• The gels decreased the unfolding rate and accelerated the folding rate of PGK compared to buffer conditions.

Conclusions:

• Alginate gel encapsulation presents both beneficial and detrimental effects on protein folding stability and dynamics.
• The observed alterations in protein stability and folding kinetics are influenced by alginate concentration and gel density.
• Phi-value analysis suggests the folding transition state of encapsulated PGK is structurally similar to the folded state, providing insights into potential stabilization mechanisms.