In this thesis we investigate the role of protein unfolding on protein aggregation and hydrogel formation in two different systems. In the context of designing protein-based hydrogels as biomaterials, we investigate how protein unfolding affects the formation dynamics of hydrogels in response to temperature changes, denaturation, and chemical reactions. In a second context we establish how microsecond to millisecond fluctuations in an amyloid forming protein, beta-2-microglobulin, correlate to the amyloid forming propensity of the protein, with an emphasis on understanding how conformational changes in the native folded state provide thermodynamic driving forces for amyloid nucleation.The work on protein hydrogel yielded two key results. First, we observed that the lifetime of dissipative hydrogels decreased and their mechanical stiffness increased with increasing denaturant concentration and constant fuel concentration. At a higher denaturant concentration, the concentration of solvent-accessible cysteines increases the stiffness of the hydrogel at the cost of a faster consumption of H_2 O_2, which is the cause of the shorter gel lifetime. This work utilizing biological macromolecules in kinetically controlled dissipative structures opens the door to future applications of such systems in which the biomolecules' structures can control the reaction kinetics. Another substantial outcome of our work is to uncover mechanisms underlying the initiation of nucleation in the initial stages of amyloid aggregate formation. The study of conformational fluctuations in the structure of the amyloid-forming protein beta 2-microglobulin (β_2 M) yielded three key results. First, β_2 M variants' aggregation propensity correlates with their conformational fluctuations rate. A longer-lived misfolded subpopulation increases the chance of aggregation initiation by increasing the collision chance of the protein's sticky regions. Second, the observed millisecond interconversions agree with the timescales required for the interconversion of a protein's structure between its subpopulations. Third, the fluctuations themselves could be a driving force for the nucleation of aggregates by decreasing the lag-time of nucleus formation by a sudden large fluctuation.