Abstract
Proteins fold and function in cellular environments that are crowded with other macromolecules. The cellular crowding effects on protein structure and stability is a key issue in molecular and structural biology. Here we report a spin-label ESR study of the T4 lysozyme protein at varying temperatures (280 ∼ 338 K) as well as crowding effects (300 ∼ 500 g/L of crowding agents, including glycerol, ficoll, and PVP). A doubly labeled T4 lysozyme (T4L-127/155), of which the two sites belong to two different but spatially-closed helical domains, is used as a model for the stability study. The corresponding cw-ESR spectra of the T4 lysozyme exhibit a substantial dipolar broadening at room temperature and confirm that the conformation is not disrupted by the mutations. By extracting the dipolar broadening from the spectra, this study shows a gradual decrease in the broadening with increasing temperature as glycerol or ficoll is used as a molecular crowder. Whereas, a drastic change is observed at temperatures around 330 K, which is about 10 K higher than the melting temperature of T4 lysozyme at a regular buffer, as the crowding agent is changed to PVP. The protein stability is found to increase in the presence of the PVP crowding; namely, as a consequence of the greater excluded volume effects of PVP (as opposed to those of glycerol and ficoll). Besides, an important mechanism underlying the crowding effects observed in this study probably is the glycerol/ficoll-mediated increase in solution viscosity that leads to protein domain motion inhibition.
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