Accessing local structural disruption of Bid protein during thermal denaturation by absorption-mode ESR spectroscopy.

Bid is a requisite protein that connects death receptors to the initiation of mitochondrial-dependent apoptosis. Its structure was determined more than a decade ago, but its structure-function relationship remains largely unexplored. Here we investigate the thermostability of Bid protein and explore how the death-promoting function of Bid is affected by thermally-induced unfolding. First, we show by circular dichroism (CD) spectroscopy that Bid remains partially folded at high temperatures (350-368 K), and that the thermal unfolding of Bid is irreversible and accompanied with intermolecular associations that lead to protein aggregation. In 3 M GdnHCl, the onset of unfolding can, however, be conveniently observed at much lower temperatures around 320 K. We employ pulsed ESR dipolar spectroscopy to show that the structure of Bid remains almost unchanged between 0 and 3 M GdnHCl before thermal denaturation. More than 30 single-labeled Bid mutants are studied using the peak-height analysis method based on ESR absorption spectroscopy, in the temperature range of 300-345 K. The ESR results provide site-specific information about the temperature dependence of the local environment of Bid, thus enabling the discrimination between the onsets of unfolding and aggregation for respective sites. Consequently, we map out the local thermostability over the Bid structure and identify a new interface between helices 3, 6, and 8 as the beginning of structural unfolding. This study also investigates the apoptotic activity of the thermally-induced Bid aggregates and shows that Bid retains the death-promoting function even when unfolded and aggregated. The applicability of the new ESR absorption peak-height method is demonstrated for protein thermostability.