title: Side-chain packing interactions stabilize an intermediate of BAX protein against chemical and thermal denaturation.
abstract: Bcl-2-associated X (BAX) protein plays a gatekeeper role in transmitting apoptotic signaling from cytosol to mitochondria. However, little is known about its stability. This study reports a comprehensive investigation on the stability of BAX using spin-label ESR, CD, and ThermoFluor methods. Point mutations covering all of the nine helices of BAX were prepared. ESR study shows that BAX can be divided into two structural regions, each responding differently to the presence of guanidine hydrochloride (GdnHCl). The N-terminal region (helices 1-3) is denatured in 6 M GdnHCl, whereas the C-terminal region (helices 4-9) is resistant to the denaturing effects. The far-UV CD spectra show an appreciable amount of helical content of BAX at high temperatures. The magnitude of the near-UV CD signal is increased with increasing temperature in either 0 or 6 M GdnHCl, indicating an enhancement of aromatic side-chain packing in the C-terminal region. Taken together with ThermoFluor results, we show that a core interior, wherein aromatic interactions are highly involved, within the C-terminal region plays an important role in stabilizing BAX against the denaturing effects. Collectively, we report a highly stable, indestructible intermediate state of BAX. Side-chain packing interactions are shown to be the major stabilizing force in determining BAX structure.
authors.affiliation: Department of Chemistry and Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Chun-Hui
authors.initials: CH
authors.lastname: Chan
authors.forename: Chia-Jung
authors.initials: CJ
authors.lastname: Tsai
authors.forename: Yun-Wei
authors.initials: YW
authors.lastname: Chiang
mesh_terms: D019791:Guanidine / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D011489:Protein Denaturation; D013696:Temperature; D051028:bcl-2-Associated X Protein / Q000737:chemistry*
chemical_list: C494826:BAX protein, human; D051028:bcl-2-Associated X Protein; D019791:Guanidine