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

Abstract

The apoptotic function of Bid does not depend on its native structure.

Protein Stability Depends Critically on the Surface Hydrogen-Bonding Network: A Case Study of Bid Protein

“…Cw-ESR spectra of the single-labeled Bid proteins were recorded on cooling temperatures from 300 to 230 K (raw spectra shown in Figure S4) and then analyzed using the ESR-based peak-height method, a recently developed approach . Briefly, this method analyzes the temperature dependence of cw-ESR spectra in the absorption mode, yielding a plot of spectral peak height versus temperature.…”

“…Heating and cooling are two common approaches to investigate protein unfolding. − While the cold-induced unfolding is usually caused by the breakdown of hydrophobic effect, ,,, the heat-induced unfolding occurs because of the increase in the thermal energy of structural fluctuation that subsequently disrupts either or both of the hydrophobic interaction and the surface hydration structure. ,, Because water molecules form a relatively ordered hydrogen-bond (H-bond) network with the surface side chains, it is believed that the surface hydration structure and dynamics play a key role in determining the structural dynamics of a protein. , Both the protein–solvent H-bonding and internal hydrophobicity are important noncovalent interactions affecting the protein stability . To experimentally study the contributions of the two interactions to protein stability, it is often necessary to conduct extensive site-directed mutational analyses of polar versus nonpolar residues. , This study identifies the two contributions using a combination of recently developed techniques including the QTY (glutamine, threonine, and tyrosine) code method and the peak-height analysis based on electron spin resonance (ESR) spectroscopy. , …”

“…The BH3 interacting-domain death agonist (Bid) protein, a proapoptotic member of the BCL2 protein family with an approximate molecular weight of 22 kDa (195 aa; Figure S1A), was previously identified to be highly resistant against thermal and chemical denaturation and is used as a model protein in the present study. − Here, we report for the first time, cold denaturation of the Bid protein. We show how the cold denaturation of Bid can be induced/inhibited by changes in the concentrations of denaturants, including guanidine hydrochloride (GdnHCl) and urea, together with glycerol that modulates the strength of protein internal and surface interactions.…”

“…The recombinant pET28a vector was transformed into the Escherichia coli BL21­(DE3) expression strain (Novagen). Recombinant proteins fused with six histidines at the N-terminal of Bid were expressed and purified by an affinity Ni column, as previously described. , Briefly, bacterial culture was grown at 37 °C in Luria-Bertani (LB) medium containing kanamycin (30 μg/mL) until OD 600 reached 0.6–0.8. Protein expression was induced by the addition of 1 mM isopropyl 1-thio-β- d -galactopyranoside (IPTG) at 30 °C for 4–6 h. The cell pellet was collected by centrifugation and resuspended in ice-cold lysis buffer [20 mM Tris, pH 7.4, 100 mM NaCl, 20 mM imidazole, and protease inhibitor tablet (cOmplete)].…”

“…The lower-field peak is selected for the peak-height plot because its change between the two temperature extremes is greater than those of the central- and higher-field peaks. This ESR-based peak-height method is a semiempirical analysis of the nitroxide spectrum, allowing one to conveniently access the temperature-dependent behaviors of individual labeling sites and identify the onset temperature for local unfolding . As previously demonstrated, the peak-height value of a freely tumbling protein decreases linearly with decreasing temperature, whereas it would change abruptly (with a change in the sign of the slope for the peak height vs temperature plot) upon structural unfolding as the rotational dynamics of spin label become (relatively) unrestricted after protein local folds disrupt.…”