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

Abstract

Understanding how proteins retain structural stability is not only of fundamental importance in biophysics but also critical to industrial production of antibodies and vaccines. Protein stability is known to depend mainly on two effects: internal hydrophobicity and H-bonding between the protein surface and solvent. A challenging task is to identify their individual contributions to a protein. Here, we investigate the structural stability of the apoptotic Bid protein in solutions containing various concentrations of guanidinium hydrochloride and urea using a combination of recently developed methods including the QTY (glutamine, threonine, and tyrosine) code and electron spin resonance-based peak-height analysis. We show that when the internal hydrophobicity of Bid is broken down using the QTY code, the surface H-bonding alone is sufficient to retain the structural stability intact. When the surface H-bonding is disrupted, Bid becomes sensitive to the temperature-dependent internal hydrophobicity such that it exhibits a reversible cold unfolding above water's freezing point. Using the combined approach, we show that the free-energy contributions of the two effects can be more reliably obtained. The surface H bonds are more important than the other effect in determining the structural stability of Bid protein.

Protein Design: From the Aspect of Water Solubility and Stability

“…However, the crystal structures of QTY proteins need to be determined to support the hypothesis. In contrast, a more recent publication showed that the soluble BH3 interacting-domain death agonist protein with similar QTY substitutions at the core region resulted in mutations with comparable secondary structures but was more prone to chemical denaturation only when surface hydrogen bond interactions were negated by a high concentration guanidine hydrochloride …”

Simple Cryoprotectant-Free Method to Advance Pulsed Dipolar ESR Spectroscopy for Capturing Protein Conformational Ensembles

“…The BH3 interacting-domain death agonist (Bid) protein (22 kDa; Figure S3A) is used as a model protein in the present work. , Wild-type (WT) Bid in a regular buffer is folded below water’s freezing point. It was recently reported that WT Bid (hereafter abbreviated only as Bid) in a 3 M guanidinium hydrochloride (GdnHCl) solution can undergo reversible cold and heat denaturation above water’s freezing temperature; it is largely unfolded at temperatures below 280 K or greater than 330 K in 3 M GdnHCl. In a 1 M GdnHCl solution, Bid is largely unfolded at temperatures below 250 K or greater than 350 K .…”

“…It was recently reported 18 that WT Bid (hereafter abbreviated only as Bid) in a 3 M guanidinium hydrochloride (GdnHCl) solution can undergo reversible cold and heat denaturation above water's freezing temperature; it is largely unfolded at temperatures below 280 K or greater than 330 K in 3 M GdnHCl. In a 1 M GdnHCl solution, Bid is largely unfolded at temperatures below 250 K or greater than 350 K. 18 We performed CD measurements on Bid in 0 and 3 M GdnHCl solutions at varying temperatures (Figure 2A,B). Consistent with the literature, 18 we found that Bid in 0 M GdnHCl manifests only heat denaturation (>360 K), whereas Bid in 3 M GdnHCl shows both cold and heat denaturation above water's freezing temperature.…”

“…The thermograms were recorded by monitoring the MRE signals at 222 nm at temperatures decreasing from 298 to 278 K, and then reversing back to 368 K, with a 5 K increment. The recorded CD spectra displayed the same thermal behaviors as those reported in the literature …”

“…The recorded CD spectra displayed the same thermal behaviors as those reported in the literature. 18 Preparation of the Rapid Freezing Samples. Approximately, a 2.5 μL solution volume of Bid (2 mM) was injected into 30 pieces (1 cm length, 100 μm i.d., and 360 ± 10 μm o.d.)…”