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.
Double
electron–electron resonance (DEER) is a powerful
technique for studying protein conformations. To preserve the room-temperature
ensemble, proteins are usually shock-frozen in liquid nitrogen prior
to DEER measurements. The use of cryoprotectant additives is, therefore,
necessary to ensure the formation of a vitrified state. Here, we present
a simple modification of the freezing process using a flexible fused
silica microcapillary, which increases the freezing rates and thus
enables DEER measurement without the use of cryoprotectants. The Bid
protein, which is highly sensitive to cryoprotectant additives, is
used as a model. We show that DEER with the simple modification can
successfully reveal the cold denaturation of Bid, which was not possible
with the conventional DEER preparations. The DEER result reveals the
nature of Bid folding. Our method advances DEER for capturing the
chemically and thermally induced conformational changes of a protein
in a cryoprotectant-free medium.
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