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Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔG). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔG, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR’s G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule–derived ΔΔGfor mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔGfor mutant V217A was 2.2-fold larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val217and a natively bound squalene lipid, highlighting the contribution of membrane protein–lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔGfor a fully folded membrane protein embedded in its native bilayer.
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Resolving the enthalpy of protein stabilization by macromolecular crowding
Abstract Proteins in the cellular milieu reside in environments crowded by macromolecules and other solutes. Although crowding can significantly impact the protein folded state stability, most experiments are conducted in dilute buffered solutions. To resolve the effect of crowding on protein stability, we use19F nuclear magnetic resonance spectroscopy to follow the reversible, two‐state unfolding thermodynamics of the N‐terminal Src homology 3 domain of theDrosophilasignal transduction protein drk in the presence of polyethylene glycols (PEGs) of various molecular weights and concentrations. Contrary to most current theories of crowding that emphasize steric protein–crowder interactions as the main driving force for entropically favored stabilization, our experiments show that PEG stabilization is accompanied by significant heat release, and entropy disfavors folding. Using our newly developed model, we find that stabilization by ethylene glycol and small PEGs is driven by favorable binding to the folded state. In contrast, for larger PEGs, chemical or soft PEG–protein interactions do not play a significant role. Instead, folding is favored by excluded volume PEG–protein interactions and an exothermic nonideal mixing contribution from release of confined PEG and water upon folding. Our results indicate that crowding acts through molecular interactions subtler than previously assumed and that interactions between solution components with both the folded and unfolded states must be carefully considered.
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- Award ID(s):
- 1909664
- PAR ID:
- 10398209
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 32
- Issue:
- 3
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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