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1. Resolving the enthalpy of protein stabilization by macromolecular crowding

   https://doi.org/10.1002/pro.4573  

   Stewart, Claire J.; Olgenblum, Gil I.; Propst, Ashlee; Harries, Daniel; Pielak, Gary J. (February 2023, Protein Science)

   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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2. Analysis of folded structure and folding thermodynamics in heterogeneous-backbone proteomimetics

   https://doi.org/10.1016/bs.mie.2021.04.009  

   Santhouse, Jacqueline R.; Rao, Shilpa R.; Horne, W. Seth (January 2021, Methods in enzymology)

   Petersson, E. James (Ed.)

   Recent years have seen a growing number of examples of designed oligomeric molecules with artificial backbone connectivity that are capable of adopting complex folded tertiary structures analogous to those seen in natural proteins. A range of experimental techniques from structural biology and biophysics have been brought to bear in the study of these proteomimetic agents. Here, we discuss some considerations encountered in the characterization of high-resolution folded structure as well as folding thermodynamics of protein-like artificial backbones. We provide an overview of the use of X-ray crystallography and NMR spectroscopy in such systems and review example applications of these methods in the primary literature. Further, we provide detailed protocols for two experiments that have proved useful in our prior and ongoing efforts to compare folding thermodynamics between natural protein domains and heterogeneous-backbone counterparts. 

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3. Quantitative entropy–enthalpy compensation in intraprotein interactions from model compound data

   https://doi.org/10.1002/pro.5013  

   Redvanly, Thomas W; Pielak, Gary J (June 2024, Protein Science)

   Abstract Many small globular proteins exist in only two states—the physiologically relevant folded state and an inactive unfolded state. The active state is stabilized by numerous weak attractive contacts, including hydrogen bonds, other polar interactions, and the hydrophobic effect. Knowledge of these interactions is key to understanding the fundamental equilibrium thermodynamics of protein folding and stability. We focus on one such interaction, that between amide and aromatic groups. We provide a statistically convincing case for quantitative, linear entropy–enthalpy compensation in forming aromatic–amide interactions using published model compound transfer‐free energy data. 

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4. The Volumetric Properties of the Transition State Ensemble for Protein Folding

   https://doi.org/10.26434/chemrxiv-2021-svw4f  

   Avagyan, Samvel and (October 2021, ChemRxiv)

   Hydrostatic pressure together with the temperature is an important environmental variable that plays an essential role in biological adaptation of extremophilic organisms. In particular, the effects of hy-drostatic pressure on the rates of the protein folding/unfolding reaction are determined by the magni-tude and sign of the activation volume changes. Here we provide computational description of the ac-tivation volume changes for folding/unfolding reaction, and compare them with the experimental data for six different globular proteins. We find that the volume of the transition state ensemble is always in-between the folded and unfolded states. Based on this, we conclude that hydrostatic pressure will invariably slow down protein folding and accelerate protein unfolding. 

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5. Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy

   https://doi.org/10.1073/pnas.2020083118  

   Jacobson, David R.; Perkins, Thomas T. (March 2021, Proceedings of the National Academy of Sciences)

   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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