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1. 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)

   null (Ed.)

   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 (ΔΔ G ). 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 ΔΔ G , 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 ΔΔ G for mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔ G for mutant V217A was 2.2-fold larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val 217 and 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 ΔΔ G for a fully folded membrane protein embedded in its native bilayer. 

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2. Elucidation of a dynamic interplay between a beta-2 adrenergic receptor, its agonist, and stimulatory G protein

   https://doi.org/10.1073/pnas.2215916120  

   Han, Yanxiao; Dawson, John R.; DeMarco, Kevin R.; Rouen, Kyle C.; Bekker, Slava; Yarov-Yarovoy, Vladimir; Clancy, Colleen E.; Xiang, Yang K.; Vorobyov, Igor (March 2023, Proceedings of the National Academy of Sciences)

   G protein-coupled receptors (GPCRs) represent the largest group of membrane receptors for transmembrane signal transduction. Ligand-induced activation of GPCRs triggers G protein activation followed by various signaling cascades. Understanding the structural and energetic determinants of ligand binding to GPCRs and GPCRs to G proteins is crucial to the design of pharmacological treatments targeting specific conformations of these proteins to precisely control their signaling properties. In this study, we focused on interactions of a prototypical GPCR, beta-2 adrenergic receptor (β 2 AR), with its endogenous agonist, norepinephrine (NE), and the stimulatory G protein (G s ). Using molecular dynamics (MD) simulations, we demonstrated the stabilization of cationic NE, NE(+), binding to β 2 AR by G s protein recruitment, in line with experimental observations. We also captured the partial dissociation of the ligand from β 2 AR and the conformational interconversions of G s between closed and open conformations in the NE(+)–β 2 AR–G s ternary complex while it is still bound to the receptor. The variation of NE(+) binding poses was found to alter G s α subunit (G s α) conformational transitions. Our simulations showed that the interdomain movement and the stacking of G s α α1 and α5 helices are significant for increasing the distance between the G s α and β 2 AR, which may indicate a partial dissociation of G s α The distance increase commences when G s α is predominantly in an open state and can be triggered by the intracellular loop 3 (ICL3) of β 2 AR interacting with G s α, causing conformational changes of the α5 helix. Our results help explain molecular mechanisms of ligand and GPCR-mediated modulation of G protein activation. 

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3. Factors That Control the Force Needed to Unfold a Membrane Protein in Silico Depend on the Mode of Denaturation

   https://doi.org/10.3390/ijms24032654  

   Faruk, Nabil F.; Peng, Xiangda; Sosnick, Tobin R. (February 2023, International Journal of Molecular Sciences)

   Single-molecule force spectroscopy methods, such as AFM and magnetic tweezers, have proved extremely beneficial in elucidating folding pathways for soluble and membrane proteins. To identify factors that determine the force rupture levels in force-induced membrane protein unfolding, we applied our near-atomic-level Upside molecular dynamics package to study the vertical and lateral pulling of bacteriorhodopsin (bR) and GlpG, respectively. With our algorithm, we were able to selectively alter the magnitudes of individual interaction terms and identify that, for vertical pulling, hydrogen bond strength had the strongest effect, whereas other non-bonded protein and membrane–protein interactions had only moderate influences, except for the extraction of the last helix where the membrane–protein interactions had a stronger influence. The up–down topology of the transmembrane helices caused helices to be pulled out as pairs. The rate-limiting rupture event often was the loss of H-bonds and the ejection of the first helix, which then propagated tension to the second helix, which rapidly exited the bilayer. The pulling of the charged linkers across the membrane had minimal influence, as did changing the bilayer thickness. For the lateral pulling of GlpG, the rate-limiting rupture corresponded to the separation of the helices within the membrane, with the H-bonds generally being broken only afterward. Beyond providing a detailed picture of the rupture events, our study emphasizes that the pulling mode greatly affects the factors that determine the forces needed to unfold a membrane protein. 

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4. Binding-Induced Stabilization Measured on the Same Molecular Protein Substrate Using Single-Molecule Magnetic Tweezers and Heterocovalent Attachments

   https://doi.org/10.1021/acs.jpcb.0c00167  

   Dahal, Narayan; Nowitzke, Joel; Eis, Annie; Popa, Ionel (January 2020, The Journal of Physical Chemistry B)

   Binding-induced mechanical stabilization plays key roles in proteins involved in muscle contraction, cellular mechanotransduction, or bacterial adhesion. Because of the vector nature of force, single-molecule force spectroscopy techniques are ideal for measuring the mechanical unfolding of proteins. However, current approaches are still prone to calibration errors between experiments and geometrical variations between individual tethers. Here, we introduce a single-molecule assay based on magnetic tweezers and heterocovalent attachment, which can measure the binding of the substrate–ligand using the same protein molecule. We demonstrate this approach with protein L, a model bacterial protein which has two binding interfaces for the same region of kappa-light chain antibody ligands. Engineered molecules with eight identical domains of protein L between a HaloTag and a SpyTag were exposed to repeated unfolding–refolding cycles at forces up to 100 pN for several hours at a time. The unfolding behavior of the same protein was measured in solution buffers with different concentrations of antibody ligands. With increasing antibody concentration, an increasing number of protein L domains became more stable, indicative of ligand binding and mechanical reinforcement. Interestingly, the dissociation constant of the mechanically reinforced states coincides with that measured for the low-avidity binding interface of protein L, suggesting a physiological role for the second binding interface. The molecular approach presented here opens the road to a new type of binding experiments, where the same molecule can be exposed to different solvents or ligands. 

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5. Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations

   https://doi.org/10.1073/pnas.2015728118  

   Edwards, Devin T.; LeBlanc, Marc-Andre; Perkins, Thomas T. (March 2021, Proceedings of the National Academy of Sciences)

   null (Ed.)

   Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to μm-scale force probes via flexible linkers? Here, we studied the folding/unfolding of α 3 D using atomic force microscopy (AFM)–based force spectroscopy. α 3 D offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed α 3 D’s configurational diffusion constant within the context of Kramers theory varies with pH. The resulting pH dependence provides a test for AFM-based force spectroscopy’s ability to track intrinsic changes in protein folding dynamics. Experimentally, however, α 3 D is challenging. It unfolds at low force (<15 pN) and exhibits fast-folding kinetics. We therefore used focused ion beam–modified cantilevers that combine exceptional force precision, stability, and temporal resolution to detect state occupancies as brief as 1 ms. Notably, equilibrium and nonequilibrium force spectroscopy data recapitulated the pH dependence measured using smFRET, despite differences in destabilization mechanism. We reconstructed a one-dimensional free-energy landscape from dynamic data via an inverse Weierstrass transform. At both neutral and low pH, the resulting constant-force landscapes showed minimal differences (∼0.2 to 0.5 k B T ) in transition state height. These landscapes were essentially equal to the predicted entropic barrier and symmetric. In contrast, force-dependent rates showed that the distance to the unfolding transition state increased as pH decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins. 

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