Search for: All records

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.

 

Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins—such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)—impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate-limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy ( Δ G unfold 0 ) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol, respectively, typical for globular proteins and similar to published Δ G unfold 0 for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by atomic force microscopy (AFM)-based force spectroscopy. SptP and SopE2 unfolded at low force ( F unfold ≤ 17 pN at 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δ x ‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust ( F unfold > 80 pN) and brittle (Δ x ‡ < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion. 

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 α₃D using atomic force microscopy (AFM)–based force spectroscopy. α₃D offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed α₃D’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, α₃D 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 0.5k_BT) 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.

 

Abstract Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that methylates histone H3 at Lysine 27. PRC2 is critical for epigenetic gene silencing, cellular differentiation and the formation of facultative heterochromatin. It can also promote or inhibit oncogenesis. Despite this importance, the molecular mechanisms by which PRC2 compacts chromatin are relatively understudied. Here, we visualized the binding of PRC2 to naked DNA in liquid at the single-molecule level using atomic force microscopy. Analysis of the resulting images showed PRC2, consisting of five subunits (EZH2, EED, SUZ12, AEBP2 and RBBP4), bound to a 2.5-kb DNA with an apparent dissociation constant ($K_{\rm{D}}^{{\rm{app}}}$) of 150 ± 12 nM. PRC2 did not show sequence-specific binding to a region of high GC content (76%) derived from a CpG island embedded in such a long DNA substrate. At higher concentrations, PRC2 compacted DNA by forming DNA loops typically anchored by two or more PRC2 molecules. Additionally, PRC2 binding led to a 3-fold increase in the local bending of DNA’s helical backbone without evidence of DNA wrapping around the protein. We suggest that the bending and looping of DNA by PRC2, independent of PRC2’s methylation activity, may contribute to heterochromatin formation and therefore epigenetic gene silencing.