Sensitivity and resolution are the two fundamental obstacles to extending solid-state nuclear magnetic resonance to even larger protein systems. Here, a novel long-observation-window band-selective homonuclear decoupling (LOW BASHD) scheme is introduced that increases resolution up to a factor of 3 and sensitivity up to 1.8 by decoupling backbone alpha-carbon (Cα) and carbonyl (C′) nuclei in U-13C-labeled proteins during direct 13C acquisition. This approach introduces short (<200 μs) pulse breaks into much longer (~8 ms) sampling windows to efficiently refocus the J-coupling interaction during detection while avoiding the deleterious effects on sensitivity inherent in rapid stroboscopic band-selective homonuclear decoupling techniques. A significant advantage of LOW BASHD detection is that it can be directly incorporated into existing correlation methods, as illustrated here for 2D CACO, NCO, and NCA correlation spectroscopy applied to the β1 immunoglobulin binding domain of protein G and 3D CBCACO correlation spectroscopy applied to the α-subunit of tryptophan synthase.

Taken together, our data suggest that two dimers of CT domain are juxtaposed around helix C3 leading to apoE3 tetramerization, and that dissociation to monomeric units is a required step in lipid binding, with helix C3 likely seeking stability via lipid interaction prior to helices C1 or C2.

Apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in cholesterol homeostasis. The N-terminal (NT) domain of apoE3 (residues 1-191) is folded into a helix bundle comprised of 4 amphipathic α-helices: H1, H2, H3 and H4, flanked by flexible helices N1 and N2, and Hinge Helix 1 (Hinge H1), at the N-and C-terminal sides of the helix bundle, respectively. The NT domain plays a critical role in binding to the low density lipoprotein receptor (LDLR), which eventually leads to lowering of plasma cholesterol levels. In order to be recognized by the LDLR, the helix bundle has to open and undergo a conformational change. The objective of the study was to understand the mechanism of opening of the helix bundle. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) revealed that apoE3 NT domain adopts several disordered and unfolded regions, with H2 exhibiting relatively little protection against exchange-in compared to H1, H3, and H4. Site-directed fluorescence labeling indicated that H2 not only has the highest degree of solvent exposure but also the most flexibility in the helix bundle. It also indicated that the lipoprotein behavior of H1 was significnatly different from that of H2, H3 and H4. These results suggest that the opening of the helix bundle is likely initiated at the flexible end of H2 and the loop linking H2/H3, and involves movement of H2/H3 away from H1/H4. Together, these observations offer mechanistic insight suggesting a regulated helix bundle opening of apoE3 NT domain can be triggered by lipid binding.

Membrane proteins are assembled through balanced interactions among protein, lipids and water. Studying their folding while maintaining the native lipid environment has been a necessary but challenging. Here we present a set of methods for analyzing key elements in membrane protein folding, including thermodynamic stability, compactness of the unfolded state and unfolding cooperativity under native conditions. The methods are based on steric trapping which couples unfolding of a doublybiotinylated protein to binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying those methods to an intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions for maintaining the stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.

Apolipoprotein E3 (apoE3) is an anti-atherogenic protein that helps maintain triglycerides and cholesterol levels in the plasma. It is responsible for binding and cellular uptake of plasma lipoproteins via the low-density lipoprotein receptor (LDLr) family of proteins. It is a highly alpha-helical protein that can exist in lipid-free and lipid-bound states, and undergoes a dramatic conformational re-organization when transitioning between the two states. The objective of this study is to understand the mechanism of chemical-induced unfolding of the 4-helix bundle N-terminal (NT) domain housing the LDLr binding sites. The rationale is that the ease of unfolding is a reflection of ease of helix bundle opening during lipid interaction. We tested the hypothesis that the 4-helix bundle undergoes a concerted opening to reveal the hydrophobic interior, analogous to opening an umbrella. Using X-ray and NMR structure guided rational approach, single cysteine-containing constructs were designed; a fluorescent probe was attached to the free -SH groups to serve as a spectroscopic reporter about the microenvironment and mobility of selected segments of the protein: the 4 main helices observed in the X-ray structure (H1 to H4) and the relatively flexible helixes flanking the helix bundle as observed in the NMR structure. We induced unfolding by chemical denaturation and monitored changes in protein secondary structure and tertiary conformation by circular dichroism and fluorescence spectroscopy, respectively. The overall global was relatively unaltered in the labeled variants; however, fluorescence emission and polarization studies revealed that during unfolding, the microenvironment of the probe on H3 and H4 is more polar and that these helices become more mobile at lower denaturant concentrations, compared to probes on H1 and H2. The data suggest that the 4-helix bundle undergoes a sequential unfolding pattern of the main helices in the following order: helix 4/helix 3, helix 2 and lastly helix 1. Our results offer insights into the mechanism of lipid and lipoprotein binding of apoE3 NT domain, with the segment towards the C-terminal end likely triggering lipid interaction.