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1. An investigation of the YidC-mediated membrane insertion of Pf3 coat protein using molecular dynamics simulations

   https://doi.org/10.3389/fmolb.2022.954262  

   Polasa, Adithya; Hettige, Jeevapani; Immadisetty, Kalyan; Moradi, Mahmoud (August 2022, Frontiers in Molecular Biosciences)

   YidC is a membrane protein that facilitates the insertion of newly synthesized proteins into lipid membranes. Through YidC, proteins are inserted into the lipid bilayer via the SecYEG-dependent complex. Additionally, YidC functions as a chaperone in protein folding processes. Several studies have provided evidence of its independent insertion mechanism. However, the mechanistic details of the YidC SecY-independent protein insertion mechanism remain elusive at the molecular level. This study elucidates the insertion mechanism of YidC at an atomic level through a combination of equilibrium and non-equilibrium molecular dynamics (MD) simulations. Different docking models of YidC-Pf3 in the lipid bilayer were built in this study to better understand the insertion mechanism. To conduct a complete investigation of the conformational difference between the two docking models developed, we used classical molecular dynamics simulations supplemented with a non-equilibrium technique. Our findings indicate that the YidC transmembrane (TM) groove is essential for this high-affinity interaction and that the hydrophilic nature of the YidC groove plays an important role in protein transport across the cytoplasmic membrane bilayer to the periplasmic side. At different stages of the insertion process, conformational changes in YidC’s TM domain and membrane core have a mechanistic effect on the Pf3 coat protein. Furthermore, during the insertion phase, the hydration and dehydration of the YidC’s hydrophilic groove are critical. These results demonstrate that Pf3 coat protein interactions with the membrane and YidC vary in different conformational states during the insertion process. Finally, this extensive study directly confirms that YidC functions as an independent insertase. 

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2. Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy

   https://doi.org/10.1073/pnas.2313818121  

   Jacobson, David R.; Perkins, Thomas T. (February 2024, Proceedings of the National Academy of Sciences)

   Ligand-induced conformational changes are critical to the function of many membrane proteins and arise from numerous intramolecular interactions. In the photocycle of the model membrane protein bacteriorhodopsin (bR), absorption of a photon by retinal triggers a conformational cascade that results in pumping a proton across the cell membrane. While decades of spectroscopy and structural studies have probed this photocycle in intricate detail, changes in intramolecular energetics that underlie protein motions have remained elusive to experimental quantification. Here, we measured these energetics on the millisecond time scale using atomic-force-microscopy-based single-molecule force spectroscopy. Precisely, timed light pulses triggered the bR photocycle while we measured the equilibrium unfolding and refolding of the terminal 8-amino-acid region of bR’s G-helix. These dynamics changed when the EF-helix pair moved ~9 Å away from this end of the G helix during the “open” portion of bR’s photocycle. In ~60% of the data, we observed abrupt light-induced destabilization of 3.4 ± 0.3 kcal/mol, lasting 38 ± 3 ms. The kinetics and pH-dependence of this destabilization were consistent with prior measurements of bR’s open phase. The frequency of light-induced destabilization increased with the duration of illumination and was dramatically reduced in the triple mutant (D96G/F171C/F219L) thought to trap bR in its open phase. In the other ~40% of the data, photoexcitation unexpectedly stabilized a longer-lived putative misfolded state. Through this work, we establish a general single-molecule force spectroscopy approach for measuring ligand-induced energetics and lifetimes in membrane proteins. 

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3. Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein

   https://doi.org/10.1073/pnas.2109169119  

   Gaffney, Kristen_A; Guo, Ruiqiong; Bridges, Michael_D; Muhammednazaar, Shaima; Chen, Daoyang; Kim, Miyeon; Yang, Zhongyu; Schilmiller, Anthony_L; Faruk, Nabil_F; Peng, Xiangda; et al (December 2021, Proceedings of the National Academy of Sciences)

   Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG ofEscherichia coli(E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron–electron resonance spectroscopy. These data, along with ourUpsidesimulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition.E. coli’s lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes. 

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4. Peptoid-based macrodiscs of variable lipid composition for structural studies of membrane proteins by oriented-sample solid-state NMR

   Galiakhmetov, Azamat R; Shah, Adit A; Lane, Addison; Davern, Carolynn M; Proulx, Caroline; Nevzorov, Alexander A (June 2024, Journal of structural biology)

   Solid-state Nuclear Magnetic Resonance (NMR) in combination with magnetically aligned discoidal lipid mimics allows for studying the conformations of membrane proteins in planar, lipid-rich bilayer environments and at the physiological temperature. We have recently demonstrated the general applicability of macrodiscs composed of DMPC lipids and peptoid belts, which yield magnetic alignment and NMR spectroscopic resolution comparable or superior to detergent-containing bicelles. Here we report on a considerable improvement in the magnetic alignment and NMR resolution of peptoid-based macrodiscs consisting of a mixture of the zwitterionic and negatively charged lipids (DMPC/DMPG at the 85% to 15% molar ratio). The resulting linewidths are about 30% sharper due to the higher orientational order parameter likely arising from the stabilizing electrostatic repulsion between the discs. Moreover, highly aligned, detergent-free macrodiscs can be formed with a longer-chain lipid, DPPC. Interestingly, the spectra of Pf1 in the two lipid mimetics are almost indistinguishable, which would mean that the overall transmembrane helix tilt might be governed not only by the hydrophobic matching but also possibly by the interactions of the flanking lysine and arginine residues at the membrane interface. 

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5. Comparing Interfacial Trp, Interfacial His and pH Dependence for the Anchoring of Tilted Transmembrane Helical Peptides

   https://doi.org/10.3390/biom10020273  

   Afrose, Fahmida; Koeppe II, Roger E. (February 2020, Biomolecules)

   Charged and aromatic amino acid residues, being enriched toward the terminals of membrane-spanning helices in membrane proteins, help to stabilize particular transmembrane orientations. Among them, histidine is aromatic and can be positively charge at low pH. To enable investigations of the underlying protein-lipid interactions, we have examined the effects of single or pairs of interfacial histidine residues using the constructive low-dynamic GWALP23 (acetyl-GG2ALW5LALALALALALALW19LAG22A-amide) peptide framework by incorporating individual or paired histidines at locations 2, 5, 19 or 22. Analysis of helix orientation by means of solid-state 2H NMR spectra of labeled alanine residues reveals marked differences with H2,22 compared to W2,22. Nevertheless, the properties of membrane-spanning H2,22WALP23 helices show little pH dependence and are similar to those having Gly, Arg or Lys at positions 2 and 22. The presence of H5 or H19 influences the helix rotational preference but not the tilt magnitude. H5 affects the helical integrity, as residue 7 unwinds from the core helix; yet once again the helix orientation and dynamic properties show little sensitivity to pH. The overall results reveal that the detailed properties of transmembrane helices depend upon the precise locations of interfacial histidine residues. 

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