GXXXG‐Mediated Parallel and Antiparallel Dimerization of Transmembrane Helices and Its Inhibition by Cholesterol: Single‐Pair FRET and 2D IR Studies

Abstract

Small-residue-mediated interhelical packings are ubiquitously found in helical membrane proteins, although their interaction dynamics and lipid dependence remain mostly uncharacterized. We used a single-pair FRET technique to examine the effect of a GXXXG motif on the association of de-novo designed (AALALAA)3 helices in liposomes. Dimerization occurred with sub-second lifetimes, which was abolished by cholesterol. Utilizing the nearly instantaneous time-resolution of 2D IR spectroscopy, parallel and antiparallel helix associations were identified by vibrational couplings across helices at their interface. Taken together, the data illustrate that the GXXXG motif controls helix packing but still allows for a dynamic and lipid-regulated oligomeric state.

Purification of recombinant SARS-CoV-2 spike, its receptor binding domain, and CR3022 mAb for serological assay

“…There are a number of GXXXG motifs in the protein sequence of HSPG ( Fig. 15B), suggesting that this protein could form a dimer [28].…”

Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules

“…Virus infection smFRET imaging assay revealed structure arrangement of critical binding domains between the membrane receptor and virus spike [124,129] Protein aggregation Intramolecular FRET showed the states of aggregation in various conditions quantitatively [141][142][143][144][145] Intermolecular FRET exhibited the aggregating principle [144,145] Protein-Lipid Cross-membrane transport smFRET with long critical transfer distance detected both parallel and antiparallel dimers of transmembrane helix regulated by cholesterol [132,133] Protein-Nucleic acid DNA synthesis Drops in FRET efficiency indicated DNA synthesis with label on polymerase or on template [146,147] Various labeling strategies can present the states of different active domains on polymerase [148]…”

“…They confirmed that cholesterol significantly stabilized the antiparallel helix dimer interpreted as hydrophobic interaction. In another study two years later, they replaced the center of (AALALAA) 3 with the guest GXXXG motif [ 133 ]. Interestingly, transient dimerizations in both parallel and antiparallel topologies achieved an equilibrium, whereas the addition of cholesterol completely abolished the Gly-mediated associations.…”

“…On the aspects of membrane proteins, other similar studies have provided a basic insight on various conformation landscapes, including membrane-protein misfolding [ 134 ] and structural dynamics of the ion channel [ 135 ]. Other tools like fluorescence correlation spectroscopy (FCS) [ 136 , 137 ], super-resolution microscopy [ 138 ] and infrared spectroscopy [ 132 , 133 ] are combined with smFRET for better analysis of the mechanism behind the phenomenon.…”

Fast slow folding of an Outer Membrane Porin

“…In contrast, continuous measurements of immobilised, confined, or encapsulated molecules permit transitions between conformational states to be measured [49,50] (typically in exchange for time resolution). Continuous smFRET measurements have been used with great effect to probe functional dynamics [51,52,53,54] and association dynamics [55,56,57,58] of membrane proteins in lipid bilayers. However, their application to membrane protein folding in a lipid bilayer environment is, to our knowledge, yet to be demonstrated; such a study was recently proposed by Krainer et al [59] and here we seek to address that call.…”