Robust membrane protein tweezers reveal the folding speed limit of helical membrane proteins

Abstract

Single-molecule tweezers, such as magnetic tweezers, are powerful tools for probing nm-scale structural changes in single membrane proteins under force. However, the weak molecular tethers used for the membrane protein studies have limited the observation of long-time, repetitive molecular transitions due to force-induced bond breakage. The prolonged observation of numerous transitions is critical in reliable characterizations of structural states, kinetics, and energy barrier properties. Here, we present a robust single-molecule tweezer method that uses dibenzocyclooctyne cycloaddition and traptavidin binding, enabling the estimation of the folding ‘speed limit’ of helical membrane proteins. This method is >100 times more stable than a conventional linkage system regarding the lifetime, allowing for the survival for ~12 hr at 50 pN and ~1000 pulling cycle experiments. By using this method, we were able to observe numerous structural transitions of a designer single-chained transmembrane homodimer for 9 hr at 12 pN and reveal its folding pathway including the hidden dynamics of helix-coil transitions. We characterized the energy barrier heights and folding times for the transitions using a model-independent deconvolution method and the hidden Markov modeling analysis, respectively. The Kramers rate framework yields a considerably low-speed limit of 21 ms for a helical hairpin formation in lipid bilayers, compared to μs scale for soluble protein folding. This large discrepancy is likely due to the highly viscous nature of lipid membranes, retarding the helix-helix interactions. Our results offer a more valid guideline for relating the kinetics and free energies of membrane protein folding.

Hidden route of protein damage through confined oxygen gas

“…This study was prompted by the unexpected observation of a rapid decline in protein foldability under blue light (λpeak = 447 nm; Extended Data Fig. 1 for the full spectrum), using a recently developed single-molecule tweezers 31 (Fig. 2a).…”

“…2a-c). This tweezer approach demonstrated high stability, enabling the capture of ~1000 reversible unfolding events in a single protein 31 . We employed this method to repetitively unfold and refold maltose binding protein (MBP), a widely-used model protein in protein folding studies, over hundreds of cycles.…”

Oxidative photocatalysis on membranes triggers non-canonical pyroptosis

“…To con rm the oxidative destabilisation of GlpG by which protein aggregation was promoted, we used a single-molecule tweezer approach that could entirely exclude the aggregation events (Fig. 2e-h) 32,[35][36][37] . In this approach, a single GlpG embedded in a lipid bilayer disc (bicelle) was tethered via DNA handles to a magnetic bead and the sample chamber surface (Fig.…”

“…In this approach, a single GlpG embedded in a lipid bilayer disc (bicelle) was tethered via DNA handles to a magnetic bead and the sample chamber surface (Fig. 2e) 37,38 . Using a pair of magnets generating the magnetic eld gradient, we applied a mechanical force of a few to tens of pN to the tethered GlpG.…”

Probing the stability and interdomain interactions in the ABC transporter OpuA, using single-molecule optical tweezers

“…Additionally, studies have been performed on the reversibility of folding of relatively simple membrane proteins in a bicelle using magnetic tweezers 13,14 . However, comparable studies on more complex transmembrane proteins are absent in literature 15 .…”