Single-molecule force spectroscopy of protein-membrane interactions

Abstract

Many biological processes rely on protein-membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach, single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2-7 pN and had binding energies of 4-14 k B T per C2 domain. Regulation by bilayer composition or Ca 2+ recapitulated known properties of both proteins. The method can be widely applied to study protein-membrane interactions.

Rearrangements under confinement lead to increased binding energy of Synaptotagmin‐1 with anionic membranes in Mg2+ and Ca2+

“…The increased binding of Syt1 in the presence of Ca 2+ is also expected from previous work , which indicates that Syt1 reorients to insert the hydrophobic residues of the Ca 2+ loop into the membrane. A recent optical tweezers study reported a binding energy of ~12 k B T in the presence of 0.5 m m free Ca 2+ for C2AB binding to an anionic membrane .…”

“…The increased binding of Syt1 in the presence of Ca 2+ is also expected from previous work , which indicates that Syt1 reorients to insert the hydrophobic residues of the Ca 2+ loop into the membrane. A recent optical tweezers study reported a binding energy of ~12 k B T in the presence of 0.5 m m free Ca 2+ for C2AB binding to an anionic membrane . A wide range of dissociation constants, and therefore binding energetics, have been measured by traditional biological assays for soluble C2AB with anionic membranes, with a maximum value of ~25 k B T .…”

“…With similar lipid compositions the dissociation constant measurements of Syt1 with membranes are reported to be in the range of~5-10 k B T in EGTA [6,7]. A recent single-molecule study reported no binding between C2AB and an anionic membrane in the absence of Ca 2+ , possibly because the Ca 2+ -independent binding requires participation of multiple molecules [20]. The value reported here (5.8 AE 0.9 k B T) is in the lower range of values reported thus far, which is perhaps expected.…”

Synaptotagmin-1 and Doc2b exhibit distinct membrane remodeling mechanisms

“…For Syt1-C2AB induced tethers, the median rupture force most likely represents proteinmembrane interactions with a median rupture force in the 50 -200 pN range, strongly dependent on the Ca 2+ (which regulates C2AB-membrane binding) and protein concentration. The binding force of one Syt1 molecule to a membrane is in the range of several pN (43). Therefore, the differences we observe in the force probably relate to a variation in the number of bound molecules.…”

A programmable DNA-origami platform for studying protein-mediated lipid transfer between bilayers

“…The shuttle model is consistent with the hydrophilic nature of the two tips of the SMP domain and with the lack of evidence for the property of the SMP domain to form tetramers or longer adducts to bind and bridge the two bilayers 10 properties 18,24 . The C2A domains of the E-Syts bind membranes in a Ca 2+ -dependent way 18,24,43 and were proposed to bind the plasma membrane 44 . However, the length of the unstructured linker that separates it from the SMP domain is too short (~ 8 nm when fully stretched) to span the ER-plasma membrane distance when the SMP domain is in close proximity of the ER during its shuttling.…”