Single-Molecule Manipulation of Macromolecules on Membranes Using High-Resolution Optical Tweezers

Despite their wide applications into soluble macromolecules, optical tweezers have rarely been used to characterize dynamics of membrane proteins, mainly due to lack of model membranes compatible with optical trapping. Here, we found that optical tweezers can stably trap giant unilamellar vesicles (GUVs) containing iodixanol with controlled membrane tension, which can potentially serve as a model membrane to study dynamics of membranes, membrane proteins, or their interactions. We also observed that small unilamellar vesicles (SUVs) are rigid enough to resist large pulling force and offer potential advantages to pull membrane proteins. To demonstrate the use of both model membranes, we pulled membrane tethers from the trapped GUVs and measured the folding or binding dynamics of a single DNA hairpin or synaptotagmin-1 C2 domain attached to the GUV or SUV with high spatiotemporal resolution. Our methodologies facilitate single-molecule manipulation studies of membranes or membrane proteins using optical tweezers.

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PRINCIPLES OF SINGLE-MOLECULE MANIPULATION AND ITS APPLICATION IN BIOLOGICAL PHYSICS

International Journal of Modern Physics B ◽

10.1142/s021797921230006x ◽

2012 ◽

Vol 26 (13) ◽

pp. 1230006 ◽

Cited By ~ 3

Author(s):

WEI-HUNG CHEN ◽

JONATHAN D. WILSON ◽

SITHARA S. WIJERATNE ◽

SARAH A. SOUTHMAYD ◽

KUAN-JIUH LIN ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Biological Molecules ◽

Force Detection ◽

Biomolecular Systems ◽

Single Molecule Level ◽

Detection Techniques ◽

Atomic Force ◽

Manipulation System ◽

Single Molecule Manipulation

Recent advances in nanoscale manipulation and piconewton force detection provide a unique tool for studying the mechanical and thermodynamic properties of biological molecules and complexes at the single-molecule level. Detailed equilibrium and dynamics information on proteins and DNA have been revealed by single-molecule manipulation and force detection techniques. The atomic force microscope (AFM) and optical tweezers have been widely used to quantify the intra- and inter-molecular interactions of many complex biomolecular systems. In this article, we describe the background, analysis, and applications of these novel techniques. Experimental procedures that can serve as a guide for setting up a single-molecule manipulation system using the AFM are also presented.

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Polybasic patches in both C2 domains of Synaptotagmin-1 are required for evoked neurotransmitter release

10.1101/2021.07.05.451149 ◽

2021 ◽

Author(s):

Zhenyong Wu ◽

Lu Ma ◽

Nicholas A Courtney ◽

Jie Zhu ◽

Yongli Zhang ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Calcium Binding ◽

Neurotransmitter Release ◽

Transmembrane Domain ◽

Snare Complex ◽

Critical Feature ◽

C2 Domains ◽

Electrophysiological Recordings ◽

Synaptotagmin 1

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release. It is composed of a single-pass transmembrane domain linked to two tandem C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is not well understood. Calcium binding to C2B, but not to C2A, is critical for synchronous release and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here we asked what critical feature of C2A may be responsible for its functional role, and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side-by-side to determine their relative contributions to Syt1 function in cultured cortical mouse neurons and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The readily releasable vesicle pool or spontaneous release were not affected, so both patches are specifically required for synchronization of release. We suggest these patches contribute to cooperative binding to membranes, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.

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Single-molecule manipulation of double-stranded DNA using optical tweezers: Interaction studies of DNA with RecA and YOYO-1

Cytometry ◽

10.1002/(sici)1097-0320(19990701)36:3<200::aid-cyto9>3.0.co;2-t ◽

1999 ◽

Vol 36 (3) ◽

pp. 200-208 ◽

Cited By ~ 104

Author(s):

Martin L. Bennink ◽

Orlando D. Sch�rer ◽

Roland Kanaar ◽

Kumiko Sakata-Sogawa ◽

Juleon M. Schins ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Double Stranded Dna ◽

Interaction Studies ◽

Single Molecule Manipulation

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Real-Time RecA Filament Disassembly in the Presence of RecX Monitored using Single-Molecule Manipulation by Optical Tweezers

Biophysical Journal ◽

10.1016/j.bpj.2014.11.413 ◽

2015 ◽

Vol 108 (2) ◽

pp. 69a

Author(s):

Georgii Pobegalov ◽

Alexandr Alekseev ◽

Anton Sabantsev ◽

Alexey Melnikov ◽

Mikhail Khodorkovskiy ◽

...

Keyword(s):

Real Time ◽

Optical Tweezers ◽

Single Molecule ◽

Single Molecule Manipulation

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Direct observation of the formation of a CRISPR–Cas12a R-loop complex at the single-molecule level

Chemical Communications ◽

10.1039/c9cc08325a ◽

2020 ◽

Vol 56 (14) ◽

pp. 2123-2126 ◽

Cited By ~ 2

Author(s):

Yang Cui ◽

Yangchao Tang ◽

Meng Liang ◽

Qinghua Ji ◽

Yan Zeng ◽

...

Keyword(s):

Optical Tweezers ◽

Direct Observation ◽

Single Molecule ◽

Single Molecule Level ◽

System P ◽

Single Molecule Manipulation

An optical tweezers-based single-molecule manipulation assay was developed to detect the formation of an R-loop complex in the CRISPR–Cas12a system.

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Single-Molecule Manipulation of Macromolecules on GUV or SUV Membranes Using High-Resolution Optical Tweezers

Biophysical Journal ◽

10.1016/j.bpj.2021.11.2884 ◽

2021 ◽

Author(s):

Yukun Wang ◽

Avinash Kumar ◽

Huaizhou Jin ◽

Yongli Zhang

Keyword(s):

High Resolution ◽

Optical Tweezers ◽

Single Molecule ◽

Single Molecule Manipulation

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Fast Diffusion of the Unassembled PetC1-GFP Protein in the Cyanobacterial Thylakoid Membrane

Life ◽

10.3390/life11010015 ◽

2020 ◽

Vol 11 (1) ◽

pp. 15

Author(s):

Radek Kaňa ◽

Gábor Steinbach ◽

Roman Sobotka ◽

György Vámosi ◽

Josef Komenda

Keyword(s):

Membrane Proteins ◽

Single Molecule ◽

Protein Interactions ◽

Thylakoid Membrane ◽

Protein Complexes ◽

Correlation Spectroscopy ◽

Membrane Microdomains ◽

Fast Diffusion ◽

Light Harvesting Complexes ◽

Term Survival

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment–protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound ‘free’ proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 − 2.95 µm2s−1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50–500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII—light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein–protein interactions in the mobility restrictions for large thylakoid protein complexes.

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