Single Molecule Force Spectroscopy Reveals the Mechanical Design Governing the Efficient Translocation of the Bacterial Toxin Protein RTX

Abstract

The efficient translocation of the bacterial toxin adenylate cyclase toxin (CyaA) from the bacterial cytosol to the extracellular environment by the type 1 secretion system (T1SS) is essential for the toxin to function. To understand the molecular features that are responsible for the efficient translocation of CyaA, here we used optical tweezers to investigate the mechanical properties and conformational dynamics of the RTX domain of CyaA at the single molecule level. Our results revealed that apo-RTX behaves like an ideal random coil. This property allows the T1SS to translocate RTX without overcoming the enthalpic resistance. In contrast, the folded holo-RTX is mechancially stable, and its folding occurs in a vectorial, cotranslocational fashion starting from its C-terminus. Moreover, our results showed that the folding of holo-RTX generates a stretching force, which can further facilitate the translocation of RTX. Our results highlight the important role played by the Ca2+-triggered folding of RTX in the translocation of RTX and provide mechanistic insights into the mechanical design that governs the efficient translocation of RTX.

Interdomain Linker Effect on the Mechanical Stability of Ig Domains in Titin

“…To examine the linker effect, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the mechanical stability of human Ig domains. SMFS can manipulate a single molecule mechanically [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ] and AFM-SMFS has been widely used to study the mechanical stability of proteins [ 23 , 24 , 25 , 26 , 27 , 28 , 29 ], protein-protein interactions [ 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ], and chemical bonds [ 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 ]. Many titin domains have been studied [ 57 , 58 , 59 ].…”

Details of Single-Molecule Force Spectroscopy Data Decoded by a Network-Based Automatic Clustering Algorithm

“…Single-molecule force spectroscopy (SMFS), including optical tweezer, magnetic tweezer, and atomic force microscopy (AFM), has been developed as a powerful methodology to probe the details of intermolecular and intramolecular interactions and thus to obtain mechanistic insights of the systems that cannot be probed using traditional methods in bulk systems. SMFS is particularly useful in studying biological systems, such as protein folding/unfolding, nucleic acid structures, protein–drug interactions, and cellular surface receptor–ligand interactions, − due to its multifunctionalities. Compared with optical and magnetic tweezers, the AFM-based SMFS (AFM-SMFS) is advantageous in several aspects, such as facile and rapid sample preparation, molecular manipulation with an AFM tip by tethering the target molecules on the AFM tip, and spatial discrimination of SMFS with the high-resolution imaging. ,,, It makes the AFM-SMFS superior in the study of biomolecular interactions, especially in complicated physiological conditions.…”

Angular Trapping of Spherical Janus Particles

“…Optical tweezers have become an indispensable tool in single molecule manipulation, allowing for the investigation of the mechanics and conformational dynamics of biomacromolecules, including nucleic acids and proteins, at the single molecule level [1][2][3][4][5][6][7] . Most single molecule manipulations by optical tweezers are realized via three-dimensional translation of the trapped micrometer sized bead to which a single biomacromolecule is attached, and the resultant force experienced by the biomacromolecule can then be measured.…”

“…DOI: 10.1002/smtd.202000565 mechanics and conformational dynamics of biomacromolecules, including nucleic acids and proteins, at the single mole cule level. [1][2][3][4][5][6][7] Most single molecule manipulations by optical tweezers are realized via 3D translation of the trapped micrometersized bead to which a single biomacromole cule is attached, and the resultant force experienced by the bio macromolecule can then be measured. It has long been recognized that endowing optical tweezers with the ability to rotate a biomacromolecule is equally useful.…”