Single-molecule investigations of single-chain cellulose biosynthesis

Abstract

Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides . Synthesis depends on uridine diphosphate glucose, Mg 2+ , and cyclic diguanosine monophosphate, with the last displaying a retention time of ∼80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.

Structural snapshots of hyaluronan formation reveal principles of length control and secretion

“…S1g). Similar biosynthesis rates have recently been reported for cellulose synthase [14]. Monitoring the length distribution of the synthesized HA under these substrate conditions (one limiting, the other in excess) shows increasing HA lengths up to a substrate concentration of 2.5 mM (Fig.…”

“…Quantification of Cv-HAS activity followed a similar workflow. Protein concentrations in CvHAS IMVs were normalized as described previously [14]. Reaction buffer consisted of 40 mM TrisHCl pH 7.5, 75 mM NaCl, 20 mM MnCl2, 0.5 mM TCEP, 5 mM UDP-GlcNAc, 5 mM UDP-GlcA and 0.01 μCi/μL [ 3 H]-UDP-GlcNAc.…”

Plant cellulose synthase membrane protein isolation directly fromPichia pastorisprotoplasts, liposome reconstitution, and its enzymatic characterization

“…Interestingly, plant cellulose synthases also had kcat in a similar range as that of G. hansenii [35,37,38], meaning they also have lower values than that of R. sphaeroides. It is difficult to assess whether the plant CesAs are slower in adding the substrate than R. sphaeroides since a recent study involving cellulose biosynthesis at a single-molecule level shows an addition of glucose every 2.5 secs at room temperature [39]. Such state-of-the-art methods may be required to determine the exact catalytic efficiency and turnover rate of the plant CesAs.…”

Probing the Stability and Interdomain Interactions in the ABC Transporter OpuA, Using Single-Molecule Optical Tweezers

“…This has been performed in a membranemimicking environment by the use of nanodiscs, the protein embedded in a phospholipid bilayer surrounded by a membrane scaffold protein (MSP). 35 This strategy to study membrane proteins with optical tweezers has only been reported once to date, 36 whereas the unfolding and interactions of a membrane protein in nanodiscs have not been studied before. By introducing a single point mutation, K521C, both SBDs of the homodimer are probed simultaneously in pulling experiments to uncover unfolding transitions and potential interactions.…”