Using the SpyTag SpyCatcher system to label smooth muscle myosin II filaments with a quantum dot on the regulatory light chain

Abstract

The regulatory light chain (RLC) of myosin is commonly tagged to monitor myosin behavior in vitro, in muscle fibers, and in cells. The goal of this study was to prepare smooth muscle myosin (SMM) filaments containing a single head labeled with a quantum dot (QD) on the RLC. We show that when the RLC is coupled to a QD at Cys‐108 and exchanged into SMM, subsequent filament assembly is severely disrupted. To address this, we used a novel approach for myosin by implementing the SpyTag002 SpyCatcher002 system to prepare SMM incorporated with RLC constructs fused to SpyTag or SpyCatcher. We show that filament assembly, actin‐activated steady‐state ATPase activities, ability to be phosphorylated, and selected enzymatic and mechanical properties were essentially unaffected if either SpyTag or SpyCatcher were fused to the C‐terminus of the RLC. Crucially for our application, we also show that a QD coupled to SpyCatcher can be covalently attached to a RLC‐Spy incorporated into a SMM filament without disrupting the filament, and that the filaments can move along actin in vitro.

Covalent Functionalization of Bioengineered Polyhydroxyalkanoate Spheres Directed by Specific Protein-Protein Interactions

“…A spontaneous covalent isopeptide bond forms between a lysine residue of the SpyCatcher domain (13 kDa) and an aspartic acid of its pairing peptide SpyTag (13 amino acid residues) in a sitespecific manner, without the need of additional reagents nor enzymes at broad ligation conditions (Reddington and Howarth, 2015). The advantageous properties of the SpyTag/SpyCatcher chemistry makes it an excellent protein ligation tool for surface functionalization of various organic and inorganic materials, such as virus-like particles (Brune et al, 2016(Brune et al, , 2017Bruun et al, 2018), protein-based scaffolds (Bae et al, 2018;Choi et al, 2018;Swartz and Chen, 2018;Zhang et al, 2018a), gold nanoparticles (Ma et al, 2018), silica (Zhang et al, 2018b,c), quantum dots (Ke et al, 2018;Brizendine et al, 2019), and crystalline graphene (Tyagi et al, 2018). We recently developed a modular PHA platform using SpyTag/SpyCatcher chemistry, where we successfully showed that purified SpyTagged proteins could ligate to SpyCatcher-coated PHA spheres in vitro with decent tunability (Wong and Rehm, 2018).…”

Evidence for S2 flexibility by direct visualization of quantum dot–labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro

“…Chicken smooth muscle RLC (UniProtKB-P02612) and SpyTag002 (RLC-Spy) construct was expressed and purified as described ( Brizendine et al, 2019 ).…”

“…Modification of QDs was performed similar to that described in Brizendine et al (2019) . Briefly, amine QDs (Invitrogen; 50 µl of 605 or 705 nm; 8 µM) were centrifuged for 3 min at 2,400× g and washed twice in a 100 kD cutoff centrifugal filter unit (Millipore) in DTT-free conjugation buffer.…”

“…1 C ). The approach requires that the QDs are attached specifically, covalently, and with the correct stoichiometry after the filament is formed, because we have previously shown that the presence of the QD disrupts normal filament assembly ( Brizendine et al, 2019 ). To monitor head motion, we used the SpyCatcher (SpyC)/SpyTag system ( Keeble et al, 2017 ) to attach a QD to the regulatory light chain (RLC) in the lever arm domain of the head, as we described previously ( Brizendine et al, 2019 ).…”

“…The approach requires that the QDs are attached specifically, covalently, and with the correct stoichiometry after the filament is formed, because we have previously shown that the presence of the QD disrupts normal filament assembly ( Brizendine et al, 2019 ). To monitor head motion, we used the SpyCatcher (SpyC)/SpyTag system ( Keeble et al, 2017 ) to attach a QD to the regulatory light chain (RLC) in the lever arm domain of the head, as we described previously ( Brizendine et al, 2019 ). To monitor filament backbone motion, we used a complementary but non–cross-reactive SnoopCatcher (SnoopC)/SnoopTag system ( Veggiani et al, 2016 ) to attach a QD to the light meromyosin, C-terminal portion of the myosin tail excluding S2 (LMM), subdomain of the rod.…”