Tag Archives: smFRET

A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding

In this project, Kankowan Champasa from Stephen Bell’s lab at MIT collaborated with other researchers from the Bell and Gelles labs to study a key process that sets the stage for replication of chromosomal DNA. They explain “licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally.”  The researchers identified a conserved motif in the Mcm4 helicase subunit essential for formation of productive replication complexes.  Single-molecule fluorescence energy transfer experiments show that mutations in the motif still allow the two hexamers to come into contact, but they prevent the formation of the stable double-hexamers that perform the extensive DNA unwinding needed for replication.

10.7554/eLife.45538
A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding.
Champasa, K., Blank, C., Friedman, L.J., Gelles, J., and Bell, S.P.
eLife (2019) 8:e40576

Conformational Cycling within the Closed State of Grp94, an Hsp90-Family Chaperone

Grp94 is a molecular chaperone that helps to fold and maintain the folded state of “client” proteins in the endoplasmic reticulum.  Acceleration of client folding is driven by conformational changes in Grp94.  However,  the sequence of conformational changes and how these changes are coupled to the cycle of ATP hydrolysis is not well understood.  Prof. Timothy Street and his lab members Bin Huang and Ming Sun, in collaboration with Larry Friedman, did single-molecule fluorescence resonance energy transfer (FRET) experiments to directly observe conformational cycling in individual Grp94 molecules.  Their studies show that ATP hydrolysis can drive repeated cycling between alternative “closed” states of Grp94, suggesting a way that enzyme might propagate structural changes to client molecules. Chemical scheme for conformational cycling of Grp94.

10.1016/j.jmb.2019.06.004
Conformational Cycling within the Closed State of Grp94, an Hsp90-Family Chaperone
Huang, B., Friedman, L.J., Gelles, J., Sun, M., and Street, T.O.
Journal of Molecular Biology 431, 3312-3323 (2019).