On-resin assembly of a linkerless lanthanide(III)-based luminescence label and its application to the total synthesis of site-specifically labeled mechanosensitive channels.
A synthesis strategy for the on-resin assembly of luminescent lanthanide chelates from commercially available compounds was developed. Advantages of the approach include the absence of spacers between the metal ion and the attachment site, and the compatibility with typical chemical protein synthesis protection schemes. Methoxycoumarin-labeled lysine and tris(tert-butyl)-DOTA were consecutively coupled with high efficiency to a free amino group in otherwise fully protected peptide segments using standard peptide synthesis methods. Addition of stoichiometric amounts of Tb(3+) to the modified, cleaved, and purified peptides yielded the desired lanthanide chelate. Incorporation of this label into a chemically synthesized, full-length mechanosensitive channel of large conductance (MscL) of E. coli and subsequent reconstitution into vesicles resulted in a functional mechanosensitive channel of comparable conductance to the wild-type channel. However, this channel required increased suction to gate. Excitation of the antenna molecule methoxycoumarin at 336 nm resulted in an emission spectrum typical for Tb(3+) and a luminescence lifetime of 0.67 ms. The location of the probe close to the backbone of this protein may provide precise information about conformational changes during channel opening from LRET studies.
Becker
Christian F W
CF
Gryphon Therapeutics, 600 Gateway Boulevard, South San Francisco, California 94080, USA.
Clayton
Daniel
D
Shapovalov
George
G
Lester
Henry A
HA
Kochendoerfer
Gerd G
GG
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D028581:Lanthanoid Series Elements / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D049408:Luminescent Agents / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008465:Mechanoreceptors / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D012117:Resins, Synthetic / Q000032:analysis / Q000737:chemistry
D028581:Lanthanoid Series Elements; D049408:Luminescent Agents; D012117:Resins, Synthetic