Date Published: February 1, 2018
Publisher: Public Library of Science
Author(s): Astrid Elisabeth Mork-Jansson, Lutz Andreas Eichacker, Andrew Webber.
The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to in vitro expressed LIL3 requires pre-incubation of reaction partners at room temperature. We show chlorophyll a but not chlorophyll b binding to LIL3 at a molar ratio of 1:1. Neither dynamic light scattering nor native PAGE, enabled a discrimination between binding of chlorophyll a and/or b to LIL3.
The light harvesting like membrane protein 3 (LIL3) is a member of the light-harvesting complex protein family (LHCP) and characterized by the presence of a common LHC sequence motif . LIL3 is classified in the group of two-helix stress enhanced proteins (SEPs). Common ancestors of SEPs are the high-light inducible proteins (HLIP) in cyanobacteria, and the one-helix proteins (OHP) in plants . LHCP genes harbor two LHC motifs which are associated with Chlorophyll (Chl) binding [1, 3, 4]. However, also proteins that harbor only one LHC motif, like LIL3, were determined to bind Chl. Microscale thermophoresis studies indicated that dimerization could provide the functional compensation for the missing LHC motif .
We show by mobility analysis of micelles that Chl a binding to LIL3 can be reconstituted in vitro in the absence of carotenoids, lipids and of Chl b. A protocol for in vitro reconstitution of Chl binding to LIL3 is presented. The method has been established on the basis of protocols developed by Plumley and Schmidt  and Paulsen  for Chl binding to LHCP. The binding of Chl to LIL3 is characterized by MST, DLS, and native PAGE. All three methods investigate changes in the mobility of solubilized LIL3 micelles in the presence and absence of an interaction with Chl micelles. Selective binding of Chl a to LIL3 is demonstrated using MST.