Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

Abstract

The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in plants self-organizes in vitro. The recombinant apoprotein, denatured in dodecyl sulfate, spontaneously folds when it is mixed with its pigments, chlorophylls, and carotenoids in detergent solution, and assembles into structurally authentic LHCII in the course of several minutes. Pulse EPR techniques, specifically double-electron-electron resonance (DEER), have been used to analyze protein folding during this process. Pairs of nitroxide labels were introduced site-specifically into recombinant LHCII and shown not to affect the stability and function of the pigment-protein complex. Interspin distance distributions between two spin pairs were measured at various time points, one pair located on either end of the second transmembrane helix (helix 3), the other one located near the luminal ends of the intertwined transmembrane helices 1 and 4. In the dodecyl sulfatesolubilized apoprotein, both distance distributions were consistent with a random-coil protein structure. A rapid freeze-quench experiment on the latter spin pair indicated that 1 s after initiating reconstitution the protein structure is virtually unchanged. Subsequently, both distance distributions monitored protein folding in the same time range in which the assembly of chlorophylls into the complex had been observed. The positioning of the spin pair spanning the hydrophobic core of LHCII clearly preceded the juxtaposition of the spin pair on the luminal side of the complex. This indicates that superhelix formation of helices 1 and 4 is a late step in LHCII assembly.assembly kinetics ͉ DEER ͉ LHCII T he major light-harvesting complex LHCII largely increases the efficiency of the photosynthesis process by collecting light energy and conducting it to a photosynthetic reaction center where light-driven charge separation takes place. The apoprotein of LHCII is one of the most abundant membrane proteins on Earth, but even so, our knowledge is fragmentary of how the LHCII apoprotein folds and assembles with pigments. For studying these questions, LHCII offers several advantages. Its crystal structure is known (1), its apoprotein can be recombinantly expressed in Escherichia coli (2), and the protein spontaneously folds and assembles with pigments in detergent solution (2, 3). This spontaneous self-organization can be triggered by mixing the apoprotein solubilized in the ionic detergent dodecyl sulfate with a non-ionic detergent solution of the pigments. The assembly process can then easily be monitored by time-resolved f luorescence spectroscopy using the Chls as built-in fluorescence labels. Such experiments showed that protein folding is dependent on the binding of pigments, and that LHCII formation in vitro occurred in at least two apparent phases, a faster one in the range of some tens of seconds and a slower one taking several minutes (4, 5). The faster step could be assigned to the binding of mostly Chl a, whereas the slower one represents Chl b binding exclus...

Rigid Core and Flexible Terminus

“…The quality of EPR samples was checked by fluorescence emission spectroscopy (31) and CD spectroscopy in the visible domain (21), as described in the supplemental material. High glycerol concentrations did not alter the functional structure of LHCII as shown previously (20).…”

“…Neither CD spectra in the visible range nor the intramolecular energy transfer from Chl b to Chl a showed any difference, demonstrating that spin labeling of the chosen sites did not affect the structure or function of LHCII (18,20). When LHCII samples were thawed after EPR measurements, they gave fluorescence and CD spectra very similar to those obtained before the measurements (supplemental Figs.…”

“…In EPR measurements, different mutant versions were used, each containing either one or two Cys replacing Ser at positions 3,11,12,14,29,34,59, 106, 123, and 160; Ala at positions 4 and 10; Lys at position 7, or Val at positions 9, 22, 90, and 196. All derivatives have either been described in earlier studies (18,20,24) or were constructed by using the QuikChange mutagenesis kit (Stratagene). Bacterial expression of the derivatives (17) and labeling of the protein with PROXYL spin labels (3-(2-iodoacetamido)-PROXYL, Aldrich) were performed as described in the supplemental material.…”

“…Distance mapping of individual protein domains in LHCII revealed a two-state structural behavior of the N-terminal domain in monomers and trimers (24). DEER recently provided insight into the refolding process of LHCII in vitro (20). Additional information about the accessibility of single residues in various domains of LHCII monomers was obtained by comparison of conventional continuous wave and pulsed EPR measurements (18).…”

“…The apoprotein of LHCII is recombinantly expressed in Escherichia coli (17) and therefore can easily be spin-labeled in specific positions (18). Adding pigments to the apoprotein in detergent solution induces the spontaneous assembly of monomeric (17,19,20) or trimeric LHCII (21). Double electron-electron resonance (DEER) spectroscopy allows the measurement of distances between two spin-labeled residues in the range of 2-8 nm (22,23).…”