Kenneth E. Eigenberg's research while affiliated with California Institute of Technology and other places

Bilayer membranes containing distearoylphosphatidylcholine and chlorophyll a have been investigated via 1H- and 31P-NMR. Measurements as a function of temperature and composition support the basic interpretation of the phase diagram for this mixed bilayer system advanced in the preceding report. The 31P-NMR spectrum of mixed vesicles above the solidus temperature of the phase diagram shows a single resonance, but below the solidus two peaks are observed. Observation of two distinct 31P resonances from the phospholipid at 46°C conclusively demonstrates that phase separation occurs at temperatures below the solidus. Observation of a 5.8 ppm upfield shift for one of the 31P resonances at low temperature provides strong evidence for an interaction between the phospholipid headgroup and chlorophyll a in one of the low-temperature phases. This conclusion is supported by the observation of increased 1H linewidths for the lipid and a longer 1H T1 for the choline N-methyl resonance of the lipid below the solidus temperature, indicating that headgroups of lipid molecules in at least one of the phases are motionally restricted. The observation of an interaction between chlorophyll a and lipid molecules in a bilayer membrane lends considerable support to the idea that chlorophyll molecules in the chloroplast antenna are physically separated from each other by strongly coordinating lipid molecules.

Several groups have introduced chlorophyll a into artificial bilayer membranes in an attempt to develop a model system for studying the behavior of chlorophyll in the photosynthetic membrane. In order to investigate the organization of chlorophyll in these model systems, mixed bilayer systems containing chlorophyll a and distearoylphosphatidylcholine under conditions of excess water have been studied by differential thermal analysis. The resulting data suggest a phase diagram for this system consisting of a double eutectic with formation of a thermodynamic compound of defined stoichiometry between chlorophyll a and phospholipid at temperatures below the liquidus. The phase diagram may be simulated to obtain thermodynamic parameters characteristic of the compound phase. It is apparent that the organization and intermolecular interactions of chlorophyll in a bilayer membrane can very widely depending on the temperature and composition of the system. In particular, phase separation can occur within the membrane over certain temperature ranges, resulting in an inhomogeneous system. Thus in interpreting the physical and spectroscopic properties of chlorophyll a in bilayer membranes, it is essential to consider the phase state of the membrane and the organization and environment of the chlorophyll in the particular phase.

The 13C-NMR spectrum at 90.5 MHz has been obtained for the photosynthetic thylakoid membrane of spinach. Specific lipid and chlorophyll resonances can be assigned in the high resolution spectrum, although protein resonances are not observed. It can be estimated from resonance intensities that at least 30% of the plant chlorophyll contributes to the high resolution 13C spectrum with the remainder broadened by incomplete motional averaging. The resonance linewidths of the observed chlorophyll phytol chains are approximately the same as those of the lipid hydrocarbon chains, indicating a similar motional state and suggesting that this particular pool of chlorophyll is lipid-bound or at most only loosely associated with proteins.

Proton nuclear magnetic resonance spectra at 360 MHz of small sonicated distearoyl phosphatidylcholine vesicles show easily distinguishable resonances due to choline N-methyl head-group protons located in the inner and outer bilayer halves. A study of the chemical shift of these resonances as a function of temperature reveals that the splitting between them increases below the phase transition. This occurs as a result of an upfield shift of the inner layer resonance at the phase transition. Consideration of the possible causes of this effect results in the conclusion that, at the phase transition, there is a change in the organization of the inner layer head-groups which does not occur for the outer layer head-groups.