Interactions of proline, serine, and leucine with isolated spinach thylakoids: solute loading during freezing is not related to membrane fluidity.

We have investigated the physical mechanisms through which amino acids influence freeze-thaw damage to isolated spinach (Spinacia oleracea L.) thylakoid membranes. The cryoprotective amino acid proline reduced osmotic membrane rupture during thawing by reducing solute loading of thylakoids during freezing for 1 h to -5 degreesC. Evidence for binding of proline to the thylakoid surface was obtained by determining the partitioning of the hydrophobicity-sensitive dye merocyanine 540 between the bulk solution and the membranes in the presence of different concentrations of proline. This binding led to a decrease in lipid fluidity at the membrane surface, as measured with fluorescence depolarization spectroscopy using the probe trimethylammonium-diphenylhexatriene (TMA-DPH). The hydrophobic core of the membranes, as probed with 1,6-diphenyl-1,3,5,-hexatriene, was not influenced. Surprisingly, cryotoxic concentrations of both serine and leucine resulted in the same amount of reduction of thylakoid lipid fluidity, without reducing solute loading during freezing. In the case of these cryotoxic amino acids, unknown additional interactions with the membranes must result in membrane destabilization during a freeze-thaw cycle.