Willem F. Wolkers

Small heat-shock proteins regulate membrane lipid polymorphism

Thermal stress in living cells produces multiple changes that ultimately affect membrane structure and function. We report that two members of the family of small heat-shock proteins (sHsp) (α-crystallin and Synechocystis HSP17) have stabilizing effects on model membranes formed of synthetic and cyanobacterial lipids. In anionic membranes of dimyristoylphosphatidylglycerol and dimyristoylphosphatidylserine, both HSP17 and α-crystallin strongly stabilize the liquid-crystalline state. Evidence from infrared spectroscopy indicates that lipid/sHsp interactions are mediated by the polar headgroup region and that the proteins strongly affect the hydrophobic core. In membranes composed of the nonbilayer lipid dielaidoylphosphatidylethanolamine, both HSP17 and α-crystallin inhibit the formation of inverted hexagonal structure and stabilize the bilayer liquid-crystalline state, suggesting that sHsps can modulate membrane lipid polymorphism. In membranes composed of monogalactosyldiacylglycerol and phosphatidylglycerol (both enriched with unsaturated fatty acids) isolated from Synechocystis thylakoids, HSP17 and α-crystallin increase the molecular order in the fluid-like state. The data show that the nature of sHsp/membrane interactions depends on the lipid composition and extent of lipid unsaturation, and that sHsps can regulate membrane fluidity. We infer from these results that the association between sHsps and membranes may constitute a general mechanism that preserves membrane integrity during thermal fluctuations.

Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro

A heat-soluble protein present in substantial quantities in Typha latifolia pollen was purified to homogeneity. The protein was subjected to cyanogen bromide cleavage, and the peptides produced were separated by HPLC chromatography and sequenced. The two sequences determined were found to be related to the putative D76 LEA protein from Brassica napus seeds and one of them to the D-7 LEA protein from upland cotton. This suggests the pollen protein to be a member of the LEA group III family of proteins. The secondary structure of the protein in solution and in the dry state was investigated using Fourier transform IR spectroscopy. Whereas the protein in solution was highly unordered, being largely in a random coil conformation, the conformation was largely alpha-helical after fast drying. Slow drying reversibly led to both alpha-helical and intermolecular extended beta-sheet structures. When dried in the presence of sucrose, the protein adopted alpha-helical conformation, irrespective of drying rate. The effect of the protein on the stability of sucrose glasses was also investigated. The dehydrated mixture of sucrose and the LEA protein had higher glass transition temperatures and average strength of hydrogen bonding than dehydrated sucrose alone. We suggest that LEA proteins may play a role together with sugars in the formation of a tight hydrogen bonding network in the dehydrating cytoplasm, thus conferring long-term stability.

Evidence for a physiological role for membrane rafts in human platelets.

We have investigated raft formation in human platelets in response to cell activation. Lipid phase separation and domain formation were detected using the fluorescent dye 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanine perchlorate (diI‐C18) that preferentially partitions into gel‐like lipid domains. We showed that when human platelets are activated by cold and physiological agonists, rafts coalesce into visible aggregates. These events were disrupted by depletion of membrane cholesterol. Using Fourier transform infrared spectroscopy (FTIR), we measured a thermal phase transition at around 30°C in intact platelets, which we have assigned as the liquid‐ordered to the liquid‐disordered phase transition of rafts. Phase separation of the phospholipid and the sphingomyelin‐enriched rafts could be observed as two phase transitions at around 15 and 30°C, respectively. The higher transition, assigned to the rafts, was greatly enhanced with removal of membrane cholesterol. Detergent‐resistant membranes (DRMs) were enriched in cholesterol (50%) and sphingomyelin (20%). The multi‐functional platelet receptor CD36 selectively partitioned into DRMs, whereas the GPI‐linked protein CD55 and the major platelet integrin αIIbβ3a did not, which suggests that the clustering of proteins within rafts is a regulated process dependent on specific lipid protein interactions. We suggest that raft aggregation is a dynamic, reversible physiological event triggered by cell activation. J. Cell. Physiol. 190: 117–128, 2002.

Stabilization of Dry Mammalian Cells: Lessons from Nature

Abstract The Center for Biostabilization at UC Davis is attempting to stabilize mammalian cells in the dry state. We review here some of the lessons from nature that we have been applying to this enterprise, including the use of trehalose, a disaccharide found at high concentrations in many anhydrobiotic organisms, to stabilize biological structures, both in vitro and in vivo. Trehalose has useful properties for this purpose and in at least in one case—human blood platelets—introducing this sugar may be sufficient to achieve useful stabilization. Nucleated cells, however, are stabilized by trehalose only during the initial stages of dehydration. Introduction of a stress protein obtained from an anhydrobiotic organism, Artemia, improves the stability markedly, both during the dehydration event and following rehydration. Thus, it appears that the stabilization will require multiple adaptations, many of which we propose to apply from studies on anhydrobiosis.

A Fourier transform infrared microspectroscopy study of sugar glasses: application to anhydrobiotic higher plant cells

Fourier transform infrared microspectroscopy (FTIR) was used to study glasses of pure carbohydrates and in the cytoplasm of desiccation tolerant plant organs. The position of the OH stretching vibration band (vOH) shifted with temperature. Two linear regression lines were observed in vOH against temperature plots. The temperature at the point of intersection between these two lines coincided with the glass transition temperature (Tg), as determined by other methods. The temperature at the intersection point decreased with increasing water content, which further validates that, indeed, Tg was observed. Tg values that were determined for dry glucose, sucrose, maltose, trehalose and raffinose glasses were 27, 57, 91, 108 and 108 degrees C, respectively. The shift of vOH with temperature, the wavenumber-temperature coefficient (WTC), was higher in sugar glasses having higher Tg. This suggests that glasses are more loosely packed when they have higher Tg. For Typha latifolia pollen and dried Craterostigma plantagineum leaves we obtained similar vOH vs. temperature plots as for carbohydrate glasses, indicating that a glass transition was observed. The Tg in dry pollen was ca. 45 degrees C and in dry plant leaves ca. 65 degrees C, with WTC values comparable to those observed in the carbohydrates. The Tg values in these tissues decreased with increasing water contents. Our data suggest that the carbohydrates that are present in the cytoplasm are primary factors contributing to the glassy state. We conclude that FTIR provides new insights in the structure of glasses in carbohydrates and in biological tissues.

Membrane Stabilization in the Dry State

Abstract We discuss current ideas of how membranes in desiccation-tolerant plant organ(ism)s are protected from the deleterious effect of complete water removal. Results of studies with model membranes showed that sugars play a major role in preventing fusion, phase transitions and most likely also phase separations. The sugars ability to form a stable glass and to interact directly with the phosphate of the phospholipid polar headgroup is the requirement for the protection of dry liposomes. Disaccharides alone fulfil these requirements. Dry membranes of desiccation tolerant plants in situ often have elevated phase transition temperatures (Tm) that are readily restored upon rehydration. Elevated Tm may point to insufficient interaction of sucrose with the polar headgroups. Attempts to observe this interaction in situ by analyzing the asymmetric phosphate stretching band failed. Thus, we suggest factors other than sugars in the suppression of Tm in intact cells and provide suggestions concerning potential roles of amphipathic compounds in this regard.

From anhydrobiosis to freeze-drying of eukaryotic cells ☆

Using what has been learned from nature, it has become possible to stabilize biological structures, including intact cells, in the dry state. Stabilization of cells or tissues in the dried state is of considerable practical significance, as is described in this review. The need for stabilization of cells in the dried state is particularly urgent in bloodbanks, where proper storage of blood cells (platelets and erythrocytes) is still a major problem. Human blood platelets are stored in blood banks for 5 days, after which they are discarded according to Federal regulation. This short lifetime has led to a chronic shortage of platelets. We report here that platelets can be preserved by freeze-drying them with trehalose, a sugar found at high concentrations in organisms that naturally survive drying. We suggest that this finding will obviate the storage problem with platelets and that the principles established here may be extended to more complex eukaryotic cells.

Stabilization of membranes in human platelets freeze-dried with trehalose.

Human blood platelets are normally stored in blood banks for 3-5 days, after which they are discarded. We have launched an effort at developing means for preserving the platelets for long term storage. In previous studies we have shown that trehalose can be used to preserve biological membranes and proteins during drying and have provided evidence concerning the mechanism. A myth has grown up about special properties of trehalose, which we discuss here and clarify some of what is fact and what is misconception. We have found a simple way of introducing this sugar into the cytoplasm of platelets and have successfully freeze-dried the trehalose-loaded platelets, with very promising results. We present evidence that membrane microdomains are maintained intact in the platelets freeze-dried with trehalose. Finally, we propose a possible mechanism by which the microdomains are preserved.

DEHYDRATION-INDUCED CONFORMATIONAL CHANGES OF POLY-L-LYSINE AS INFLUENCED BY DRYING RATE AND CARBOHYDRATES

The conformation of hydrated and air-dried poly-L-lysine in thin films was studied using Fourier transform IR spectroscopy in the amide-I region. Hydrated poly-L-lysine has a random coil conformation. Upon slow drying of small droplets of the polypeptide solution over a period of several hours, an extended beta-sheet conformation is adopted. This conformational transition can be prevented by fast air-drying within 2-3 min. Slow air-drying in the presence of sucrose also preserves the aqueous conformation and results in the formation of a glassy state. Comparison of shifts of the OH band with temperature indicates that sucrose/poly-L-lysine mixtures form a molecularly more densely packed glassy matrix, having a higher glass transition temperature (Tg), than sucrose alone. Whether direct interaction of sugar and polypeptide or glass formation is involved in the stabilization during slow air-drying was studied by drying in the presence of glucose or dextran. Compared with dextran (and sucrose to a lesser extent), glucose gives superior protection. Dried glucose has the lowest Tg and the best interacting properties. We conclude that either immobilization by fast air-drying or sufficient interaction with a protectant through hydrogen bonding (slow drying) plays the leading role in the preservation of the aqueous protein structure.

Effect of Sucrose and Maltodextrin on the Physical Properties and Survival of Air‐Dried Lactobacillus bulgaricus: An in Situ Fourier Transform Infrared Spectroscopy Study

The effect of sucrose, maltodextrin and skim milk on survival of L. bulgaricus after drying was studied. Survival could be improved from 0.01% for cells that were dried in the absence of protectants to 7.8% for cells dried in a mixture of sucrose and maltodextrin. Fourier transform infrared spectroscopy (FTIR) was used to study the effect of the protectants on the overall protein secondary structure and thermophysical properties of the dried cells. Sucrose, maltodextrin and skim milk were found to have minor effects on the membrane phase behavior and the overall protein secondary structure of the dried cells. FTIR was also used to show that the air‐dried cell/protectant solutions formed a glassy state at ambient temperature. 1‐Palmitoyl 2‐oleoyl phosphatidyl choline (POPC) was used in order to determine if sucrose and maltodextrin have the ability to interact with phospholipids during drying. In addition, the glass transition temperature and strength of hydrogen bonds in the glassy state were studied using this model system. Studies using poly‐l‐lysine were done in order to determine if sucrose and maltodextrin are able to stabilize protein structure during drying. As expected, sucrose depressed the membrane phase transition temperature (Tm) of POPC in the dried state and prevented conformational changes of poly‐l‐lysine during drying. Maltodextrin, however, did not depress the Tm of dried POPC and was less effective in preventing conformational changes of poly‐l‐lysine during drying. We suggest that when cells are dried in the presence of sucrose and maltodextrin, sucrose functions by directly interacting with biomolecules, whereas maltodextrin functions as an osmotically inactive bulking compound causing spacing of the cells and strengthening of the glassy matrix.