Fluorescence ratio intrinsic basis states analysis: a novel approach to monitor and analyze protein unfolding by fluorescence.

Fluorescence ratio intrinsic basis states analysis (FRIBSTA) is a novel method allowing quantitative estimation of the stability of proteins in aqueous solution as a function of temperature. In FRIBSTA emission fluorescence spectra are repeatedly recorded while ramping temperature from < or =-15 to > or =100 degrees C. Subsets of these are identified as reference spectra of the protein in either its folded or in its heat denatured configuration. Each reference spectrum of both sets is normalized by its own integrated fluorescence intensity to give a fractional area spectrum. Linear extrapolations of these normalized reference spectral shapes over the entire temperature range of measurement are then used to deconvolute each experimental emission spectrum to give a fraction of emission from native state and a fraction from denatured state. Additionally, the integrated emission fluorescence intensity for the native configuration is fitted and extrapolated over the temperature range of measurement. Division of the deconvoluted native integrated fluorescence intensity by the fitted-extrapolated integrated emission fluorescence intensity yields the fraction folded. The free energy functions derived from fraction unfolded are presented for beta-lactoglobulin and phosphoglycerate kinase. According to these results both proteins are considerably less stable than heretofore assumed at ambient temperatures and partially denatured at temperatures < or =0 degrees C. The method is employed to study the effect of denaturants on these proteins as well. The major usefulness of FRIBSTA is that one can directly measure the protein stability at ambient and subambient temperatures in the absence of denaturants rather than predicting it by extrapolation from heat denaturation data.

Phase behaviour of distearoylphosphatidylethanolamine in glycerol--a thermal and X-ray diffraction study.

The phase behaviour of 1,2-distearoylphosphatidylethanolamine in glycerol has been examined using differential scanning calorimetry and real-time synchrotron X-ray diffraction methods. Dry phospholipid and phospholipid dispersed in glycerol over the concentration range 2.4%-90% (w/w) was equilibrated for 30 min at 20 degrees C and thermal and structural parameters on the temperature range 60 degrees C to 110 degrees C recorded during an initial heating and subsequent reheating. The characteristic feature of the initial heating scan was a direct lamellar crystalline to inverted hexagonal phase transition. In the subsequent cooling scan a lamellar gel structure was formed from the non-lamellar phase which transformed, on reheating, to a lamellar crystalline phase in which the acyl chain packing was titled with respect to the bilayer plane. The mechanism of the formation of the two crystalline phases was examined in the context of a relaxation model, where the liquid-crystal phase below the transition temperature from lamellar crystalline phase is metastable. A binary phase diagram over the temperature range 60 degrees C to 110 degrees C has been constructed.

The effects of glycerol on the phase behaviour of hydrated distearoylphosphatidylethanolamine and its possible relation to the mode of action of cryoprotectants.

A phase diagram for 1,2-distearoylphosphatidylethanolamine (DSPE) dispersed in glycerol/water mixtures was constructed using data obtained from differential scanning calorimetry and time-resolved X-ray diffraction measurements. The phase sequence seen on heating the lipid remains the same for samples containing up to 70 wt% glycerol. Depending on the hydration conditions, the samples are either in a metastable lamellar gel (L beta) or one or other of two possible sub-gel phases (Lc and Lc') at low temperatures. These phases convert first to a lamellar liquid crystalline (L alpha) and then to an inverted hexagonal (HII) phase on heating. On cooling, the samples revert first to the L alpha and then to the L beta phase. Although the phase sequence is preserved, marked changes are seen in the transition temperatures between the different phases. The temperature of the transition between the L alpha and the HII phases decreases strongly with increasing glycerol concentration while that of the Lc and Lc' phases to L alpha, and to a lesser extent that of the L beta to L alpha transition, increases. Substantial changes in phase behaviour are seen if the glycerol concentration is increased above 70 wt%. Under these conditions, the Lc and Lc' phases transform directly into the HII phase on heating (a similar direct transition from the L beta to the HII phase is seen above 80 wt% glycerol). An exothermic transition from the L beta phase to the Lc' phase is observed and there is also an increasing tendency for the samples to revert to the Lc or Lc' phases on storage. These changes in relative stability of the different phases are discussed in terms of a possible membrane Hofmeister effect and their relevance to the mode of action of cryoprotectants is explored.

Effect of trehalose on the phase properties of hydrated and lyophilized dipalmitoylphosphatidylcholine multilayers.

The structure and thermal behavior of hydrated and lyophilized dipalmitoylphosphatidylcholine (DPPC) multilayers in the presence of trehalose were investigated by differential scanning calorimetry and X-ray diffraction methods. Trehalose enters the aqueous space between hydrated bilayers and increases the interbilayer separation (from 0.36 to 1.37 nm in the different DPPC phases at 1 M trehalose). It does not affect the lipid chain packing and also the slow isothermal conversion at 4 degrees C of the metastable L beta' phase into the equilibrium crystalline Lc phase. Addition of trehalose leads to a slight upward shift (about 1 degrees C at 1 M trehalose) of the three phase transitions (sub-, pre-, and main transition) in fully hydrated DPPC while their other properties (enthalpy, excess specific heat, and transition width) remain unchanged. The effect of trehalose on the thermal behavior of DPPC multilayers freeze-dried from an initially completely hydrated state is qualitatively similar to that of water. These data support the "water replacement" hypothesis about trehalose action. It is suggested that trehalose prevents the formation of direct interbilayer hydrogen bonds in states of low hydration.