Arimatti Jutila

Evidence for the Lack of a Specific Interaction between Cholesterol and Sphingomyelin

The putative specific interaction and complex formation by sphingomyelin and cholesterol was investigated. Accordingly, low contents (1 mol % each) of fluorescently labeled derivatives of these lipids, namely 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PyrPC), n-[10-(1-pyrenyl)decanoyl]sphingomyelin (PyrSM), and increasing concentrations of cholesterol (up to 5 mol %), were included in large unilamellar vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC), and the excimer/monomer fluorescence emission ratio (I(e)/I(m)) was measured. In DNPC below the main phase transition, the addition of up to 5 mol % cholesterol reduced I(e)/I(m) significantly. Except for this, cholesterol had only a negligible effect in both matrices and for both probes. We then compared the efficiency of resonance energy transfer from PyrPC and PyrSM to 22-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBDchol). An augmenting colocalization of the latter resonance energy transfer pair with temperature was observed in a DMPC matrix below the main phase transition. In contrast, compared to PyrSM the colocalization of PyrPC with NBDchol was more efficient in the longer DNPC matrix. These results could be confirmed using 5,6-dibromo-cholestan-3beta-ol as a collisional quencher for the pyrene-labeled lipids. The results indicate lack of a specific interaction between sphingomyelin and cholesterol, and further imply that hydrophobic mismatch between the lipid constituents could provide the driving force for the cosegregation of sphingomyelin and cholesterol in fluid phospholipid bilayers of thicknesses comparable to those found for biomembranes.

Comparison of the effects of clozapine, chlorpromazine, and haloperidol on membrane lateral heterogeneity

The interactions of three neuroleptic drugs, clozapine (CLZ), chlorpromazine (CPZ), and haloperidol (HPD) with phospholipids were compared using DSC and Langmuir balance. Main emphasis was on the drug-induced effects on the lateral organization of lipid mixtures of the saturated zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and the unsaturated acidic phosphatidylserine, brainPS. In multilamellar vesicles (MLV) phase separation was observed by DSC at X(PS)> or =0.05. All three drugs bound to these MLVs, abolishing the pretransition at X(drug)> or =0.03. The main transition temperature (T(m)) decreased almost linearly with increasing contents of the drugs, CLZ having the smallest effect. In distinction from the other two drugs, CLZ abolished the phase separation evident in the endotherms for DPPC/brainPS (X(PS)=0.05) MLVs. Compression isotherms of DPPC/brainPS/drug (X(PS)=X(drug)=0.05) monolayers revealed the neuroleptics to increase the average area/molecule, CLZ being the most effective. Penetration into brainPS monolayers showed strong interactions between the three drugs and this acidic phospholipid (in decreasing order CPZ>HPD>CLZ). Hydrophobic interactions demonstrated using neutral eggPC monolayers decreased in a different order, CLZ>CPZ>HPD. Fluorescence microscopy revealed domain morphology of DPPC/brainPS monolayers to be modulated by these drugs, increasing the gel-fluid domain boundary length in the phase coexistence region. To conclude, our data support the view that membrane-partitioning drugs could exert part of their effects by changing the lateral organization and thus also the functions of biomembranes.

Impact of the tryptophan residues of Humicola lanuginosa lipase on its thermal stability.

Thermal stability of wild type Humicola lanuginosa lipase (wt HLL) and its two mutants, W89L and the single Trp mutant W89m (W117F, W221H, and W260H), were compared. Differential scanning calorimetry revealed unfolding of HLL at T(d)=74.4 degrees C whereas for W89L and W89m this endotherm was decreased to 68.6 and 62 degrees C, respectively, demonstrating significant contribution of the above Trp residues to the structural stability of HLL. Fluorescence emission spectra revealed the average microenvironment of Trps of wt HLL and W89L to become more hydrophilic at elevated temperatures whereas the opposite was true for W89m. These changes in steady-state emission were sharp, with midpoints (T(m)) at approx. 70.5, 61.0, and 65.5 degrees C for wt HLL, W89L, and W89m, respectively. Both steady-state and time resolved fluorescence spectroscopy further indicated that upon increasing temperature, the local movements of tryptophan(s) in these lipases were first attenuated. However, faster mobilities became evident when the unfolding temperatures (T(m)) were exceeded, and the lipases became less compact as indicated by the increased hydrodynamic radii. Even at high temperatures (up to 85 degrees C) a significant extent of tertiary and secondary structure was revealed by circular dichroism. Activity measurements are in agreement with increased amplitudes of conformational fluctuations of HLL with temperature. Our results also indicate that the thermal unfolding of these lipases is not a two-state process but involves intermediate states. Interestingly, a heating and cooling cycle enhanced the activity of the lipases, suggesting the protein to be trapped in an intermediate, higher energy state. The present data show that the mutations, especially W89L in the lid, contribute significantly to the stability, structure and activity of HLL.

Effects of i-propanol on the structural dynamics of Thermomyces lanuginosa lipase revealed by tryptophan fluorescence

Influence of isopropanol (iPrOH) on the structural dynamics of Thermomyces lanuginosa lipase (TLL) was studied by steady‐state, time‐resolved, and stopped‐flow fluorescence spectroscopy, monitoring the intrinsic emission of Trp residues. The fluorescence of the four Trps of the wild‐type enzyme report on the global changes of the whole lipase molecule. To monitor the conformational changes in the so‐called “lid,” an α‐helical surface loop, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Circular dichroism (CD) spectra revealed that iPrOH does not cause major alterations in the secondary structures of the wild‐type TLL and W89m. With increasing [iPrOH], judged by the ratio of emission intensities at 350 nm and 330 nm, the average microenvironment of the Trps in the wild‐type TLL became more hydrophobic, whereas Trp89 of W89m moved into a more hydrophilic microenvironment. Time‐resolved fluorescence measurements revealed no major changes to be induced by iPrOH neither in the shorter fluorescence lifetime component (τ1 = 0.5–1.2 ns) for the wild‐type TLL nor in the longer fluorescence lifetime component (τ2 = 4.8–6.0 ns) in the wild‐type TLL and the W89m mutant. Instead, for W89m on increasing iPrOH from 25% to 50% the value for τ1 increased significantly, from 0.43 to 1.5 ns. The shorter correlation time ϕ1 of W89m had a minimum of 0.08 ns in 25% iPrOH. Judged from the residual anisotropy r∞ the amplitude of the local motion of Trp89 increased upon increasing [iPrOH] 10%. Stopped‐flow fluorescence spectroscopy measurements suggested the lid to open within ≈2 ms upon transfer of W89m into 25% iPrOH. Steady‐state anisotropies and longer correlation times revealed increasing concentrations of iPrOH to result also in the formation of dimers as well as possibly also higher oligomers by TLL.

Binding of Adriamycin to Liposomes as a Probe for Membrane Lateral Organization

A stopped-flow spectrofluorometer equipped with a rapid scanning emission monochromator was utilized to monitor the binding of adriamycin to phospholipid liposomes. The latter process is evident as a decrease in fluorescence emission from a trace amount of a pyrene-labeled phospholipid analog (PPDPG, 1-palmitoyl-2-[(6-pyren-1-yl)]decanoyl-sn-glycero-3-phospho-rac-++ +glyce rol) used as a donor for resonance energy transfer to adriamycin. For zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes, fluorescence decay was slow, with a half-time t1/2 of approximately 2 s. When the mole fraction of the acidic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), was increased to XPG >/= 0.04, the decay of fluorescence became double exponential, and an additional, significantly faster process with t1/2 in the range between 2 and 4 ms was observed. Subsequently, as XPG was increased further, the amplitude of the fast process increased, whereas the slower process was attenuated, its t1/2 increasing to 20 s. Increasing [NaCl] above 50 mM or [CaCl2] above 150 microM abolished the fast component, thus confirming this interaction to be electrostatic. The critical dependence of the fast component on XPG allows the use of this process to probe the organization of acidic phospholipids in liposomes. This was demonstrated with 1, 2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes incorporating PPDPG (XPPDPG = 0.03), i.e., conditions where XPG in fluid bilayers is below the required threshold yielding the fast component. In keeping with the presence of clusters of PPDPG, the fast component was observed for gel-state liposomes. At approximately 34 degreesC (i.e., 6 degrees below Tm), the slower fluorescence decay also appeared, and it was seen throughout the main phase transition region as well as in the liquid-crystalline state. The fluorescence decay behavior at temperatures below, above, and at the main phase transition temperature is interpreted in terms of thermal density fluctuations and an intermediate state between gel and liquid-crystalline states being involved in the phospholipid main phase transition. This is the first observation of a cluster constituted by acidic phospholipids controlling the membrane association of a drug.

Effects of betaine on the structural dynamics of Thermomyces (Humicola) lanuginosa lipase

Abstract The impact of betaine on the structural dynamics of a soluble protein, Thermomyces (Humicola) lanuginosa lipase (HLL) was studied using differential scanning calorimetry (DSC), surface tension measurements, and both steady-state and time-resolved fluorescence spectroscopy. Betaine increased the thermal stability of wild type HLL and protected this enzyme from unfolding by the chaotrope, guanidine hydrochloride. Time-resolved fluorescence anisotropy measurements revealed betaine to decrease the mean hydrodynamic volume of the single Trp HLL mutant, W89m, and to reduce the movements of the α-helical segment constituting the so-called ‘lid’. In the presence of betaine, the average environment of Trp89 became more hydrophobic with decreased accessibility to this residue for the water soluble quencher I−, the bimolecular collisional rate constant, kq decreasing from 6.86×108 to 5.06×108 M−1 s−1 in 1.0 M betaine. The above results are in keeping with an augmented packing of the lipase in the presence of betaine. Betaine increases surface tension (γ) of water and enhances the denaturation of HLL into the air–water interface. The increased stability of HLL and increased γ by betaine suggest low or no binding of betaine to the surface of protein. The effects of betaine on the structural dynamics of HLL are readily explained by the increased surface tension of water caused by this solute.