Sashka Krumova

Microcalorimetry of Blood Serum Proteome: A Modified Interaction Network in the Multiple Myeloma Case

Hereby we report on a novel approach in the study of multiple myeloma (MM), namely, differential scanning calorimetry (DSC) combined with serum protein electrophoresis. Distinct thermodynamic signatures describe the DSC thermograms of MM blood sera, in contrast to the unique profile found for healthy individuals. The thermal behavior of MM sera reflects a complex interplay between the serum concentration and isotype of the M protein and of albumin, and modified ligand- and/or protein-protein interactions, resulting in stabilization of globulins and at least a fraction of albumin. In all MM cases the 85 °C, transferrin-assigned transition is missing. A distinct feature of IgG isotype (κ and λ) DSC profiles only is the presence of a transition at 82 °C. A DSC-based classification of MM depicts two sets of melting patterns (MMt2 and MMt3 with two or three successive thermal transitions), and subsets within each set (MMt2(i) or MMt3(i), the subscript i = 1, 2 or 3 denotes the main transition being one of the three transitions). The results demonstrate the potential of DSC to monitor MM-related modifications of the serum proteome, even at low M protein concentrations, Bence Jones and importantly nonsecretory multiple myeloma cases, and prove DSC as a versatile tool for oncohematology.

Calorimetry-based profiling of blood plasma from colorectal cancer patients

BACKGROUND Differential scanning calorimetry (DSC), a highly sensitive technique for resolving thermally-induced protein folding/unfolding transitions, recently was recognized as a novel tool for disease diagnosis and monitoring. To further elaborate this approach we have applied DSC in a study of blood plasma from patients with colorectal cancer (CRC) at different stages of tumor development and localization. METHODS Blood plasma from patients diagnosed with CRC was analyzed by DSC. The CRC thermograms were compared to those of healthy individuals and patients with gastric cancer and non-cancerous soft tissue inflammation. The thermodynamic parameters: excess heat capacity and enthalpy of the transitions corresponding to the most abundant plasma proteins, as well as transition and first moment temperatures were determined from the calorimetric profiles. RESULTS The calorimetric profiles of blood plasma from CRC patients are found to be distinct from those of healthy individuals and those of patients with soft tissue, non-cancerous inflammation. Generally the CRC thermograms exhibit reduced heat capacity of the major albumin/globulin-assigned thermal transitions, lower enthalpy and a featureless high temperature region compared to healthy individuals. CONCLUSIONS A classification of blood plasma proteome from patients with colorectal cancer (CRC1, CRC2 and CRC3 groups, and subgroups within each group CRC1(1-2), CRC2(1-2) and CRC3(1-2)) is proposed based on the derived thermodynamic parameters. GENERAL SIGNIFICANCE The presented data demonstrate a proof of principle and confirm that the DSC technique has a potential to monitor changes in the CRC blood plasma proteome. This study is a further step toward the validation of calorimetry as a diagnostic tool.

Digalactosyl-diacylglycerol-deficiency lowers the thermal stability of thylakoid membranes.

We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the chirally organized macrodomains containing the main chlorophyll a/b light-harvesting complexes. The mutation also brings about changes in the overall chlorophyll fluorescence lifetimes, measured in whole leaves as well as in isolated thylakoids. As shown by time-resolved measurements, using the lipophylic fluorescence probe Merocyanine 540 (MC540), the altered lipid composition affects the packing of lipids in the thylakoid membranes but, as revealed by flash-induced electrochromic absorbance changes, the membranes retain their ability for energization. Thermal stability measurements revealed more significant differences. The disassembly of the chiral macrodomains around 55°C, the thermal destabilization of photosystem I complex at 61°C as detected by green gel electrophoresis, as well as the sharp drop in the overall chlorophyll fluorescence lifetime above 45°C (values for the wild type—WT) occur at 4–7°C lower temperatures in dgd1. Similar differences are revealed in the temperature dependence of the lipid packing and the membrane permeability: at elevated temperatures MC540 appears to be extruded from the dgd1 membrane bilayer around 35°C, whereas in WT, it remains lipid-bound up to 45°C and dgd1 and WT membranes become leaky around 35 and 45°C, respectively. It is concluded that DGDG plays important roles in the overall organization of thylakoid membranes especially at elevated temperatures.

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by 31P-NMR.

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing. In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for 31P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 degrees C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 degrees C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.

Monitoring Photosynthesis in Individual Cells of Synechocystis sp. PCC 6803 on a Picosecond Timescale

Picosecond fluorescence kinetics of wild-type (WT) and mutant cells of Synechocystis sp. PCC 6803, were studied at the ensemble level with a streak-camera and at the cell level using fluorescence-lifetime-imaging microscopy (FLIM). The FLIM measurements are in good agreement with the ensemble measurements, but they (can) unveil variations between and within cells. The BE mutant cells, devoid of photosystem II (PSII) and of the light-harvesting phycobilisomes, allowed the study of photosystem I (PSI) in vivo for the first time, and the observed 6-ps equilibration process and 25-ps trapping process are the same as found previously for isolated PSI. No major differences are detected between different cells. The PAL mutant cells, devoid of phycobilisomes, show four lifetimes: ∼20 ps (PSI and PSII), ∼80 ps, ∼440 ps, and 2.8 ns (all due to PSII), but not all cells are identical and variations in the kinetics are traced back to differences in the PSI/PSII ratio. Finally, FLIM measurements on WT cells reveal that in some cells or parts of cells, phycobilisomes are disconnected from PSI/PSII. It is argued that the FLIM setup used can become instrumental in unraveling photosynthetic regulation mechanisms in the future.

Effects of exogenous 24-epibrassinolide on the photosynthetic membranes under non-stress conditions

In the present work the effects of exogenous 24-epibrassinolide (EBR) on functional and structural characteristics of the thylakoid membranes under non-stress conditions were evaluated 48 h after spraying of pea plants with different concentrations of EBR (0.01, 0.1 and 1.0 mg.L(-1)). The results show that the application of 0.1 mg.L(-1) EBR has the most pronounced effect on the studied characteristics of the photosynthetic membranes. The observed changes in 540 nm light scattering and in the calorimetric transitions suggest alterations in the structural organization of the thylakoid membranes after EBR treatment, which in turn influence the kinetics of oxygen evolution, accelerate the electron transport rate, increase the effective quantum yield of photosystem II and the photochemical quenching. The EBR-induced changes in the photosynthetic membranes are most probably involved in the stress tolerance of plants.

Temperature dependence of the lipid packing in thylakoid membranes studied by time- and spectrally resolved fluorescence of Merocyanine 540.

The lipid packing of thylakoid membranes is an important factor for photosynthetic performance. However, surprisingly little is known about it and it is generally accepted that the bulk thylakoid lipids adopt the liquid-crystalline phase above -30 degrees C and that a phase transition occurs only above 45 degrees C. In order to obtain information on the nature of the lipid microenvironment and its temperature dependence, steady-state and time-resolved fluorescence measurements were performed on the fluorescence probe Merocyanine 540 (MC540) incorporated in isolated spinach thylakoids and in model lipid systems (dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) adopting different phases. It is demonstrated that the degree and way of incorporation differs for most lipid phases--upon selective excitation at 570 nm, the amplitude of the fluorescence component that corresponds to membrane-incorporated MC540 is about 20% in gel-, 60% in rippled gel-, and 90% in liquid-crystalline and inverted hexagonal phase, respectively. For thylakoids, the data reveal hindered incorporation of MC540 (amplitude about 30% at 7 degrees C) and marked spectral heterogeneity at all temperatures. The incorporation of MC540 in thylakoids strongly depends on temperature. Remarkably, above 25 degrees C MC540 becomes almost completely extruded from the lipid environment, indicating major rearrangements in the membrane.

Brassinosteroids regulate the thylakoid membrane architecture and the photosystem II function

Brassinosteroids (BRs) are plant steroid hormones known to positively affect photosynthesis. In this work we investigated the architecture and function of photosynthetic membranes in mature Arabidopsis rosettes of BR gain-of-function (overexpressing the BR receptor BR INSENSITIVE 1 (BRI1), BRI1OE) and loss-of-function (bri1-116 with inactive BRI1 receptor, and constitutive photomorphogenesis and dwarfism (cpd) deficient in BR biosynthesis) mutants. Data from atomic force microscopy, circular dichroism, fluorescence spectroscopy and polarographic determination of oxygen yields revealed major structural (enlarged thylakoids, smaller photosystem II supercomplexes) and functional (strongly inhibited oxygen evolution, reduced photosystem II quantum yield) changes in all the mutants with altered BR response compared to the wild type plants. The recorded thermal dependences showed severe thermal instability of the oxygen yields in the BR mutant plants. Our results suggest that an optimal BR level is required for the normal thylakoid structure and function.

Calorimetric markers of Bence Jones and nonsecretory multiple myeloma serum proteome.

The present work provides a thermodynamic description of blood serum from patients diagnosed with Bence Jones myeloma (BJMM) and nonsecretory myeloma (NSMM) by means of differential scanning calorimetry (DSC), serum protein electrophoresis, and free light chain assay. Specific alterations in the thermodynamic behavior of both BJMM and NSMM proteome have been revealed. On the basis of the transition temperature of the main transition in the calorimetric profiles and the shape similarity criterion, we defined BJMM and NSMM sets/subsets of thermograms with very similar thermodynamic features. We show that some of the BJMM and NSMM subsets correlate with previously defined secretory myeloma subsets (Todinova et al. Anal. Chem. 2011, 83, 7992). The established analogies strongly suggest that common molecular markers contribute to the calorimetric profiles of the different, secretory and nonsecretory, myeloma types; our data show robust evidence that these are ligands stabilizing the major serum proteins. We demonstrate that the DSC approach might be highly beneficial, especially for NSMM patients, since the characteristic modifications in the DSC profiles might serve as calorimetric markers when no monoclonal proteins can be detected in the bloodstream and the diagnosis heavily relies on invasive methods.

Interaction of biological membranes with substituted 1,4-anthraquinones

Abstract The interaction of the substituted 1,4-anthraquinones with purple membranes (PM) is studied by the fluorescence and electric light scattering techniques. Substituted 1,4-anthraquinones are shown to quench bacteriorhodopsin tryptophan fluorescence. The quenching efficiency of 1,4-anthraquinone derivatives is smaller than that of 1,4-anthraquinone. It is the smallest at di-substituted 1,4-anthraquinones (R9=OCOCH 3 and R3=Cl or Br). The type of the halogen atom as substituent does not affect the quenching efficiency of di-substituted 1,4-anthraquinones (R9=OH or OCOCH 3 and R3=Cl or Br). The quenching efficiency depends on the position of the Cl atom for di-substituted 1,4-anthraquinone derivatives (R9=OH and R3=Cl or R9=OH and R10=Cl). 9-Hydroxy-1,4-anthraquinones induce decrease in purple membrane charge asymmetry distribution whereas mono-substituted 1,4-anthraquinones containing R9=OCH 3 or R9=OCOCH 3 induce increase in the membrane charge asymmetry. The membrane electric polarizability, related to the structure and dynamics of the electric double layer, decreases in the presence of 1,4-anthraquinone derivatives, the greatest effect being observed for the 9-hydroxy-1,4-anthraquinones. The energy transfer and the quenching of bR tryptophan fluorescence from 9-hydroxy-1,4-anthraquinones correlate with the variation of the surface electric properties of PM in the presence of relatively low concentrations of substituted 1,4-anthraquinones.