Vladimir V. Shubin

Trimeric forms of the photosystem I reaction center complex pre-exist in the membranes of the cyanobacterium Spirulina platensis

Oligomeric and monomeric forms of chlorophyll—protein complexes of photosystem I (PSI) have been isolated from the mesophilic cyanobacterium Spirulina [(1992) FEBS Lett. 309, 340–342]. Electron microscopic analysis of the complexes showed that the oligomeric form is a trimer of the shape and dimensions similar to those of the trimer from thermophilic cyanobacteria. The chlorophyl ratio in the isolated trimer and monomer was found to be 7:3. The trimeric form of PSI complex in contrast to the monomeric one contains the chlorophyll emitting at 760 nm (77K), which is also found in Spirulina membranes and therefore could be used as an intrinsic probe for the trimeric complex. The 77K circular dichroism spectrum of the trimeric form is much more similar to that of Spirulina membranes than the spectrum of the monomer. Thus, the trimeric PSI complexes exist and dominate in the Spirulina membranes.

Isolation from Spirulina membranes of two photosystem I-type complexes, one of which contains chlorophyll responsible for the 77 K fluorescence band at 760 nm

Two types of chlorophyll‐protein complexes of photosystem 1 (PSIa, PSIc) have been isolated from the membranes of Spirulina platensis using a Triton X‐100 treatment and chromatography on DEAE‐Toyopearl. The complexes are equally enriched with P700 (Chl: P700=100–110) but show different electrophoretic molecular masses ‐ 140 (PSIa) and 320 kDa (PSIc) ‐ and differ in the content of long‐wavelength absorbing Chl. PSIa has a typical PSI fluorescence band at 730 nm (F730) as the main band at 77 K, whereas PSIc is responsible for F760, the intensity of which depends on the redox state of P700. PSIc only shows 77 K light‐induced variable fluorescence at 760 typical of Spirulina membranes and cells.

Efficient energy transfer from the long-wavelength antenna chlorophylls to P700 in photosystem I complexes from Spirulina platensis

Abstract To study the role of the long-wavelength chlorophylls (Chl) in photosystem I (PSI), the action spectra of P700 photooxidation at 293 and 77 K have been measured for PSI trimeric and monomeric complexes isolated from Spirulina platensis . The long-wavelength Chls which absorb in the region 710dash740 nm transfer excitation energy to the reduced P700 with the same efficiency as bulk antenna Chls, causing the oxidation of P700. The relative quantum yield of P700 photooxidation is about unity (293-77 K) even under the direct excitation of Chl absorbing at 735 nm (Chl735). At 77 K Chl735 exhibits a fluorescence band at 760 nm (F760) whose intensity is quenched under illumination of the PSI trimeric complexes from Spirulina . The relative quantum yield of F760 quenching is not dependent on the wavelength of excitation in the region 620–750 nm. Since the value of the overlap integral between the band of F760 and the absorption band of the cation radical of P700 (P700 + ) is higher than that of the P700 band, it is suggested that Chl735 transfers energy to P700 + more efficiently than to reduced P700; energy transfer to P700 + causes the quenching of F760. A linear relationship between the photooxidation rate of P700 and the fraction of P700 + at 293 K indicates that the energy exchange between PSI subunits of the trimer is negligible. Thus, the antenna of PSI trimers of Spirulina is organized in separate photosynthetic units.

High molecular weight pea leaf protein similar to the groE protein of escherichia coli

Abstract A high molecular weight protein which in its structure and some properties is similar to the gro E protein of Escherichia coli , has been found in pea leaves. Using electron microscopy we established that the protein consisted of 14 identical monomers which are arranged with point 72 symmetry in two layers, seven monomers in each layer. The molecular weight of the protein, determined by gel filtration and sedimentation equilibrium techniques, was found to be 900000 ∓ 150000 and 950000 ∓ 50000, respectively. The sedimentation coefficient was 24.3 ∓ 1.0S. During polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the protein dissociated into identical polypeptide chains of M r 67000 ∓ 3000. On the basis of the circular dichroism spectrmn file share of α-helical portions was found to be 0.47∓0.06, that of β-structures 0.13∓0.03, that of β-turn 0.16∓0.03, and that of irregular portions 0.24∓0.07. The protein had a low ATPase (EC 3.6.1.3) activity. The protein content in the leaves was found to vary with their growth stage.

Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin

Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, Uhr) is characterized by a high rate of aggregation. Aggregation of Uhr leads to the formation of primary aggregates with the hydrodynamic radius (Rh,1) of 10.3 nm. The second form (low reactive unfolded form, Ulr) participates in the aggregation process by its attachment to the primary aggregates produced by the Uhr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (Ast). At complete exhaustion of Ulr, secondary aggregates with the hydrodynamic radius (Rh,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.

Energy exchange between the chlorophyll antennae of monomeric subunits within the Photosystem I trimeric complex of the cyanobacterium Spirulina

The energy exchange between antenna chlorophylls of the monomeric subunits within a Photosystem I trimeric complex of the cyanobacterium Spirulina was studied, comparing the kinetics of the light-induced decrease of Photosystem I fluorescence at 760 nm, emitted by the longwave chlorophyll absorbing at 735 nm and P700 photooxidation. Both kinetics have been measured at 77 K simultaneously using a fiberoptic system. The light-induced decrease of fluorescence at 760 nm in trimers was faster than P700 oxidation, although the decrease of fluorescence at 760 nm was ascribed to the quenching effect of oxidized P700 that is a stronger quencher of fluorescence at 760 nm than P700 reduced [Shubin et al. (1995) J Photochem. Photobiol 27B: 153–160]. The proportionality of the reciprocal value of the half time for the fluorescence decrease at 760 nm in trimers to the light intensity indicates the photochemical nature of the fluorescence quenching. The nonlinear relationship between the variable part of fluorescence at 760 nm and the fraction of reduced P700 is considered to be a result of an energetic connectivity of the antennae of monomeric subunits within a trimer. When one P700 in PS I trimers of Spirulina is oxidized, the energy from an antenna of monomeric subunit(s) with reduced P700 may migrate to a subunit with oxidized P700 and quenched there. This may explain the slower rate of P700 photooxidation in trimers as compared with monomers. The analytical description of cooperativity processes in Photosystem I coincides well with the measured data. A model is presented describing the energetic interaction of chlorophyll antennae of monomeric subunits within the trimer via the extreme long-wavelength chlorophyll form. This intersubunit interaction may stimulate the dissipation of excess energy as heat and, therefore, protect the pigment–protein complex against photodestruction.

Switching from blue to yellow : altering the spectral properties of a high redox potential laccase by directed evolution

Abstract During directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae, the characteristic maximum absorption at the T1 copper site (Abs610T1Cu) was quenched, switching the typical blue colour of the enzyme to yellow. To determine the molecular basis of this colour change, we characterized the original wild-type laccase and its evolved mutant. Peptide printing and MALDI-TOF analysis confirmed the absence of contaminating protein traces that could mask the Abs610T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochemical redox titrations. Both wild-type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R-478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-expressing the mutant laccase was the generation of a six-residue N-terminal acidic extension, which was associated with the failure of the STE13 protease in the Golgi compartment giving rise to alternative processing. Removal of the N-terminal tail had a negative effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra analysis suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of laboratory evolution appeared to mediate the alterations to Abs610T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates.

Glutamine synthetases of pea leaf and seed cytosol. Structure and properties

Abstract Methods for purifying to homogeneity glutamine synthetases ( l -glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) of pea seed cytosol (GScs) and leaf cytosol (GScl) have been developed. The gel filtration method was used to estimate GScs molecular weight as 384 000 and that of GScl, 520 000. Under electrophoresis in polyacrylamide gel in the presence of SDS, GScs and GScl dissociated into monomers of molecular weight 48 000 and 64 000, respectively. The quaternary structures of GScs and GScl were investigated by electron microscopy. The two enzymes were found to consist of eight identical monomers each arranged with dihedral point group symmetry 422 (D4) at the vertices of two squares. These squares are twisted about the 4-fold axis relative to each other. Unlike GScl; GScs is characterized by annular projection of particles in micrographs due to filling of the interior portion of the protein molecule with a stain. GScs and GScl secondary structures, calculated on the basis of the circular dichoroism spectra, also differed. The nature of the effects of substrates, M2+, and methionine sulphoximine on GScs secondary structure was investigated. The isoelectric point of GScs was situated at pH 5.15, and that of GScl at pH 4.8. The N-terminal amino acid of GScl was alanine, and that of GScs, glycine. The kinetic characteristics of GScl and GScs also differ. GScs and GScl, and also leaf chloroplast glutamine synthetase and root glutamine synthetase of pea plant were compared by means of rabbit antibodies against GScs. The conclusion was made that the multiple molecular forms of glutamine synthetase of pea plant may be isoenzymes.

Chlorophyll-water interaction during oxygen photoevolution at the octane-water interface

Abstract The interaction between water and chlorophyll a dissolved in octane has been studied. Factors governing the formation of the hydrated chlorophyll oligomer with the absorption maximum at 742 nm have been identified. It has been revealed that the water photooxidation reaction sensitized by chlorophyll absorbed at the interface requires the presence of water in the octane phase during self-assemblage of the catalytic complex responsible for the reaction.

Structural changes in R-phycoerythrin upon CdS quantum dot synthesis in tunnel cavities of protein molecules.

Structural changes in R-phycoerythrin used as a matrix for the synthesis of CdS quantum dots have been analyzed by circular dichroism spectrometry. In deionized water, quantum dot synthesis in the tunnel cavity of the R-phycoerythrin molecule proved to be accompanied by uncoiling of α-helices and changes in the conformation of its chromophore groups, with consequent decay of protein fluorescence. Since R-phycoerythrin fluorescence is important for practical applications, conditions for quantum dot synthesis have been optimized by replacing deionized water with 0.01 M MES buffer, pH 5.7. Under such conditions, the size of the CdS quantum dots (determined from atomic force microscopy images) remains the same as in deionized water, but quantum dots cause only minor structural changes in protein molecules, as follows from circular dichroism and absorption spectra. The thermostability of R-phycoerythrin is enhanced, as indicated by an increase in the experimental activation energy for denaturation (from 140.8 to 149.9 kJ/mol) and the intensity of R-phycoerythrin fluorescence is also enhanced approximately twofold.