Structural basis for the ligand promiscuity of the neofunctionalized, carotenoid-binding fasciclin domain protein AstaP.

Fasciclins (FAS1) are ancient adhesion protein domains with no common small ligand binding reported. A unique microalgal FAS1-containing astaxanthin (AXT)-binding protein (AstaP) binds a broad repertoire of carotenoids by a largely unknown mechanism. Here, we explain the ligand promiscuity of AstaP-orange1 (AstaPo1) by determining its NMR structure in complex with AXT and validating this structure by SAXS, calorimetry, optical spectroscopy and mutagenesis. α1-α2 helices of the AstaPo1 FAS1 domain embrace the carotenoid polyene like a jaw, forming a hydrophobic tunnel, too short to cap the AXT ß-ionone rings and dictate specificity. AXT-contacting AstaPo1 residues exhibit different conservation in AstaPs with the tentative carotenoid-binding function and in FAS1 proteins generally, which supports the idea of AstaP neofunctionalization within green algae. Intriguingly, a cyanobacterial homolog with a similar domain structure cannot bind carotenoids under identical conditions. These structure-activity relationships provide the first step towards the sequence-based prediction of the carotenoid-binding FAS1 members.

Structural basis for the carotenoid binding and transport function of a START domain.

STARD3, a steroidogenic acute regulatory lipid transfer protein, was identified as a key xanthophyll-binding protein in the human retina. STARD3 and its homologs in invertebrates are known to bind and transport carotenoids, but this lacks structural elucidation. Here, we report high-resolution crystal structures of the apo- and zeaxanthin (ZEA)-bound carotenoid-binding protein from silkworm Bombyx mori (BmCBP). Having a STARD3-like fold, BmCBP features novel elements, including the Ω1-loop that, in the apoform, is uniquely fixed on the α4-helix by an R173-D279 salt bridge. We exploit absorbance, Raman and dichroism spectroscopy, and calorimetry to describe how ZEA and BmCBP mutually affect each other in the complex. We identify key carotenoid-binding residues, confirm their roles by ZEA-binding capacity and X-ray structures of BmCBP mutants, and also demonstrate that markedly different carotenoid-binding capacities of BmCBP and human STARD3 stem from differences in the structural organization of their carotenoid-binding cavity.

The mechanism of thermal aggregation of glutamate dehydrogenase. The effect of chemical chaperones.

Chemical chaperones are low-molecular compounds counteracting protein aggregation. Understanding of the mechanism of their effects is key to their potential use in biotechnology. The aggregation of bovine liver glutamate dehydrogenase (GDH) was studied at 40 °C and 50 °C using dynamic light scattering, analytical ultracentrifugation, size-exclusion chromatography and differential scanning calorimetry. At 40 °C the GDH aggregation proceeds through the slow stages of hexamer dissociation and formation of small oligomeric aggregates. At 50 °C these stages are transient. The rate-limiting stage of the overall aggregation process is unfolding of the protein molecule; the order of aggregation with respect to protein, n = 1. The test system based on GDH aggregation at 50 °C was used to quantify the anti-aggregation activity of chemical chaperones by comparing their half-saturation concentrations [L]0.5. Arginine ethyl ester had the highest anti-aggregation activity, with [L]0.5 = 4 ± 1 mM. For other additives, [L]0.5 was 22 ± 1 mM (arginine), 18 ± 1 mM (argininamide) and 95 ± 12 mM (proline). Arginine at concentrations up to 300 mM, argininamide at concentrations higher than 300 mM and arginine ethyl ester at concentrations higher than 500 mM enhance aggregate-aggregate sticking. These results explain the mechanism of heat-induced GDH aggregation and its peculiarities at different temperatures or in the presence of chemical chaperones.

Kinetic regime of Ca2+ and Mg2+-induced aggregation of phosphorylase kinase at 40 °C.

Many functions of phosphorylase kinase (PhK) are regulated by Ca2+ and Mg2+ ions. Ca2+ and Mg2+ ions stimulate activity of PhK, induce the changes in the tertiary and quaternary structure of the hexadecameric enzyme molecule, provoke association/aggregation of PhK molecules, enhance PhK binding to glycogen. To establish the kinetic regime of Ca2+ and Mg2+-induced aggregation of PhK from rabbit skeletal muscles at 40 °C, in the present work the kinetics of aggregation was studied at various protein concentrations using the dynamic light scattering. The proposed mechanism of aggregation involves the stage of unfolding of the protein molecule with retention of the integrity of its oligomeric structure, the nucleation stage and stages of the growth of protein aggregates. The initial rate of the aggregation process at the stage of aggregate growth depends linearly on the protein concentration. This means that the order of aggregation with respect to the protein is equal to unity and the aggregation rate is limited by the rate of protein unfolding. The rate constant of the first order characterizing the stage of protein unfolding was found to be equal to 0.071 min-1 (40 mM Hepes, pH 6.8, 100 mM NaCl, 0.1 mM Ca2+, 10 mM Mg2+).

Structural plasticity and thermal stability of the histone-like protein from Spiroplasma melliferum are due to phenylalanine insertions into the conservative scaffold.

The histone-like (HU) protein is one of the major nucleoid-associated proteins of the bacterial nucleoid, which shares high sequence and structural similarity with IHF but differs from the latter in DNA-specificity. Here, we perform an analysis of structural-dynamic properties of HU protein from Spiroplasma melliferum and compare its behavior in solution to that of another mycoplasmal HU from Mycoplasma gallisepticum. The high-resolution heteronuclear NMR spectroscopy was coupled with molecular-dynamics study and comparative analysis of thermal denaturation of both mycoplasmal HU proteins. We suggest that stacking interactions in two aromatic clusters in the HUSpm dimeric interface determine not only high thermal stability of the protein, but also its structural plasticity experimentally observed as slow conformational exchange. One of these two centers of stacking interactions is highly conserved among the known HU and IHF proteins. Second aromatic core described recently in IHFs and IHF-like proteins is considered as a discriminating feature of IHFs. We performed an electromobility shift assay to confirm high affinities of HUSpm to both normal and distorted dsDNA, which are the characteristics of HU protein. MD simulations of HUSpm with alanine mutations of the residues forming the non-conserved aromatic cluster demonstrate its role in dimer stabilization, as both partial and complete distortion of the cluster enhances local flexibility of HUSpm.

A thermal after-effect of UV irradiation of muscle glycogen phosphorylase b.

Different test systems are used to characterize the anti-aggregation efficiency of molecular chaperone proteins and of low-molecular-weight chemical chaperones. Test systems based on aggregation of UV-irradiated protein are of special interest because they allow studying the protective action of different agents at physiological temperatures. The kinetics of UV-irradiated glycogen phosphorylase b (UV-Phb) from rabbit skeletal muscle was studied at 37°C using dynamic light scattering in a wide range of protein concentrations. It has been shown that the order of aggregation with respect to the protein is equal to unity. A conclusion has been made that the rate-limiting stage of the overall process of aggregation is heat-induced structural reorganization of a UV-Phb molecule, which contains concealed damage.

A change in the aggregation pathway of bovine serum albumin in the presence of arginine and its derivatives.

Chemical chaperones including arginine and its derivatives are widely used by biochemists working on the design of agents, which are able to efficiently suppress protein aggregation. To elucidate the mechanisms of anti-aggregation activity of chemical chaperones, methods based on registration of the increment in light scattering intensity must be supplemented with methods for direct detection of the portion of aggregated protein (γagg). For this purpose asymmetric flow field-flow fractionation was used in the present work. It was shown that heat-induced aggregation of bovine serum albumin (BSA) followed the kinetics of the reaction of the second order (0.1 M sodium phosphate buffer, pH 7.0, 70 °C). It was proposed to use R h vs γagg plots to characterize the aggregation pathway (R h is the hydrodynamic radius of the protein aggregates, which was calculated from the dynamic light scattering data). The changes in the shape of R h vs γagg plots in the presence of arginine, arginine amide and arginine ethyl ester are indicative of the changes in the aggregation pathway of BSA aggregation. A conclusion has been made that larger aggregates are formed in the presence of arginine hydrochloride and its derivatives.

A change in the pathway of dithiothreitol-induced aggregation of bovine serum albumin in the presence of polyamines and arginine.

When studying the anti-aggregation activity of chemical chaperones, a kinetic regime of the aggregation process for selected test systems should be established. To elucidate the mechanism of suppression of protein aggregation by polyamines (putrescine, spermidine) and arginine, we used a test system based on dithiothreitol (DTT)-induced aggregation of bovine serum albumin (BSA) at 45°C (0.1M Na-phosphate buffer, pH 7.0; [DTT]=2mM). The rate-limiting stage of DTT-induced aggregation of BSA under the studied conditions is that of unfolding of the protein molecule. The kinetics of BSA aggregation was monitored by dynamic light scattering and asymmetric flow field-flow fractionation. On the basis of the obtained data a mechanism of DTT-induced aggregation of BSA in the presence of polyamines and arginine has been proposed. It is assumed that the chemical chaperones under study stabilize the native form of protein with a subsequent decrease in the aggregation rate. However, they stimulate the sticking of aggregates formed in the aggregation process. To prove this mechanism, plots of the hydrodynamic radius of protein aggregates versus the portion of aggregated protein have been constructed.

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.

Quantification of anti-aggregation activity of UV-irradiated α-crystallin.

Ultraviolet radiation is a risk factor for cataractogenesis. It is believed that enhanced rates of lens opacification and cataract formation are the results of gradual loss of chaperone-like efficiency of α-crystallin upon exposure to UV light. To characterize chaperone-like activity of α-crystallin damaged by UV irradiation, a test system based on dithiothreitol-induced aggregation of holo-α-lactalbumin from bovine milk was used. The adsorption capacity of α-crystallin (AC0) with respect to the target protein (α-lactalbumin) was used as a measure of anti-aggregation activity of α-crystallin. The data on SDS-PAGE testify that UV irradiation of α-crystallin results in covalent cross-linking of subunits in α-crystallin oligomers. The dependence of AC0 value on the irradiation dose was compared with the UV-induced diminution of the portion of native α-crystallin estimated from the data on differential scanning calorimetry. On the basis of such comparison a conclusion has been made that the loss in chaperone-like activity is mainly due to UV-induced denaturation of α-crystallin subunits. Cross-linking of remaining native subunits leads to an additional decrease in anti-aggregation activity.

Dual effect of arginine on aggregation of phosphorylase kinase.

Arginine is widely used in biotechnology as a folding enhancer and aggregation suppressor. However, its action on the stability of complexly organized oligomeric proteins, on the one hand, and its role in the formation of supramolecular structures, on the other hand, are poorly known. The investigation is concerned with the effects of arginine on protein-protein interactions using phosphorylase kinase (PhK) as an example. PhK, a 1.3MDa (αßγδ)4 hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme that catalyzes phosphorylation and activation of glycogen phosphorylase b. On the basis of light scattering measurements it was shown that arginine induced aggregation of Ca(2+)-free PhK. On the contrary, when studying Ca(2+), Mg(2+)-induced aggregation of PhK at 37°C, the protective effect of arginine was demonstrated. The data on analytical ultracentrifugation are indicative of disruption of PhK hexadecameric structure under the action of arginine. Though HspB6 and HspB5 suppress aggregation of PhK they do not block the disruption effect of arginine with respect to both forms of PhK (Ca(2+)-free and Ca(2+), Mg(2+)-bound conformers). The dual effect of arginine has been interpreted from view-point of dual behaviour of arginine, functioning both like an osmolyte and a protein denaturant.

Thermal denaturation and aggregation of apoform of glycogen phosphorylase b. Effect of crowding agents and chaperones.

The effect of protein and chemical chaperones and crowders on thermal stability and aggregation of apoform of rabbit muscle glycogen phosphorylase b (apoPhb) has been studied at 37°C. Proline suppressed heat-induced loss in ability of apoPhb to reconstitution at 37°C, whereas α-crystallin did not reveal a protective action. To compare the antiaggregation activity of intact and crosslinked α-crystallins, an adsorption capacity (AC) of a protein chaperone with respect to a target protein was estimated. This parameter is a measure of the antiaggregation activity. Crosslinking of α-crystallin results in 11-fold decrease in the initial AC. The nonlinear character of the relative initial rate of apoPhb aggregation versus the [intact α-crystallin]/[apoPhb] ratio plot is indicative of the decrease in the AC of α-crystallin with increasing the [α-crystallin]/[apoPhb] ratio and can be interpreted as an evidence for dynamic chaperone structure and polydispersity of α-crystallin-target protein complexes. As for chemical chaperones, a semisaturation concentration of the latter was used as a characteristic of the antiaggregation activity. A decrease in the semisaturation concentration for proline was observed in the presence of the crowders (polyethylene glycol and Ficoll-70).

Engineering catalytic properties and thermal stability of plant formate dehydrogenase by single-point mutations.

The analysis of the 3D model structure of the ternary complex of recombinant formate dehydrogenase from soya Glycine max (EC 1.2.1.2., SoyFDH) with bound NAD+ and an inhibitor azide ion revealed the presence of hydrophobic Phe290 in the coenzyme-binding domain. This residue should shield the enzyme active site from solvent. On the basis of the alignment of plant FDHs sequences, Asp, Asn and Ser were selected as candidates to substitute Phe290. Computer modeling indicated the formation of two (Ser and Asn) or three (Asp) new hydrogen bonds in such mutants. The mutant SoyFDHs were expressed in Escherichia coli, purified and characterized. All amino acid substitutions increased K(м)(HCOO-) from 1.5 to 4.1-5.0 mM, whereas the K(м)(NAD+) values remained almost unchanged in the range from 9.1 to 14.0 µM, which is close to wt-SoyFDH (13.3 µM). The catalytic constants for F290N, F290D and F290S mutants of SoyFDH equaled 2.8, 5.1 and 4.1 s⁻¹, respectively; while that of the wild-type enzyme was 2.9 s⁻¹. The thermal stability of all mutant SoyFDHs was much higher compared with the wild-type enzyme. The differential scanning calorimetry data were in agreement with the results of thermal inactivation kinetics. The mutations F290S, F290N and F290D introduced into SoyFDH increased the T(m) values by 2.9°C, 4.3°C and 7.8°C, respectively. The best mutant F290D exhibited thermal stability similar to that of FDH from the plant Arabidopsis thaliana and exceeded that of the enzymes from the yeast Candida boidinii and the bacterium Moraxella sp. C1.

Kinetics of aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle. Effect of agents possessing chaperone-like activity.

An aggregation test system based on the aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle has been proposed. On the basis of the measurements of the enzyme activity and differential scanning calorimetry data a conclusion has been made that UV radiation results in formation of damaged protein molecules with lower thermostability. It was shown that the order of aggregation rate for UV-irradiated GAPDH with respect to the protein was close to 2. This means that such a test system allows detecting the effect of various agents exclusively on the stage of aggregation of unfolded protein molecules. The influence of α-crystallin and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on aggregation of UV-irradiated GAPDH was studied. Despite the fact that HP-ß-CD accelerates thermal aggregation of non-irradiated GAPDH, in the case of aggregation of UV-irradiated GAPDH HP-ß-CD reveals a purely protective effect.

Does the crowded cell-like environment reduce the chaperone-like activity of α-crystallin?

The effect of crowding on the chaperone-like activity of α-crystallin has been studied using aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle as an aggregation test system. The merit of this test system is the possibility of testing agents that directly affect the stage of aggregation of the protein molecules. It was shown that the solution of Phb denatured by UV contained aggregates with a hydrodynamic radius of 10.4 nm. These aggregates are relatively stable at 20 °C; however, they reveal a tendency to stick further in the presence of crowding agents. The study of the effect of α-crystallin on the aggregation of UV-irradiated Phb in the presence of the crowding agents by dynamic light scattering at 37 °C showed that under crowding conditions the antiaggregation ability of α-crystallin was weakened. On the basis of the analytical ultracentrifugation, size-exclusion chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the scheme of interaction of UV-irradiated Phb and α-crystallin has been proposed. It is assumed that chaperone-target protein complexes of two types are formed, namely, the complexes of dissociated forms of α-crystallin with a protein substrate and high-mass α-crystallin-denatured protein complexes. The complexes of the first type reveal a weak propensity to aggregate even under crowding conditions. The complexes of the second type are characterized by the lower rate of aggregation in comparison with that of original UV-irradiated Phb. However, crowding stimulates the rate of aggregation of these complexes, resulting in the above-mentioned decrease in the chaperone-like activity of α-crystallin.

Mechanism of aggregation of UV-irradiated ß(L)-crystallin.

Thermal denaturation and aggregation of UV-irradiated ß(L)-crystallin from eye lenses of steers have been studied. The data on size-exclusion chromatography and SDS-PAGE indicated that UV irradiation of ß(L)-crystallin at 10 °Ð¡ resulted in fragmentation of the protein molecule and formation of cross-linked aggregates. Fluorescence data showed that tryptophan fluorescence in the irradiated protein decreased exponentially with the UV dose. Decrease in tryptophan fluorescence is a result of photochemical destruction, but not of conformational changes of protein, because there is no red shift in the fluorescence maximum. The differential scanning calorimetry (DSC) profiles of the samples of UV-irradiated and wild type ß(L)-crystallin were registered. The area under curves, which is proportional to the amount of the native protein, decreased exponentially with increasing the irradiation dose. The shape of the DSC profiles for the samples of UV-irradiated ß(L)-crystallin was identical to that for wild type ß(L)-crystallin. The DSC data allowed estimating the portion of UV-denatured ß(L)-crystallin, which is not registered by DSC, and the portion of the combined fraction consisting of native and UV-damaged molecules retaining the native structure. A conclusion has been made that UV-induced denaturation of ß(L)-crystallin follows the one-hit model. The study of the kinetics of thermal aggregation of UV-irradiated ß(L)-crystallin at 37 °Ð¡ using dynamic light scattering showed that the initial stage of aggregation was that of formation of the start aggregates with the hydrodynamic radius of 20 nm. Further sticking of the start aggregates proceeded in the regime of reaction-limited cluster-cluster aggregation. Splitting of the aggregate population into two components occurred above a definite point in time.

Effect of 2-hydroxypropyl-ß-cyclodextrin on thermal stability and aggregation of glycogen phosphorylase b from rabbit skeletal muscle.

The study of the kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscles by dynamic light scattering at 48°C showed that 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) accelerated the aggregation process and induced the formation of the larger protein aggregates. The reason of the accelerating effect of HP-ß-CD is destabilization of the protein molecule under action of HP-ß-CD. This conclusion was supported by the data on differential scanning calorimetry and the kinetic data on thermal inactivation of Phb. It is assumed that destabilization of the Phb molecule is due to preferential binding of HP-ß-CD to intermediates of protein unfolding in comparison with the original native state. The conclusion regarding the ability of the native Phb for binding of HP-ß-CD was substantiated by the data on the enzyme inhibition by HP-ß-CD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 986-993, 2010.

Effect of alpha-crystallin on thermostability of mitochondrial aspartate aminotransferase.

Effect of alpha-crystallin on thermal inactivation, denaturation and aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been in the focus of this study. Acceleration of heat-induced inactivation of mAAT was demonstrated in the presence of alpha-crystallin. According to the data of differential scanning calorimetry, alpha-crystallin induces destabilization of the mAAT molecule. The size of protein aggregates formed at heating of mAAT at a constant rate (1 degree C/min) has been defined by dynamic light scattering. The obtained data show that aggregation of mAAT in the presence of alpha-crystallin proceeds in the regime of reaction-limited cluster-cluster aggregation.

Effect of proline on thermal inactivation, denaturation and aggregation of glycogen phosphorylase b from rabbit skeletal muscle.

It has been shown that the relatively low concentrations of proline (0.1 M) have a slight accelerating effect on thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle registered by the accumulaton of the aggregated protein. The suppression of Phb aggregation at high proline concentrations is mainly due to the protective action of proline on the stage of unfolding of the Phb molecule. The enhancement of Phb stability in the presence of the high concentrations of proline was demonstrated by the data on differential scanning calorimetry, analytical ultracentrifugation and thermoinactivation kinetics. The construction of the protein aggregate size versus time plots allowed the acceleration of the stage of Phb aggregation in the presence of high concentrations of proline to be demonstrated. The obtained results are consistent with the predictions of the crowding theory.