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1.
Analyses of unimolecular electron self-exchange reactions provide a comparatively simple and direct approach to understanding biological electron transfer. Such studies are currently limited by a lack of well characterised aggregating systems. In the presence of sodium hexametaphosphate, cytochrome c forms stable protein aggregates as a result of binding hexametaphosphate at a single site on its surface (preceding paper in this issue of the journal). Here we report the location of the principal polyphosphate binding site on the surface of cytochrome c for both hexametaphosphate and a second polyphosphate, tripolyphosphate determined using 1H-NMR spectroscopy in conjunction with the relaxation probe potassium hexacyanochromium(III). Addition of either hexametaphosphate or tripolyphosphate to ferricytochrome c in the presence of the relaxation probe causes a decrease in intensity of several resonances in the paramagnetic difference spectrum, including Phe82 ortho/meta, Ile85 delta methyl and Ile9 gamma methyl. Together these effects put the site of polyphosphate binding close to lysines 13, 86, and 87. Additionally the effect of sodium tripolyphosphate and sodium trimetaphosphate on cytochrome c aggregation is described. The potential role of this site in anion-induced cytochrome c aggregation is discussed. 相似文献
2.
1H-NMR spectroscopy has been used to measure the rate of unimolecular electron exchange between cytochrome c molecules in protein aggregates stabilised by the addition of sodium hexametaphosphate. The average intracomplex electron exchange rate is measured from line broadening of hyperfine-shifted resonances of ferricytochrome c in an equimolar mixture of reduced and oxidised protein. The line-broadening due to electron exchange is significantly greater than that due to protein aggregation and reaches a maximum value between 1-2 mol hexametaphosphate/mol protein. Significantly the exchange-induced broadening is a first-order process and is directly proportional to the size of the cytochrome c oligomer. From the temperature dependence of exchange broadening the activation enthalpy was estimated to be 75.8 kJ mol-1 whereas the activation entropy was 295 J mol-1 K-1 for a dimer of cytochrome c at a hexametaphosphate/protein molar ratio of 1. Both activation parameters decrease in magnitude as the order of the cytochrome c oligomer increases. The rates of intracomplex electron exchange in Saccharomyces cerevisiae iso-2 and Candida krusei cytochromes c are lower than that of the horse protein, implying that primary sequence plays a fundamental part in determining the rate of exchange. The relevance of these observations is discussed in terms of the function of cytochrome c. 相似文献
3.
Roman SG Chebotareva NA Eronina TB Kleymenov SY Makeeva VF Poliansky NB Muranov KO Kurganov BI 《Biochemistry》2011,50(49):10607-10623
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. 相似文献
4.
Markossian KA Khanova HA Kleimenov SY Levitsky DI Chebotareva NA Asryants RA Muronetz VI Saso L Yudin IK Kurganov BI 《Biochemistry》2006,45(44):13375-13384
Thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and analytical ultracentrifugation. The maximum of the protein thermal transition (T(m)) increased with increasing the protein concentration, suggesting that the denaturation process involves the stage of reversible dissociation of the enzyme tetramer into the oligomeric forms of lesser size. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. The DLS data support the mechanism of protein aggregation that involves a stage of the formation of the start aggregates followed by their sticking together. The hydrodynamic radius of the start aggregates remained constant in the temperature interval from 37 to 55 degrees C and was independent of the protein concentration (R(h,0) approximately 21 nm; 10 mM sodium phosphate, pH 7.5). A strict correlation between thermal aggregation of GAPDH registered by the increase in the light scattering intensity and protein denaturation characterized by DSC has been proved. 相似文献
5.
Kira A. Markossian Nikolay V. Golub Natalia A. Chebotareva Regina A. Asryants Irina N. Naletova Vladimir I. Muronetz Konstantin O. Muranov Boris I. Kurganov 《The protein journal》2010,29(1):11-25
Effects of α-crystallin and GroEL on the kinetics of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) have been studied using dynamic light scattering and analytical ultracentrifugation. The analysis of the initial parts
of the dependences of the hydrodynamic radius of protein aggregates on time shows that in the presence of α-crystallin or
GroEL the kinetic regime of GAPDH aggregation is changed from the regime of diffusion-limited cluster–cluster aggregation
to the regime of reaction-limited cluster–cluster aggregation, wherein the sticking probability for the colliding particles
becomes lower the unity. In contrast to α-crystallin, GroEL does not interfere with formation of the start aggregates which
include denatured GAPDH molecules. On the basis of the analytical ultracentrifugation data the conclusion has been made that
the products of dissociation of GAPDH and α-crystallin or GroEL play an important role in the interactions of GAPDH and chaperones
at elevated temperatures. 相似文献
6.
We studied the thermal denaturation of eglin c by using CD spectropolarimetry and differential scanning calorimetry (DSC). At low protein concentrations, denaturation is consistent with the classical two-state model. At concentrations greater than several hundred microM, however, the calorimetric enthalpy and the midpoint transition temperature increase with increasing protein concentration. These observations suggested the presence of intermediates and/or native state aggregation. However, the transitions are symmetric, suggesting that intermediates are absent, the DSC data do not fit models that include aggregation, and analytical ultracentrifugation (AUC) data show that native eglin c is monomeric. Instead, the AUC data show that eglin c solutions are nonideal. Analysis of the AUC data gives a second virial coefficient that is close to values calculated from theory and the DSC data are consistent with the behavior expected for nonideal solutions. We conclude that the concentration dependence is caused by differential nonideality of the native and denatured states. The nondeality arises from the high charge of the protein at acid pH and is exacerbated by low buffer concentrations. Our conclusion may explain differences between van't Hoff and calorimetric denaturation enthalpies observed for other proteins whose behavior is otherwise consistent with the classical two-state model. 相似文献
7.
Kinetics of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle: mechanism of protective action of alpha-crystallin 总被引:1,自引:0,他引:1
The kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle have been studied by dynamic light scattering (0.08M Hepes, pH 6.8, containing 0.1M NaCl; 48 degrees C). The hydrodynamic radius of the start aggregates determined from the initial linear parts of the dependences of the hydrodynamic radius (R(h)) on time was found to be 16.7 +/- 1.0 nm. At rather high values of time, the R(h) value for the protein aggregates becomes proportional to t(1/1.8) = t(0.56) suggesting that the aggregation process proceeds in the regime of diffusion-limited cluster-cluster aggregation. In the presence of alpha-crystallin, a protein possessing the chaperone-like activity, the process of protein aggregation switches to the regime of reaction-limited cluster-cluster aggregation as indicated by the exponential dependence of the R(h) value on time. It was shown that the addition of alpha-crystallin raises the rate of thermal inactivation of Phb. These data in combination with the results of the study of interaction of Phb with alpha-crystallin by analytical ultracentrifugation suggest that alpha-crystallin interacts with the intermediates of unfolding of the Phb molecule. 相似文献
8.
The mechanism of IgG heat aggregation was studied using IgG aggregates complexed with azo dyes to increase their solubility and stability. Heat dependent and heat independent steps of aggregation were differentiated. On heating IgG at the dye concentration exceeding 100 times that of protein, mainly dimers are formed, as judged from ultracentrifugation and chromatographic analysis, whereas high molecular weight derivatives appear at room temperature when the protein/dye ratio is decreased. The analysis of spectral changes following either the attachment or removal of the dye from IgG aggregates implies that only a part of the dye molecules is bound firmly and directly to the protein binding sites. These dye molecules which are easily removed by adsorption to cellulose or reduced by dithionate but migrate together with IgG aggregates on chromatography and electrophoresis, are supposed to constitute that part of the micelle which extrudes from the binding site and, hence, is fixed indirectly to protein. Various proteins with predominant beta-structure were also found to bind azo dyes when heated. 相似文献
9.
Pivovarova AV Chebotareva NA Chernik IS Gusev NB Levitsky DI 《The FEBS journal》2007,274(22):5937-5948
Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548-1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27-3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin-Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of approximately 16 nm, a sedimentation coefficient of 17-20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions. 相似文献
10.
Jayaraman M Kodali R Sahoo B Thakur AK Mayasundari A Mishra R Peterson CB Wetzel R 《Journal of molecular biology》2012,415(5):881-899
The 17-amino-acid N-terminal segment (htt(NT)) that leads into the polyglutamine (polyQ) segment in the Huntington's disease protein huntingtin (htt) dramatically increases aggregation rates and changes the aggregation mechanism, compared to a simple polyQ peptide of similar length. With polyQ segments near or above the pathological repeat length threshold of about 37, aggregation of htt N-terminal fragments is so rapid that it is difficult to tease out mechanistic details. We describe here the use of very short polyQ repeat lengths in htt N-terminal fragments to slow this disease-associated aggregation. Although all of these peptides, in addition to htt(NT) itself, form α-helix-rich oligomeric intermediates, only peptides with Q(N) of eight or longer mature into amyloid-like aggregates, doing so by a slow increase in β-structure. Concentration-dependent circular dichroism and analytical ultracentrifugation suggest that the htt(NT) sequence, with or without added glutamine residues, exists in solution as an equilibrium between disordered monomer and α-helical tetramer. Higher order, α-helix rich oligomers appear to be built up via these tetramers. However, only htt(NT)Q(N) peptides with N=8 or more undergo conversion into polyQ β-sheet aggregates. These final amyloid-like aggregates not only feature the expected high β-sheet content but also retain an element of solvent-exposed α-helix. The α-helix-rich oligomeric intermediates appear to be both on- and off-pathway, with some oligomers serving as the pool from within which nuclei emerge, while those that fail to undergo amyloid nucleation serve as a reservoir for release of monomers to support fibril elongation. Based on a regular pattern of multimers observed in analytical ultracentrifugation, and a concentration dependence of α-helix formation in CD spectroscopy, it is likely that these oligomers assemble via a four-helix assembly unit. PolyQ expansion in these peptides appears to enhance the rates of both oligomer formation and nucleation from within the oligomer population, by structural mechanisms that remain unclear. 相似文献
11.
We have used microcalorimetry and analytical ultracentrifugation to test the model proposed in Pettigrew et al. [(1999) J. Biol. Chem. 274, 11383-11389] for the binding of small cytochromes to the cytochrome c peroxidase of Paracoccus denitrificans. Both methods reveal complexity in behavior due to the presence of a monomer/dimer equilibrium in the peroxidase. In the presence of either Ca(2+), or higher ionic strength, this equilibrium is shifted to the dimer. Experiments to study complex formation with redox partners were performed in the presence of Ca(2+) in order to simplify the equilibria that had to be considered. The results of isothermal titration calorimetry reveal that the enzyme can bind two molecules of horse cytochrome c with K(d) values of 0.8 microM and 2.5 microM (at 25 degrees C, pH 6.0, I = 0.026) but only one molecule of Paracoccus cytochrome c-550 with a K(d) of 2.8 microM, molar binding ratios confirmed by ultracentrifugation. For both horse cytochrome c and Paracoccus cytochrome c-550, the binding is endothermic and driven by a large entropy change, a pattern consistent with the expulsion of water molecules from the interface. For horse cytochrome c, the binding is weakened 3-fold at I = 0.046 M due to a smaller entropy change, and this is associated with an increase in enzyme turnover. In contrast, neither the binding of cytochrome c-550 nor its oxidation rate is affected by raising the ionic strength in this range. We propose that, at low ionic strength, horse cytochrome c is trapped in a nonproductive orientation on a broad capture surface of the peroxidase. 相似文献
12.
Gutmann DA Mizohata E Newstead S Ferrandon S Postis V Xia X Henderson PJ van Veen HW Byrne B 《Protein science : a publication of the Protein Society》2007,16(7):1422-1428
One key to successful crystallization of membrane proteins is the identification of detergents that maintain the protein in a soluble, monodispersed state. Because of their hydrophobic nature, membrane proteins are particularly prone to forming insoluble aggregates over time. This nonspecific aggregation of the molecules reduces the likelihood of the regular association of the protein molecules essential for crystal lattice formation. Critical buffer components affecting the aggregation of membrane proteins include detergent choice, salt concentration, and presence of glycerol. The optimization of these parameters is often a time- and protein-consuming process. Here we describe a novel ultracentrifugation dispersity sedimentation (UDS) assay in which ultracentrifugation of very small (5 microL) volumes of purified, soluble membrane protein is combined with SDS-PAGE analysis to rapidly assess the degree of protein aggregation. The results from the UDS method correlate very well with established methods like size-exclusion chromatography (SEC), while consuming considerably less protein. In addition, the UDS method allows rapid screening of detergents for membrane protein crystallization in a fraction of the time required by SEC. Here we use the UDS method in the identification of suitable detergents and buffer compositions for the crystallization of three recombinant prokaryotic membrane proteins. The implications of our results for membrane protein crystallization prescreening are discussed. 相似文献
13.
Study of the chaperoning mechanism of bovine lens alpha-crystallin, a member of the alpha-small heat shock superfamily
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We have studied the interaction between lysozyme, destabilized by reducing its -S-S- bonds, and bovine eye lens alpha-crystallin, a member of the alpha-small heat shock protein superfamily. We have used gel filtration, photon correlation spectroscopy, and analytical ultracentrifugation to study the binding of lysozyme by alpha-crystallin at 25 degrees C and 37 degrees C. We can conclude that alpha-crystallin chaperones the destabilized protein in a two-step process. First the destabilized proteins are bound by the alpha-crystallin so that nonspecific aggregation of the destabilized protein is prevented. This complex is unstable, and a reorganization and inter-particle exchange of the peptides result in stable and soluble large particles. alpha-Crystallin does not require activation by temperature for the first step of its chaperone activity as it prevents the formation of nonspecific aggregates at 25 degrees C as well as at 37 degrees C. The reorganization of the peptides, however, gives rise to smaller particles at 37 degrees C than at 25 degrees C. Indirect evidence shows that the association of several alpha-crystallin/substrate protein complexes leads to the formation of very large particles. These are responsible for the increase of the light scattering. 相似文献
14.
The cytoplasmic membrane of Mycoplasma laidlawii strain B is solubilized by anionic and nonionic detergents, succinylation, phospholipase A, alkaline phosphatase, trypsin, and chymotrypsin. Cationic detergents are without effect, as are chelating agents, even in the presence of high concentrations of monovalent cation. The detergent-solubilized membrane exhibits one peak in the analytical ultracentrifuge, but the sedimentation coefficient is dependent upon concentration of detergent. Simple dialysis does not remove all of the sodium dodecylsulfate except from lipid-depleted membrane particles. Membranes bind sodium dodecylsulfate but acetone powders of membranes do not. Sulfated alcohols with chain lengths of C(14) and C(16) are more tightly bound than dodecylsulfate. A constant amount of di- and trivalent cation is bound by the membrane upon aggregation. Only a portion of this cation is removable with chelating agents. No chelating agent is bound by these aggregates. A portion of the lipid-depleted membrane particles is solubilized by negatively charged lipids and detergents, giving rise to aggregates in the presence of divalent cation. Fractionations of detergent-solubilized membranes by preparative gel electrophoresis and ammonium sulfate were inconclusive. Density gradient centrifugation of succinylated membranes yielded at least five fractions which exhibited homogeneity by ultracentrifugation. Analytical gel electrophoresis of these fractions demonstrated heterogeneity. The composition of these five fractions suggested separation of protein from lipid. 相似文献
15.
B W K?nig L T Schilder M J Tervoort B F Van Gelder 《Biochimica et biophysica acta》1980,621(2):283-295
A large-scale isolation method for cytochrome c1 from beef heart is presented, based in principle on the procedure of Yu et al. (Yu, C.A., Yu, L. and King, T.E. (1972) J. Biol. Chem. 247, 1012--1019). Optimal solubilization of cytochrome c1 from succinate-cytochrome c oxidoreductase was achieved with 15% beta-mercaptoethanol, 1.5% cholate, 0.5% deoxycholate in 8% saturated ammoniun sulphate. The protein is purfied to a higher degree by chromatography on DEAE-cellulose and Ultrogel AcA 44. The method is reproducible and gives highly purified cytochrome c1 with a yield from succinate-cytochrome c oxidoreductase of 40%. The purified cytochrome c1 contains 32 nmol of heme/mg protein and has a spectral heme-to-protein ratio (Ared417nm/Ax276nm) of 2.7. Reduced cytochrome c1 is oxidized very rapidly by ferricytochrome c (k = 3 . 10(7) M-1 . S-1 at 10 degrees C, 100 mM potassium phosphate (pH 7.0) and 1% Tween 20). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate shows that the isolated protein consists of one peptide, with a molecular weight of 31 000, carrying the chromophore. In the presence of 1% sodium cholate or 1% Tween 80, cytochrome c1 is in the monomeric state, whereas at lower concentrations of detergent the protein aggregates. The aggregation of cytochrome c1 is found to be reversible. 相似文献
16.
Electrostatic forces contribute to interactions between trp repressor dimers. 总被引:1,自引:0,他引:1
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K S Martin C A Royer K P Howard J Carey Y C Liu K Matthews E Heyduk J C Lee 《Biophysical journal》1994,66(4):1167-1173
The trp repressor of Escherichia coli (TR), although generally considered to be dimeric, has been shown by fluorescence anisotropy of extrinsically labeled protein to undergo oligomerization in solution at protein concentrations in the micromolar range (Fernando, T., and C. A. Royer 1992. Biochemistry. 31:3429-3441). Providing evidence that oligomerization is an intrinsic property of TR, the present studies using chemical cross-linking, analytical ultracentrifugation, and molecular sieve chromatography demonstrate that unmodified TR dimers form higher order aggregates. Tetramers and higher order species were observed in chemical cross-linking experiments at concentrations between 1 and 40 microM. Results from analytical ultracentrifugation and gel filtration chromatography were consistent with average molecular weight values between tetramer and dimer, although no plateaus in the association were evident over the concentration ranges studied, indicating that higher order species are populated. Analytical ultracentrifugation data in presence of corepressor imply that corepressor binding destabilizes the higher order aggregates, an observation that is consistent with the earlier fluorescence work. Through the investigation of the salt and pH dependence of oligomerization, the present studies have revealed an electrostatic component to the interactions between TR dimers. 相似文献
17.
Pettigrew GW Pauleta SR Goodhew CF Cooper A Nutley M Jumel K Harding SE Costa C Krippahl L Moura I Moura J 《Biochemistry》2003,42(41):11968-11981
According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently. 相似文献
18.
Aggregation of proteins is a problem with serious medical implications and economic importance. To develop strategies for preventing aggregation, the mechanism(s) and pathways by which proteins aggregate must be characterized. In this study, the thermally induced aggregation processes of three alpha-helix proteins (myoglobin, cytochrome c, and lysozyme) in the presence and absence of 1.0 m guanidine hydrochloride (GdnHCl) were investigated by means of infrared spectroscopy. In the absence of GdnHCl, intensities of the alpha-helix bands (approximately 1656 cm(-1)) decrease as a function of temperature at above 50 degrees C. With myoglobin and cytochrome c, the loss of helix bands was accompanied by the appearance of two new bands at 1694 and 1623 cm(-1), indicative of the formation of intermolecular beta-sheet aggregates. For lysozyme, bands indicative of intermolecular beta-sheet aggregates did not appear in any significant intensity. In the presence of 1.0 m GdnHCl, two major intermediate states rich in 3(10)-helix (represented by the band at 1663 cm(-1)) and beta-turn structure (represented by the band at 1667 cm(-1)), respectively, were observed. These findings demonstrated that IR spectroscopic studies of protein aggregation using a combination of thermal and chemical denaturing factors could provide a means to populate and characterize aggregation intermediates. 相似文献
19.
A. M. Polyanichko N. V. Mikhailov N. M. Romanov Yu. G. Baranova E. V. Chikhirzhina 《Cell and Tissue Biology》2017,11(1):9-15
The mechanisms of intermolecular protein complex formation were studied by the example of monomers, oligomers and aggregates of bovine serum albumin (BSA) depending on the protein concentration, pH and urea concentration. Using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and PAG electrophoresis we have shown the existence of dynamic equilibrium between monomers and aggregates in BSA solution. Decreasing pH of the solution (4.0–1.0) resulted in increasing sizes of the aggregates. In the solutions with low urea concentrations (below 2 M) the sizes of aggregates decreased, while higher urea concentrations (2–8 M) induced formation of larger aggregates due to the unfolding of the protein. 相似文献
20.
Schüler J Frank J Behlke J Saenger W Georgalis Y 《Biochimica et biophysica acta》2000,1480(1-2):211-221
The colloidal properties of transferrin receptor, isolated from human placenta, in detergent free solution has been investigated by light scattering techniques and analytical ultracentrifugation. In detergent free solution at 293.2 K, hTfR forms stable aggregates with an apparent hydrodynamic radius of 17 nm. The molecular mass was determined by ultracentrifugation to lie between (1722+/-87) kDa (sedimentation equilibrium) and (1675+/-46) kDa (sedimentation velocity). This implies that the aggregates are build up from nine hTfR dimers. Based on model calculations, which are in good agreement with the experimental data, we propose a torus-like structure for the aggregates. Upon pH shift from pH 7.5 to 5.0 or removal of the N-linked carbohydrate chains, formation of larger aggregates is induced. These aggregates can be described in terms of porous fractal structures. We propose a simple model, which accounts for that behaviour assuming that the aggregation is mainly due to the reduction of negative surface charge. 相似文献