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1.
Measurements of the anisotropy of protein fluorescence as a function of an added collisional quencher, such as acrylamide, are used to construct Perrin plots. For single tryptophan containing proteins, such plots yield an apparent rotational correlation time for the depolarization process, which, in most cases, is approximately the value expected for Brownian rotation of the entire protein. Apparent limiting fluorescence anisotropy values, which range from 0.20 to 0.32 for the proteins studied, are also obtained from the Perrin plots. The lower values for the limiting anisotropy found for some proteins are interpreted as indicating the existence of relatively rapid, limited (within a cone of angle 0 degrees--30 degrees) motion of the tryptophan side chains that is independent of the overall rotation of the protein. Examples of the use of this fluorescence technique to study protein conformational changes are presented, including the monomer in equilibrium dimer equilibrium of beta-lactoglobulin, the monomer in equilibrium tetramer equilibrium of melittin, the N in equilibrium F transition of human serum albumin, and the induced change in the conformation of cod parvalbumin caused by the removal of Ca+2. Because multitryptophan-containing proteins have certain tryptophans that are accessible to solute quencher and others that are inaccessible, this method can be used to determine the steady state anisotropy of each class of tryptophan residues.  相似文献   

2.
Dependency of optical rotation of di- and triphosphoinositide water solutions on pH is investigated. The inversion of myo-inosite ring in triphosphoinositide molecule is found to take place at pH range corresponding to the transition of phosphomonoesters from monoanionic into dianionic form. Stabilization of optical rotation is observed at pH range, where one monoester phosphate group is in monoanionic form and the other--in dianionic form. Probably, triphosphoinositide is in the conformation (distorted boat) at this pH range. Diphosphoinositide does not change its optical rotation under pH changes of water or organic solutions. A contribution is estimated of free conformational energy into standard free energy of splitting triphosphoinositide phosphate group depending on pH value. It is concluded that low energy phosphate bond becomes high energy bond due to the free electrostatic interaction of dianionic phosphate group with other negatively charged group in sin-clynal conformation.  相似文献   

3.
The relaxation time of thyroglobulin has been determined in water at. neutral pH, in concentrated urea and guanidine solutions, at alkaline pH, both before and after reduction with β-mercaptoethanol. The structure of thyroglobulin in concentrated urea solutions is markedly affected by the pH, Time-dependent changes occur in thyroglobulin in concentrated urea or guanidine solutions which arc observable by polarization of fluorescence but not by optical rotation or viscosity. The reduction of the disulfide crosslinks of thyroglobulin in urea at high pH or in guanidine produces linear polypeptide chains with few if any permanent contacts between segments.  相似文献   

4.
The effect of pH on the conformation of ricin and its A- and B-chains has been studied by measuring their intrinsic fluorescence. At pH 5.0 and 7.5, the structural stability of toxin and subunits was estimated according to the denaturing action of guanidine hydrochloride. It was demonstrated that the fluorescence of native toxin and catalytic A-subunit does not depend significantly on pH in the range pH 3-8, whereas ricin B-chain undergoes a structural transition at pH less than 5.0. The structural stability of ricin and isolated chains differs significantly at pH 7.5 and 5.0; the structural stability of ricin and the A-chain increases, whereas that of the B-chain decreases.  相似文献   

5.
The changes in F-actin conformation in myosin-free single ghost fiber induced by the binding of heavy meromyosin (HMM) with dephosphorylated or phosphorylated light chains-2 (LC2) have been studied by measuring intrinsic tryptophan polarized fluorescence of F-actin. It has been found that at low concentrations of Ca2+ (pCa greater than or equal to 8), the binding of HMM with dephosphorylated LC2 to F-actin in ghost fibres increases, whereas the binding of HMM with phosphorylated LC2 decreases the anisotropy of polarized tryptophan fluorescence. The effect is reversed at high concentrations of Ca2+ (pCa = 5). It has been assumed that this effect of myosin light chains phosphorylation may be due to its influence on the type of myosin head binding to F-actin.  相似文献   

6.
M J Smerdon  I Isenberg 《Biochemistry》1976,15(19):4233-4242
This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.  相似文献   

7.
Arabinoxylan preparations from sugar cane show temperature-induced shifts of optical rotation in aqueous methyl sulphoxide solution, the sign and magnitude of which depend on the content of arabinofuranose side-groups. The shift has a sigmoidal form and shows a distinct hysteresis loop on heating and cooling. This evidence, together with the sign and magnitude, is interpreted in terms of a conformation change of the backbone of β-(1→4)-linked D-xylose residues, from the random coil to an ordered, ribbon-like conformation similar to that which is known to exist in the solid state. The driving force for this change is not intramolecular but derives from an aggregation that occurs simultaneously and can be detected by other methods. The arabinofuranose side-groups can be incorporated into the ordered assembly and contribute to the optical rotation shift, and they therefore have an unusual role compared with other polysaccharide side-chains. We conclude that the optical rotation shifts show a “melting” and re-formation of ordered associations which may imitate the natural biological cohesion between hemicellulose chains.  相似文献   

8.
Values of four conformational properties, namely unperturbed dimension [r2]0, dipole moment [mu 2], mean squared optical anisotropy [gamma 2], and molar Kerr constant [mK], have been calculated for polyglycine chains allowing several combinations of the secondary structure with the aim of studying the dependence of these magnitudes on the secondary structure of the chain. Two different approaches to the secondary structure have been used. In the first, chains with all their units in a given conformation (random coil, alpha-helix or beta-sheet) are interrupted at several positions by one unit in a different conformation. In the second, chains with varying composition of two conformations alpha-helix/beta-sheet and beta-sheet/random coil were allowed and the results obtained compared with previous work for alpha-helix/random coil chains.  相似文献   

9.
Intrinsic protein fluorescence of native plant toxin and its isolated subunits were studied. The effect of pH was studied on: conformation of ricin and its A- and R-chains; affinity to galactose of ricin and its binding B-subunit. At two pH 5.0 and 7.0, the structural stability of toxin and subunits was estimated according to denaturational action of guanidine chloride. It was demonstrated that position of maximum and the spectrum shape of fluorescence of native toxin and catalytical A-subunit insignificantly depends on pH in the range of 3-8, whereas sufficient changes of the separameters for the ricin B-chain reveal structural transition at pH 4-5. The affinity of galactose of ricin and its isolated B-chain depends on pH, the maximal binding is observed at pH 7. The structural stability of ricin and isolated chains significantly differs at pH 7.5 and 5.0, thus the structure stability of ricin and A-chain increases, and that of B-chain decreases at pH 5.0.  相似文献   

10.
Sugars, which function as osmolytes within cells, retard the amyloid fibril formation of the amyloidosis peptides and proteins. To examine the mechanism of this retardation in detail, we analyzed the effect of sugars (trehalose, sucrose, and glucose) on the polypeptide chains in 3Hmut Wil, which is formed by the mutation of three His residues in Wil mutant as a cause of amyloid light‐chain (AL) amyloidosis, at pH 2, a pH condition under which 3Hmut Wil was almost denatured. Sugars caused the folding of 3Hmut Wil so that its polypeptide chains adopted a native‐like rather than a denatured conformation, as suggested by tryptophan fluorescence, CD spectroscopy, and heteronuclear NMR. Furthermore, these sugars promoted the folding to a native‐like conformation according to the effect of preferential hydration rather than direct interaction. However, the type of sugar had no effect on the elongation of amyloid fibrils. Therefore, it was concluded that sugar affected the thermodynamic stability of 3Hmut Wil but not the elongation of amyloid fibrils.  相似文献   

11.
A chiroptical method of conformational analysis is applied to linear (1 → 3)-β-D -glucans and the dimeric analogues β- and α-laminaribioside. The method is based on a recently developed semiempirical calculational model for saccharide optical activity. We conclude that disaccharide conformational energy maps in the literature represent the effective potential energy surface in aqueous solution well. The positive optical rotation observed with long chains in dilute alkaline solution is not characteristic of any single–chain conformation, and must reflect chain association.  相似文献   

12.
The fluorescence properties of human milk bile salt-activated lipase (BAL) in aqueous solution at various pH and in the presence of denaturing reagents and bile salts have been studied by measuring the accessibility of tryptophan side chains to the iodide ion. The fluoresence quenching studies of BAL demonstrated that the BAL conformation was pH sensitive. At pH 7.5, in the presence of denaturing reagents, all of the BAL tryptophan became accessible to iodide, suggesting the presence of random conformation in this medium. The decrease in tryptophan accessibility to iodide with various bile salt activators was found to correlate with the corresponding activity of BAL with long chain triacylglycerol substrate.  相似文献   

13.
We have used fluorescence spectroscopy techniques such as fluorescence correlation spectroscopy and fluorescence anisotropy decay on a wide time range, from nanoseconds to seconds, to investigate the unfolding kinetics induced by guanidinium chloride of GFPMut2 and its point mutation H148G, which has proved to be relevant for GFP photochemistry and photophysics. The mutation affects the unfolding kinetics of GFP leading to a much faster process at alkaline pH values, where protonation dynamics is negligible, that can be ascribed to a twofold role of His148, either as a proton shutter towards the chromophore and as a conformation stabiliser. For both mutants a soft region located near beta-strand 3 is found that starts to gain flexibility in the ns range at denaturant concentrations far lower than those required to turn off the chromophore fluorescence, as derived from the anisotropy decay of an extrinsic probe covalently bound to the proteins.  相似文献   

14.
The primary structures of the four bacterial polysaccharides gellan, welan, S-657, and rhamsan are the same with respect to their backbones, but have different side-chains. This difference has a profound influence on their behavior in aqueous media. Solutions of gellan gum form stable aqueous gels under appropriate ionic conditions. By contrast, welan, S-657, and rhamsan do not gel but give very viscous solutions over a wide range of thermal, pH, and salt conditions. X-Ray fiber diffraction analysis and computer modeling of these branched polysaccharides demonstrate that they all have the same half-staggered, double-helical conformations as in the unbranched gellan, suggesting, therefore, that the side chains are responsible for diminishing gelling behavior. Depending on the size and location, the side chains shield the carboxylate groups to varying degrees; this shielding is substantial in welan and S-657, but less in rhamsan. In all cases, side-chain-main-chain interactions within the double helix prevent the carboxylate-mediated aggregation of double helices that is necessary for the gelation.  相似文献   

15.
Role of substituents on the properties of some polysaccharides   总被引:3,自引:0,他引:3  
Rinaudo M 《Biomacromolecules》2004,5(4):1155-1165
This paper concerns the influence of the chemical structure on the physical properties of some polysaccharides. Especially, we proposed to discuss the role of the substituents on these properties. In some cases, non-carbohydrate substituents play a minor role on rheological properties in the presence of a salt excess as shown on xanthan and succinoglycan. The rheology of aqueous solution of these stereoregular polysaccharides is controlled by the conformation (helical conformation) whose stability is not largely influenced by these substituents. On the other hand, the interaction between galactomannan and xanthan depends on the presence of acetyl substituents on xanthan but also on the xanthan conformation. However, for polymers such as gellan, XM-6 or BEC 1615, complete deacetylation induces the ability to form physical gels in given thermodynamic conditions. The presence of carbohydrate substituents or short side chains was also examined. Especially in the gellan family, the role of position of substitution (position 3 on the glucose unit C or position 6 on the A glucose) was presented. It is concluded that the substituents giving the higher stability for the helical conformation (higher DeltaH and Tm values) also cause a lower salt sensitivity for the helical stability. The role of the substituents on the properties is also described for natural polymers and their chemically or enzymatically modified derivatives.  相似文献   

16.
T Hirao  T Sato  A Teramoto  T Matsuo  H Suga 《Biopolymers》1990,29(14):1867-1876
A triple helical polysaccharide schizophyllan in aqueous solution exhibited a highly cooperative transition between ordered and disordered states associated with the conformation of its side chains and nearby water molecules. The transition was followed by optical rotation and calorimetry using water containing additives such as NaOH and DMSO as solvents. The ordered state was stabilized or destabilized depending on the kind and amount of the additive employed; in particular, the addition of DMSO had a remarkable stabilizing effect. This effect was analyzed by means of a statistical mechanical theory of linear cooperative transitions, where DMSO was assumed to interact favorably with the ordered side chains. A small amount of NaOH in a solvent mixture stabilized the ordered state and made the transition curve very gradual. No molecular mechanism was elucidated to account for the role of NaOH.  相似文献   

17.
Single-chain equilibrium conformation and dimerization of the three types of ionic EAK16 peptide are studied under three pH conditions using all-atom molecular dynamics simulations. It is found that both the single-chain conformation and the dimerization process of EAK16-IV are considerably different from those of the two other types, EAK16-I and EAK16-II. The value of pH is found to have a stronger effect on the single-chain conformation and dimerization of EAK16-IV. It is shown that in addition to the charge pattern on the peptide chains, the size of the side chains of the charged amino acids plays role in the conformation of the peptide chains and their dimerization. The results shed light on the pH-dependent self-assembly behavior of EAK16 peptide in the bulk solution, which has been reported in the literature.  相似文献   

18.
Monellin is a protein that tastes sweet. In the native state it is a dimer composed of two dissimilar noncovalently associated polypeptides. The conformation of the protein is a determinant of its sweetness, and the present investigation takes advantage of the fluorescence spectrum being a sensitive index of its conformation. The emission spectrum is used to evaluate the ability of temperature and pH to alter the conformation and the sweetness of the protein. When monellin dissolved in water is heated in discrete steps from 25 to 100 degrees C, its sweetness decreases. The halfwidth of the fluorescence emission band increases in parallel with the loss of sweetness. The increase in halfwidth is due primarily to an increase in the intensity of tyrosine emission that may be the result of the two dissimilar polypeptides of monellin beginning to separate. Tyrosine residues are present in both chains, while the single tryptophan occurs in only one. Monellin is less susceptible to denaturation by increasing temperature when dissolved in sodium acetate buffer at pH 4 than it is at pH 3 or 7. When the pH of a solution containing monellin in 0.1 M KC1 is varied from 2 to 13, there is a broad pH range (pH 4 to 9) where monellin's conformation is not markedly altered. Below pH 3.5 and above pH 10.5, however, the emission spectra indicate that substantial denaturation occurs. However, monellin can be partially renatured following pH 12 treatment with only minimal loss of sweetness. The sweetness of monellin under these two types of denaturing conditions, temperature and pH, can be predicted by the fluorescence emission spectrum of the protein. In addition, this study confirms that the biological activity of monellin, its sweetness, is a function of quaternary structure of the protein.  相似文献   

19.
Effects of internal rotation on the fluorescence decay functions and time-dependent anisotropies of fluorophores bound to a spherical macromolecule are theoretically investigated in the presence of the intramolecular energy transfer interaction by solving relevant rotational diffusion equations. The model system examined is one in which the energy donor is internally rotating around an axis fixed at the macromolecule and the acceptor is fixed at a definite position in the macromolecule. The effect of internal rotation in the system is described by Hill's functions with two cosine terms. The fluorescence decay function and anisotropy decay are functions of the ratio of energy-transfer probability averaged over the internal rotation angle to the rotary diffusion co-efficient. When the internal rotation is much faster than energy transfer, the decay function of the donor is predicted to be a single exponential, and the anisotropy decay is essentially described by the expression derived by Gotlieb and Wahl (1963. J. Chim. Phys. 60:849-856). However, deviation from it becomes pronounced as the rotation becomes slower. Methods of numerical analysis are presented for decay function and anisotropy decay, as well as relative quantum yield and polarization anisotropy under steady-state excitation, and examined for a simplified system under the variation of the diffusion coefficient.  相似文献   

20.
The circular polarization of the luminescence of a chromophore, in addition to its circular dichroism and optical rotatory dispersion, is a manifestation of its asymmetry. In the study of proteins, the circular polarization of luminescence yields more specific information than circular dichroism or optical rotatory dispersion since nonfluorescent chromophores do not contribute, and the spectra of the tyrosine and the tryptophan residues are much better resolved in emission than in absorption. The circular polarization of the fluorescence of the tyrosine and tryptophan residues in derivatives of subtilisin Carlsberg and subtilisin Novo were indeed resolved in this study. The tyrosine residues in the Carlsberg protein, and both tyrosine and tryptophan residues in the Novo protein, were found to be heterogeneous with respect to their optical activity and emission spectra. Changes in the environment of the emitting tyrosine residues in both proteins and in the tryptophan residues in the Novo protein were found on changing the pH from 5.0 to 8.3. The pH dependence of the enzymatic activity of these proteins may thus be due, at least in part, to conformational changes in the molecules. Fluorescence circular polarization also revealed that covalently bound inhibitors at the active site of subtilisin Novo affect the environment of the emitting aromatic side chains, presumably via changes in conformation.  相似文献   

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