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21.
Interactions of bisANS and ANS to tubulin in the presence and absence of GTP were investigated, and the binding and thermodynamic parameters were determined using isothermal titration calorimetry. Like bisANS binding to tubulin, we observed a large number of lower affinity ANS binding sites (N1 = 1.3, K1 = 3.7 x 10(5) M(-1), N2 = 10.5, K2 = 7 x 10(4)/M(-1)) in addition to 1-2 higher affinity sites. Although the presence of GTP lowers the bisANS binding to both higher and lower affinity sites (N1 = 4.3, N2 = 11.7 in absence and N1 = 1.8, N2 = 3.6 in presence of GTP), the stoichiometries of both higher and lower affinity sites of ANS remain unaffected in the presence of GTP. BisANS-induced structural changes on tubulin were studied using site-specific proteolysis with trypsin and chymotrypsin. Digestion of both alpha and beta tubulin with trypsin and chymotrypsin, respectively, has been found to be very specific in presence of GTP. GTP has dramatic effects on lowering the extent of nonspecific digestion of beta tubulin with trypsin and stabilizing the intermediate bands produced from both alpha and beta. BisANS-treated tubulin is more susceptible to both trypsin and chymotrypsin digestion. At higher bisANS concentration (>20 microM) both alpha and beta tubulins are almost totally digested with enzymes, indicating bisANS-induced unfolding or destabilization of tubulin structure. Again, the addition of GTP has remarkable effect on lowering the bisANS-induced enhanced digestion of tubulin as well as stabilizing effect on intermediate bands. These results of isothermal titration calorimetry, proteolysis and the DTNB-kinetics data clearly established that the addition of GTP makes tubulin compact and rigid and hence the GTP-induced stabilization of tubulin structure. No such destabilization of tubulin structure has been noticed with ANS, although, like bisANS, ANS possesses a large number of lower affinity binding sites. On the basis of these results, we propose that the unique structure of bisANS, which in absence of GTP can bind tubulin as a bifunctional ligand (through its two ANS moieties), is responsible for the structural changes of tubulin. 相似文献
22.
We studied the equilibrium binding of two hydrophobic fluorescent dyes, ANS and bisANS, to four members of a family of intracellular lipid-binding proteins: IFABP, CRABP I, CRABP II, and ILBP. The spectral and binding parameters for the probes bound to the proteins were determined. Typically, there was a single binding site on each protein for the ligands. However, IFABP cooperatively bound a second bisANS molecule in the binding pocket. Comparative analysis of affinities and spectral characteristics for the two probes allowed us to examine the contributions of electrostatic and hydrophobic interactions to the binding process, and to address some aspects of the internal structure of the studied proteins. 相似文献
23.
Farruggia B Nerli B Picó G 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,798(1):25-33
The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase. 相似文献
24.
Evidence showing an intermolecular interaction between KChIP proteins and Taiwan cobra cardiotoxins 总被引:2,自引:0,他引:2
Lin YL Lin SR Wu TT Chang LS 《Biochemical and biophysical research communications》2004,319(3):720-724
Direct protein-protein interaction between Taiwan cobra cardiotoxin3 (CTX3) and potassium channel-interacting proteins (KChIPs) was investigated in the present study. It was found that KChIPs bound with CTX3, in which KChIP and CTX3 formed a 1:1 complex as evidenced by the results of chemical cross-linking. Pull-down assay revealed that the intact EF-hands 3 and 4 of KChIP1 were critical for CTX3-binding. Likewise, removal of EF-hands 3 and 4 distorted the ability of KChIP1 to bind with Kv4.2 N-terminal fragment (KvN) as well as fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS). In contrast to the interaction between KChIP1 and KvN, the binding of CTX3 to KChIP1 showed a Ca(2+)-independent manner. Fluorescence measurement revealed that CTX3 affected the binding of ANS to Ca(2+)-bound KChIP1, but not Ca(2+)-free KChIP1. Alternatively, KChIP1 simultaneously bound with KvN and CTX3, and the interaction between KChIP1 and KvN was enhanced by CTX3. In terms of the fact that KChIPs regulate the electrophysiological properties of Kv K(+) channel, the potentiality of CTX for this biomedical application could be considered. 相似文献
25.
TRAP (trp RNA-binding attenuation protein) is an 11 subunit RNA-binding protein that regulates expression of genes involved in tryptophan metabolism (trp) in Bacillus subtilis in response to changes in intracellular tryptophan concentration. When activated by binding up to 11 tryptophan residues, TRAP binds to the mRNAs of several trp genes and down-regulates their expression. Recently, a TRAP mutant was found that binds RNA in the absence of tryptophan. In this mutant protein, Thr30, which is part of the tryptophan-binding site, is replaced with Val (T30V). We have compared the RNA-binding properties of T30V and wild-type (WT) TRAP, as well as of a series of hetero-11-mers containing mixtures of WT and T30V TRAP subunits. The most significant difference between the interaction of T30V and WT TRAP with RNA is that the affinity of T30V TRAP is more dependent on ionic strength. Analysis of the hetero-11-mers allowed us to examine how subunits interact within an 11-mer with regard to binding to tryptophan or RNA. Our data suggest that individual subunits retain properties similar to those observed when they are in homo-11-mers and that individual G/UAG triplets within the RNA can bind to TRAP differently. 相似文献
26.
Chou CY Lin YL Huang YC Sheu SY Lin TH Tsay HJ Chang GG Shiao MS 《Biophysical journal》2005,88(1):455-466
Human apolipoprotein E (apoE) is a 299-amino-acid protein with a molecular weight of 34 kDa. The difference between the apoE3 and apoE4 isoforms is a single residue substitution involving a Cys-Arg replacement at residue 112. ApoE4 is positively associated with atherosclerosis and late-onset and sporadic Alzheimer's disease (AD). ApoE4 and its C-terminal truncated fragments have been found in the senile plaques and neurofibrillary tangles in the brain of AD patients. However, detail structural information regarding isoform and domain interaction remains poorly understood. We prepared full-length, N-, and C-terminal truncated apoE3 and apoE4 proteins and studied their structural variation. Sedimentation velocity and continuous size distribution analysis using analytical ultracentrifugation revealed apoE3(72-299) as consisting of a major species with a sedimentation coefficient of 5.9. ApoE4(72-299) showed a wider and more complicated species distribution. Both apoE3 and E4 N-terminal domain (1-191) existed with monomers as the major component together with some tetramer. The oligomerization and aggregation of apoE protein increased when the C-terminal domain (192-271) was incorporated. The structural influence of the C-terminal domain on apoE is to assist self-association with no significant isoform preference. Circular dichroism and fluorescence studies demonstrated that apoE4(72-299) possessed a more alpha-helical structure with more hydrophobic residue exposure. The structural variation of the N-terminal truncated apoE3 and apoE4 protein provides useful information that helps to explain the greater aggregation of the apoE4 isoform and thus has implication for the involvement of apoE4 in AD. 相似文献
27.
Jiahn-Shing Lee Jiahn-Haur Liao Shih-Hsiung Wu Shyh-Horng Chiou 《Journal of Protein Chemistry》1997,16(4):283-289
-Crystallin, a major protein of the eye lens, is known to have chaperone activity in preventing heat-induced aggregation of enzymes and other crystallins. In this study, we investigate the ability of -crystallin to inhibit UV-light-induced aggregation of other lens proteins and the effect of exposure of -crystallin to UV irradiation on its chaperone activity. The chaperone activities of -crystallin preincubated at different temperatures were found to be different and could be correlated with its change in quaternary structure as determined by the fluorescence probe ANS (8-anilo-1-naphthalene sulfonate). -Crystallin can inhibit the aggregation of -crystallin from UV irradiation at room temperature, and the preheated -crystallins provide more protection than the native one. Upon irradiation by UV light, -crystallin gradually lost its ability to protect -crystallin against thermal aggregation. The loss of the chaperone efficacy of -crystallin to protect other lens proteins may shed light on human cataract formation induced by long-term exposure to UV irradiation. 相似文献
28.
Two steps in the transition between the native and acid states of bovine α-lactalbumin detected by circular polarization of luminescence: Evidence for a premolten globule state? 下载免费PDF全文
E. E. Gussakovsky E. Haas 《Protein science : a publication of the Protein Society》1995,4(11):2319-2326
A few studies indirectly support the existence of an intermediate in the transition of Ca(2+)-saturated bovine alpha-lactalbumin (alpha-LA) from the native (N) to the acidic (A) state, known as the molten globule state. However, direct experimental evidence for the appearance of this intermediate has not been obtained. The signal of circular polarization of luminescence (CPL) is sensitive to fine conformational transitions because of its susceptibility to changes in the environmental asymmetry of fluorescent chromophores in their excited electronic states. In the present study, CPL measurements were applied using the intrinsic tryptophan fluorescence of alpha-LA as well as the fluorescence of 8-anilino-1-naphthalenesulfonic acid (ANS) bound to alpha-LA. CPL of tryptophan and ANS was measured in the pH range of 2.5-6 in order to find direct experimental evidence for the proposed intermediate. CPL (characterized by the emission anisotropy factor, g(em)) depends on the asymmetry of the protein molecular structure in the environment of the tryptophan and the ANS chromophores in the excited electronic state. The pH dependence of both the gab, absorption anisotropy factor determined by CD, and the ANS steady state fluorescence, showed a single transition at pH 3-3.7 as already reported elsewhere. This transition was interpreted as being a result of a change of the alpha-LA tertiary structure, which resulted in a loss of asymmetry of the environment of both the tryptophan residues and the ANS hydrophobic binding sites. The pH dependence of the tryptophan and ANS g(em) showed an additional conformational transition at pH 4-5, which coincided with the pKa of Ca2+ dissociation (pKa 5), as predicted by Permyakov et al. (1981, Biochem Biophys Res Commun 100:191-197). The titration curve showed that there is a pH range between 3.7 and 4.1 in which alpha-LA exists in an intermediate state between the N- and A-state. We suggest that the intermediate is the premolten globule state characterized by a reduced Ca2+ binding to the alpha-LA, native-like tertiary structure, and reduced asymmetric fluctuation of the tertiary structure on the nanosecond time scale. This intermediate resembles the "critical activated state" theoretically deduced by Kuwajima et al. (1989, J Mol Biol 206:547-561). The present study demonstrates the power of CPL measurements for the investigation of folding/unfolding transitions in proteins. 相似文献
29.
Yury V. Matsuka Deborah A. Dilts Susan Hoiseth Rasappa Arumugham 《The protein journal》1998,17(7):719-728
An outer membrane PIA protein fromNeisseria gonorrhoeae strain FA19 was expressed inEscherichia coli and refoldedin vitro in the presence of zwitterionic detergent. Its proper folding and subunit organization was confirmed by comparison with the
native counterpart. The unfolding of PIA has been investigated using fluorescence spectroscopy and analytical size-exclusion
chromatography methods. Analysis of the denaturation pathway of the PIA revealed that it forms an unusually labile quaternary
structure. In the presence of 1 M guanidinium chloride (GdmCl) or upon heating up to 50°C, dissociation of the PIA oligomer
was observed resulting in the formation of folded monomeric intermediates. Unfolding of monomers occurs at 80°C or in the
presence of 4.3 M GdmCl, indicating high intrinsic stability toward both GdmCl and elevated temperatures. Both oligomeric
and monomeric forms of PIA exhibited affinity to the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and bind
withK
d=80 and 130 μM, respectively. Denaturation of the PIA completely abolished affinity to ANS, suggesting that hydrophobicity
is a property of the folded state of the porin. 相似文献
30.