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1.
The small-angle X-ray scattering technique was used to characterize the overall structural change as well as the state of aggregation of troponin C upon binding various amount of Ca2+ ions: in the Ca2+-free state and at pCa 6.5 and 4.0. Under these conditions, the forward scattering intensities of troponin C are not much different from each other: i.e., they coincide within 4%. From these intensities, the Ca2+-facilitated dimerization of troponin C was not verified, and no appreciable aggregation of troponin C molecules was detected below pCa 4.0. Thus, the small-angle X-ray scattering profiles from troponin C solutions were analyzed assuming a monomeric molecule. The radii of gyration of troponin C were 27.8 +/- 0.3 A, 23.8 +/- 0.2 A, and 22.6 +/- 0.1 A for the Ca2+-free state and at pCa 6.5 and 4.0, respectively. The maximum dimension of the molecule decreases from 111 to 98 A with increasing Ca2+ concentration. These results indicate that the troponin C molecule shrinks remarkably as Ca2+ ions bind to the high affinity sites of the molecule. Ca2+ binding to the low affinity sites, on the other hand, leads to a less pronounced change. Following the interpretation of scattering from the dumbbell-shaped structure (Fujisawa, T., Ueki, T., Inoko, Y., & Kataoka, M. [1987] J. Appl. Cryst. 20, 349-355), the two domains of the molecule move closer to each other. The distance between the centers of the two domains decreases from 46 to 35 A.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
Ca2+-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering
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Priddy TS MacDonald BA Heller WT Nadeau OW Trewhella J Carlson GM 《Protein science : a publication of the Protein Society》2005,14(4):1039-1048
Phosphorylase kinase (PhK), a 1.3-MDa (alphabetagammadelta)(4) hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (alphabetagammadelta)(2) octameric lobes that are oriented back-to-back with overall D(2) symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca(2+) by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca(2+) in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca(2+)-activated PhK. Although the overall dimensions of the complex were not affected by Ca(2+), the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca(2+)-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca(2+)-activated conformers of PhK are consistent with Ca(2+)-induced conformational changes in both the lobes and the interlobal bridges. 相似文献
3.
A small-angle X-ray scattering study on troponin C showed that two domains of the molecule move closer to each other and the molecule shrinks along its long axis upon Ca2+ binding in the absence of Mg2+ ions (Fujisawa, T., Ueki, T., & Iida S. (1988) J. Biochem. 105, 377-383). When Mg2+ ions bind to troponin-C, the radius of gyration changes from 27.8 to 24.3 A and the average radius of gyration of the two domains is estimated to be 15.1 A. These radii indicate that the distance between the centers of the two domains is 38.1 A. Such a change is analogous to the previous result for troponin C with two Ca2+ ions bound at the high-affinity sites. Thus, the structural behavior of troponin C molecule is essentially the same when Ca2+/Mg2+ ions bind to its high-affinity sites. On the other hand, the effect of Ca2+ binding to the low-affinity sites in the presence of Mg2+ ions is quite different from the previous result. The binding of Ca2+ ions causes a dimerization of troponin C molecules with an apparent constant of 511 M-1. Such a characteristic behavior, implying the occurrence of a surface property change, may be related to the physiological role of troponin C molecule in the muscle. The scattering experiments on the tryptic fragments of troponin C also had interesting and important results: the C-domain shrinks, with the radius of gyration changing from 17.0 to 14.9 A while the N-domain swells from 13.9 to 15.0 A upon Ca2+ binding. Such an opposite change is consistent with the results of circular dichroism and spectroscopic studies of the domains. 相似文献
4.
Yang W Yan J Springsteen G Deeter S Wang B 《Bioorganic & medicinal chemistry letters》2003,13(6):1019-1022
In this paper we report 8-quinolineboronic acid as a novel type of fluorescent probe for carbohydrates. This boronic acid responds to the binding of a carbohydrate with over 40-fold increases in fluorescence intensity and shows optimal fluorescence change at physiological pH in aqueous solution. 相似文献
5.
The interaction between the calcium binding and inhibitory components of troponin is central to the regulation of muscle contraction. In this work, two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance (2D-?1H,15N?-HSQC NMR) spectroscopy was used to determine the stoichiometry, affinity, and mechanisms for binding of Ca2+ and two synthetic TnI peptides [TnI1-40 (or Rp40) and TnI96-115] to the isolated C-domain of skeletal troponin C (CTnC). The Ca2+ titration revealed that 2 equiv of Ca2+ binds to sites III and IV of CTnC with strong positive cooperativity and high affinity [dissociation constant (KD) = 0.1 microM]. In this process, CTnC folds from a largely unstructured state to a compact domain capable of interacting with TnI. Titration of CTnC x 2Ca2+ with Rp40 occurs with a 1:1 stoichiometry and a KD of 2 +/- 1 microM. Titration of CTnC x 2Ca2+ with a peptide corresponding to the inhibitory region of TnI (TnI96-115) also reveals a 1:1 ratio, but weaker affinity (KD = 47 +/- 7 microM). Both Rp40- and TnI96-115-induced backbone amide chemical shift changes of CTnC x 2Ca2+ are similarly distributed along the sequence, indicating that these two regions of TnI may compete for the same binding site on CTnC x 2Ca2+. The changes induced by Rp40 are much larger, however, and define the interaction sites on TnC and regions where the flexibility of hinge and terminal residues is altered. To investigate the possibility of direct competition, TnI(96-115) was titrated into the CTnC x 2Ca(2+) x Rp40 complex, whereas Rp40 was titrated into the CTnC x 2Ca2+. TnI96-115 complex. The results show that Rp40 can displace TnI96-115 completely, while TnI96-115 has no effect on CTnC x 2Ca2+ x Rp40. Recent proposals for the mechanism of muscle regulation [Tripet, B. P., Van Eyk, J. E., and Hodges, R. S. (1997) J. Mol. Biol. 271, 728-750] suggest that the N-terminal and inhibitory regions of TnI competitively bind the structural domain of TnC. The findings presented here indicate that additional factors, such as interactions between the N-domain of TnC with the C-domain of TnI or the C-domain of TnT, are required, if the inhibitory region is going to successfully compete for the structural domain of TnC. 相似文献