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(1/531) Opposite behavior of two isozymes when refolding in the presence of non-ionic detergents.

GroEL has a greater affinity for the mitochondrial isozyme (mAAT) of aspartate aminotransferase than for its cytosolic counterpart (cAAT) (Mattingly JR Jr, Iriarte A, Martinez-Carrion M, 1995, J Biol Chem 270:1138-1148), two proteins that share a high degree of sequence similarity and an almost identical spatial structure. The effect of detergents on the refolding of these large, dimeric isozymes parallels this difference in behavior. The presence of non-ionic detergents such as Triton X-100 or lubrol at concentrations above their critical micelle concentration (CMC) interferes with reactivation of mAAT unfolded in guanidinium chloride but increases the yield of cAAT refolding at low temperatures. The inhibitory effect of detergents on the reactivation of mAAT decreases progressively as the addition of detergents is delayed after starting the refolding reaction. The rate of disappearance of the species with affinity for binding detergents coincides with the slowest of the two rate-limiting steps detected in the refolding pathway of mAAT. Limited proteolysis studies indicate that the overall structure of the detergent-bound mAAT resembles that of the protein in a complex with GroEL. The mAAT folding intermediates trapped in the presence of detergents can resume reactivation either upon dilution of the detergent below its CMC or by adding beta-cyclodextrin. Thus, isolation of otherwise transient productive folding intermediates for further characterization is possible through the use of detergents.  (+info)

(2/531) Effect of the hemolytic lectin CEL-III from Holothuroidea Cucumaria echinata on the ANS fluorescence responses in sensitive MDCK and resistant CHO cells.

The addition of CEL-III to sensitive MDCK cells preincubated with 8-anilino-1-naphthalenesulfonate (ANS) caused an increase in the fluorescence intensity of the probe. The increase in the ANS fluorescence caused by CEL-III was Ca2+-dependent and strongly inhibited by 0.1 M lactose, indicating that Ca2+-dependent binding of CEL-III to specific carbohydrate receptors on the plasma membrane is responsible for this phenomenon. In contrast, no significant effect of CEL-III on the ANS fluorescence was observed in CHO cells, which are highly resistant to CEL-III cytotoxicity. In MDCK cells, energy transfer from tryptophan residues to bound ANS molecules was observed in the presence of CEL-III, but not in CHO cells. Furthermore, the amount of ANS bound to MDCK cells increased as the concentration of CEL-III increased. Therefore, a simple interpretation is that the CEL-III-induced increase in ANS fluorescence is attributable to an increase of the hydrophobic region in the plasma membrane where ANS could bind. Immunoblotting analysis of proteins from cells treated with CEL-III indicated that CEL-III oligomers were irreversibly bound to the cells, and the amount of oligomer bound to MDCK cells was much greater than that bound to CHO cells under any conditions tested. The oligomerization may be accompanied by an enhancement of the hydrophobicity of CEL-III molecules, which in turn provides new ANS-binding sites. The difference in susceptibility of MDCK and CHO cells to CEL-III cytotoxicity may be due to a difference in oligomerization of bound CEL-III.  (+info)

(3/531) The Mycobacterium tuberculosis small heat shock protein Hsp16.3 exposes hydrophobic surfaces at mild conditions: conformational flexibility and molecular chaperone activity.

Hsp16.3, the alpha-crystallin-related small heat shock protein of Mycobacterium tuberculosis that is maximally expressed during the stationary phase and is a major membrane protein, has been reported to form specific trimer-of-trimers structure and to act as an effective molecular chaperone (Chang Z et al., 1996, J. Biol Chem 271:7218-7223). However, little is known about its action mechanism. In this study, Hsp16.3 conformational intermediates with dramatically increased chaperone activities were detected after treatment with very low concentrations of guanidine hydrochloride (0.05 M), urea (0.3 M), or mild heating (30 degrees C). The intermediates showed a significant increase in their capacity to bind the hydrophobic probe 1-anilino-8-naphthalene sulfonate (ANS), indicating an increased exposure of hydrophobic surfaces. Interestingly, the greatest chaperone activities of Hsp16.3 were observed in the presence of 0.3 M guanidine HCl or when heated to 35 degrees C. CD spectroscopy studies revealed no significant changes in protein secondary and tertiary structures at these mild treatments. Our in vitro studies also indicate that long-time-heated Hsp16.3, heated even to temperatures as high as 85 degrees C, has almost the same, if not a slightly greater, chaperone activities as the native protein when cooled to room temperature and its secondary structures also almost recovered. Together, these results suggest that Hsp16.3 modulates its chaperone activity by exposing hydrophobic surfaces and that the protein structure is highly stable and flexible, thus highly adapted for its function.  (+info)

(4/531) Fluorescence measurements detect changes in scallop myosin regulatory domain.

Ca2+-induced conformational changes of scallop myosin regulatory domain (RD) were studied using intrinsic fluorescence. Both the intensity and anisotropy of tryptophan fluorescence decreased significantly upon removal of Ca2+. By making a mutant RD we found that the Ca2+-induced fluorescence change is due mainly to Trp21 of the essential light chain which is located at the unusual Ca2+-binding EF-hand motif of the first domain. This result suggests that Trp21 is in a less hydrophobic and more flexible environment in the Ca2+-free state, supporting a model for regulation based on the 2 A resolution structure of scallop RD with bound Ca2+ [Houdusse A. and Cohen C. (1996) Structure 4, 21-32]. Binding of the fluorescent probe, 8-anilinonaphthalene-1-sulphonate (ANS) to the RD senses the dissociation of the regulatory light chain (RLC) in the presence of EDTA, by energy transfer from a tryptophan cluster (Trp818, 824, 826, 827) on the heavy chain (HC). We identified a hydrophobic pentapeptide (Leu836-Ala840) at the head-rod junction which is required for the effective energy transfer and conceivably is part of the ANS-binding site. Extension of the HC component of RD towards the rod region results in a larger ANS response, presumably indicating changes in HC-RLC interactions, which might be crucial for the regulatory function of scallop myosin.  (+info)

(5/531) Conformational intermediates and fusion activity of influenza virus hemagglutinin.

Three strains of influenza virus (H1, H2, and H3) exhibited similar characteristics in the ability of their hemagglutinin (HA) to induce membrane fusion, but the HAs differed in their susceptibility to inactivation. The extent of inactivation depended on the pH of preincubation and was lowest for A/Japan (H2 subtype), in agreement with previous studies (A. Puri, F. Booy, R. W. Doms, J. M. White, and R. Blumenthal, J. Virol. 64:3824-3832, 1990). While significant inactivation of X31 (H3 subtype) was observed at 37 degrees C at pH values corresponding to the maximum of fusion (about pH 5.0), no inactivation was seen at preincubation pH values 0.2 to 0.4 pH units higher. Surprisingly, low-pH preincubation under those conditions enhanced the fusion rates and extents of A/Japan as well as those of X31. For A/PR 8/34 (H1 subtype), neither a shift of the pH (to >5.0) nor a decrease of the temperature to 20 degrees C was sufficient to prevent inactivation. We provide evidence that the activated HA is a conformational intermediate distinct from the native structure and from the final structure associated with the conformational change of HA, which is implicated by the high-resolution structure of the soluble trimeric fragment TBHA2 (P. A. Bullough, F. M. Hughson, J. J. Skehel, and D. C. Wiley, Nature 371:37-43, 1994).  (+info)

(6/531) Role of regulatory exosite I in binding of thrombin to human factor V, factor Va, factor Va subunits, and activation fragments.

The blood coagulation proteinase, thrombin, converts factor V into factor Va through a multistep activation pathway that is regulated by interactions with thrombin exosites. Thrombin exosite interactions with human factor V and its activation products were quantitatively characterized in equilibrium binding studies based on fluorescence changes of thrombin covalently labeled with 2-anilinonaphthalene-6-sulfonic acid (ANS) linked to the catalytic site histidine residue by Nalpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl ([ANS]FPR-thrombin). Exosite I was shown to play a predominant role in the binding of factor V and factor Va from the effect of the exosite I-specific ligand, hirudin54-65, on the interactions. Factor V and factor Va bound to exosite I of [ANS]FPR-thrombin with similar dissociation constants of 3.4 +/- 1.3 and 1.1 +/- 0.4 microM and fluorescence enhancements of 182 +/- 41 and 127 +/- 17%, respectively. Native thrombin and labeled thrombin bound with similar affinity to factor Va. Among factor V activation products, the factor Va heavy chain was shown to contain the site of exosite I binding, whereas exosite I-independent, lower affinity interactions were observed for activation fragments E and C1, and no detectable binding was observed for the factor Va light chain. The results support the conclusion that the factor V activation pathway is initiated by exosite I-mediated binding of thrombin to a site in the heavy chain region of factor V that facilitates the initial cleavage at Arg709 to generate the heavy chain of factor Va. The results further suggest that binding of thrombin through exosite I to factor V activation intermediates may regulate their conversion to factor Va and that similar binding of thrombin to the factor Va produced may reflect a mode of interaction involved in the regulation of prothrombin activation.  (+info)

(7/531) The membrane topology of proton-pumping Escherichia coli transhydrogenase determined by cysteine labeling.

The membrane topology of proton-pumping nicotinamide-nucleotide transhydrogenase from Escherichia coli was determined by site-specific chemical labeling. A His-tagged cysteine-free transhydrogenase was used to introduce unique cysteines in positions corresponding to potential membrane loops. The cysteines were reacted with fluorescent reagents, fluorescein 5-maleimide or 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid, in both intact cells and inside-out vesicles. Labeled transhydrogenase was purified with a small-scale procedure using a metal affinity resin, and the amount of labeling was measured as fluorescence on UV-illuminated acrylamide gels. The difference in labeling between intact cells and inside-out vesicles was used to discriminate between a periplasmic and a cytosolic location of the residues. The membrane region was found to be composed of 13 helices (four in the alpha-subunit and nine in the beta-subunit), with the C terminus of the alpha-subunit and the N terminus of the beta-subunit facing the cytosolic and periplasmic sides, respectively. These results differ from previous models with regard to both number of helices and the relative location and orientation of certain helices. This study constitutes the first in which all transmembrane segments of transhydrogenase have been experimentally determined and provides an explanation for the different topologies of the mitochondrial and E. coli transhydrogenases.  (+info)

(8/531) Evidence of heterogeneous 1-anilinonaphthalene-8-sulfonate binding to beta-lactoglobulin from fluorescence spectroscopy.

Steady-state and dynamic fluorescence titrations show that: (a) the complex between beta-lactoglobulin (BLG) and 1-anilinonaphthalene-8-sulfonate (ANS) displays a heterogeneous equilibrium with large changes in the binding strength vs. pH and ion concentration; and (b) the fluorescence response of bound ANS reveals two separate lifetimes that suggest two different sites (or binding modes). While steady-state fluorescence titrations yield effective values of the binding constant and of the bound ANS quantum efficiency, it is shown that, by combining steady-state fluorescence and lifetime decay of ANS, it is possible to give quantitative estimates of the association constants for each site. When heading from the acid (pH approximately 2) to the native state (pH approximately 6) the main result is a very large reduction of the effective binding constant. This and the results of titrations vs. ionic strength suggest that electrostatic interactions are a major contribution to ANS binding to BLG.  (+info)