[Show abstract][Hide abstract] ABSTRACT: The mineralocorticoid receptor (MR) binds the steroid hormones aldosterone and cortisol and has an important physiological role in the control of salt homeostasis. Regions of the protein important for gene regulation have been mapped to the amino-terminal domain (NTD) and termed activation function (AF)1a, AF1b, and middle domain (MD). In the present study, we used a combination of biophysical and biochemical techniques to investigate the folding and function of the MR-NTD transactivation functions. We demonstrate that MR-AF1a and MR-MD have relatively little stable secondary structure but have the propensity to form α-helical conformation. Induced folding of the MR-MD enhanced protein-protein binding with a number of coregulatory proteins, including the coactivator cAMP response element-binding protein-binding protein and the corepressors SMRT and RIP140. By contrast, the MR-AF1b domain appeared to have a more stable conformation consisting predominantly of β-secondary structure. Furthermore, MR-AF1b specifically interacted with the TATA-binding protein, via an LxxLL-like motif, in the absence of induced folding. Together, these data suggest that the MR-NTD contains a complex transactivation system made up of distinct structural and functional domains. The results are discussed in the context of the induced folding paradigm for steroid receptor NTDs.
[Show abstract][Hide abstract] ABSTRACT: Caged versions of the most common mitochondrial uncouplers (proton translocators) have been prepared that sense the reactive oxygen species (ROS) hydrogen peroxide to release the uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) from caged states with second order rate constants of 10 (+/-0.8) M(-1) s(-1) and 64.8 (+/-0.6) M(-1) s(-1), respectively. The trigger mechanism involves conversion of an arylboronate into a phenol followed by fragmentation. Hydrogen peroxide-activated uncouplers may be useful for studying the biological process of ageing.
[Show abstract][Hide abstract] ABSTRACT: Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature midpoint (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (DeltaHeq) to its ability to function over various temperature ranges. In the present study, we show that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independent of enzyme reaction or structure, based at or near the active site, by which enzymes lose activity as temperature rises, as opposed to denaturation which is global. Results show that activity losses below Teq may lead to significant errors in the determination of DeltaG*cat made on the basis of the two-state ('Classical') model, and the measured kcat will then not be a true indication of an enzyme's catalytic power. Overall, the results provide a molecular rationale for observations that the active site tends to be more flexible than the enzyme as a whole, and that activity losses precede denaturation, and provide a general explanation in molecular terms for the effect of temperature on enzyme activity.
[Show abstract][Hide abstract] ABSTRACT: In order to increase understanding of the basis of the stability of the native conformational state of porcine pepsin A, a strategy based on induction and monitoring of protein denaturation was developed. Structural perturbation was achieved by adding acetonitrile (MeCN) to the protein-solvent system. MeCN was found to induce non-coincident disruption of the secondary and tertiary structural features of pepsin A. It is proposed that gross unfolding is prompted by disruption of the protein hydration pattern induced by the organic co-solvent. It should be noted that the functional properties and thermal stability of the protein were already impaired before the onset of global unfolding. Low and intermediate contents of MeCN in the protein-solvent system affected the sharpness of the thermal transition and the degree of residual structure of the heat-denatured state. The importance of hydration to the conformational stability of pepsin A in its biologically active state is discussed.
International journal of biological macromolecules 06/2009; 45(3):213-20. · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm(-1) to 1632 cm(-1), indicating a shift from alpha-helical structure to beta-sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to beta-sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.
[Show abstract][Hide abstract] ABSTRACT: A comparison was made of the cytotoxic activity and secondary structural features of four recombinant forms of adenylate cyclase toxin (CyaA). These forms were fully functional CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*. At a toxin concentration>1 microg/ml, CyaA* was as cytotoxic towards J774.2 cells as CyaA and mediated cell killing at a faster rate than CyaA. At concentrations<0.5 microg/ml, CyaA* was less cytotoxic than CyaA and, at <0.1 microg/ml of CyaA*, no activity was detected. CyaA, but not CyaA*, was able to induce caspase 3/7 activity, a measure of apoptosis. ProCyaA and proCyaA* had no detectable cytotoxic or apoptotic activity. CyaA caused 50% inhibition of the zymosan-stimulated oxidative burst at 0.003 microg/ml, whereas a approximately 500-fold greater toxin concentration of CyaA* or proCyaA was needed for 50% inhibition. ProCyaA* was inactive. CyaA is a calcium-binding protein and far UV circular dichroism (CD), near UV CD and fluorescence spectra analyses showed that all the forms of CyaA had similar overall structures at different calcium concentrations up to 5.0 mM. At 7.5 mM CaCl2, the far UV spectrum of CyaA altered significantly, indicating a change in secondary structure associated with high beta-sheet content or a beta-aggregated state, whereas the spectrum of CyaA* showed only a slight alteration at this calcium concentration. Near UV CD and fluorescence studies were consistent with a rearrangement of secondary structural elements in the presence of CaCl2 for all CyaA forms. There was a marked dependence on protein concentration of the far UV spectra of these CyaA forms, implying an interaction between individual molecules at higher protein concentrations.
[Show abstract][Hide abstract] ABSTRACT: Poly-amino acid repeats, especially long stretches of glutamine (Q), are common features of transcription factors and cell-signalling proteins and are prone to expansion, resulting in neurodegenerative diseases. The amino-terminal domain of the androgen receptor (AR-NTD) has a poly-Q repeat between 9 and 36 residues, which when it expands above 40 residues results in spinal bulbar muscular atrophy. We have used spectroscopy and biochemical analysis to investigate the structural consequences of an expanded repeat (Q45) or removal of the repeat (DeltaQ) on the folding of the AR-NTD. Circular dichroism spectroscopy revealed that in aqueous solution, the AR-NTD has a relatively limited amount of stable secondary structure. Expansion of the poly-Q repeat resulted in a modest increase in alpha-helix structure, while deletion of the repeat resulted in a small loss of alpha-helix structure. These effects were more pronounced in the presence of the structure-promoting solvent trifluoroethanol or the natural osmolyte trimethylamine N-oxide. Fluorescence spectroscopy showed that the microenvironments of four tryptophan residues were also altered after the deletion of the Q stretch. Other structural changes were observed for the AR-NTDQ45 polypeptide after limited proteolysis; in addition, this polypeptide not only showed enhanced binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonic acid but was more sensitive to urea-induced unfolding. Taken together, these findings support the view that the presence and length of the poly-Q repeat modulate the folding and structure of the AR-NTD.
Journal of Molecular Endocrinology 10/2008; 41(5):301-14. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The enzymatic activity of phenoloxidase is assayed routinely in the presence of SDS. Similar assay conditions elicit phenoloxidase activity in another type 3 copper protein, namely hemocyanin, which normally functions as an oxygen carrier. The nature of the conformational changes induced in type 3 copper proteins by the denaturant SDS is unknown. This comparative study demonstrates that arthropod hemocyanins can be converted from being an oxygen carrier to a form which exhibits phenoloxidase activity by incubation with SDS, with accompanying changes in secondary and tertiary structure. Structural characterisation, using various biophysical methods, suggests that the micellar form of SDS is required to induce optimal conformational transitions in the protein which may result in opening a channel to the di-copper centre allowing bulky phenolic substrates access to the catalytic site.
Biochimica et Biophysica Acta 12/2007; 1774(11):1380-94. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circular dichroism (CD) is a powerful technique for studying the structures of proteins in solution, as well as structural changes that may occur when proteins bind to ligands. Changes in CD signals accompanying complex formation can be used to determine the strength of the interaction and to provide information about the nature and extent of the conformational changes involved. This unit outlines the type of information available from CD studies and describes how such experiments should be carried out to ensure that reliable information is obtained.
Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 01/2007; Chapter 20:Unit 20.10.
[Show abstract][Hide abstract] ABSTRACT: Immobilised enzymes are widely used in industry, but the reasons for loss of activity of such biocatalysts are usually not known. We have used circular dichroism (CD) to investigate the structure of one such system, i.e., subtilisin Carlsberg (SC) immobilised on silica gel particles (60 microm). A number of technical problems have to be overcome in order to obtain appropriate data from which conclusions can be drawn. A rotating cell holder has been developed to avoid sedimentation of the silica particles during the collection of spectra. By moving the cell holder as close as possible to the detector window, the effects of differential scattering can be minimised. However, the effects of absorption flattening limit the extent to which reliable quantitative information on secondary structure content can be obtained from far UV CD studies. We have used an empirical approach based on absorbance units derived from the high-tension voltage to correct for absorption flattening effects. After applying the correction there was satisfactory agreement with the solution spectra. Comparison of the fresh and used (inactive) SC-silica gel spectra in organic media reveals substantial change in the secondary structure. Additional evidence for loss of native conformation is provided by the significant decrease in the near UV CD spectrum. These results for the first time clearly demonstrate the origin of enzyme instability in the immobilised state.
Biochimica et Biophysica Acta 07/2006; 1764(6):1119-25. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) is upregulated following treatment of the mouse mammary epithelial cell line HC11 with lactogenic hormones (dexamethasone, insulin, and prolactin-DIP). In addition, we have also shown that IGFBP-5 is upregulated in mammary epithelial cells in vivo during involution of the rodent mammary gland. We have, therefore, postulated that there may be a dual regulation of IGFBP-5 expression during the temporally separated processes of differentiation and apoptosis of mammary epithelial cells. To test this hypothesis further, we have used a phenotypically differentiated model, which comprises primary cultures of mouse mammary epithelial cells grown on a layer of EHS (Engelbreth-Holm-Swarm) extracellular matrix. We show that lactogenic hormone treatment (hydrocortisone, insulin, and prolactin-HIP) of these cultures induces the upregulation of IGFBP-5 thus replicating the results obtained with the HC11 cell line. In addition, following the induction of apoptosis in primary cultures of mammary epithelial cells by treatment with TGFbeta-3, IGFBP-5 expression is also upregulated. In parallel with this upregulation of IGFBP-5, there is also an increase in the levels of cleaved caspase-3, a well-characterized marker of cellular apoptosis. These findings confirm previous in vivo work demonstrating an increase in IGFBP-5 expression during involution of the mouse mammary gland. When HC11 cells are cultured under serum-free conditions (a well-characterized apoptotic insult in cell culture), there is also an increase in cleaved caspase-3 levels. Unexpectedly, in the presence of TGFbeta-3, caspase-3 levels are attenuated. In the presence of DIP, caspase-3 levels are also decreased in HC11 cells. As described previously, TGFbeta-3 inhibits beta-casein synthesis in HC11 cells. In the HC11 cell line (in contrast to primary cultures of mammary epithelial cells), there is no evidence for TGFbeta-3 induction of IGFBP-5 under either serum-free or DIP-supplemented conditions. We believe our data with primary cultures of mammary epithelial cells support the hypothesis of dual regulation of IGFBP-5 expression during both differentiation and apoptosis in the mammary gland and emphasizes the importance of using appropriate cell culture models to investigate such phenomena in this tissue. We discuss the possible implications of our observations in relation to the physiological processes of pregnancy, lactation, and involution in the mammary gland and the associated changes in mammary epithelial cell function.
Journal of Cellular Physiology 06/2006; 207(2):471-9. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interaction of immunoglobulin E (IgE) with its cellular receptor FcepsilonRIalpha is a central regulator of allergy. Structural studies have identified the third domain (Cepsilon3) of the constant region of epsilon heavy chain as the receptor binding region. The isolated Cepsilon3 domain is a "molten globule" that becomes structured upon binding of the FcepsilonRIalpha ligand. In this study, fluorescence and nuclear magnetic resonance spectroscopies are used to characterise the role of soluble FcepsilonRIalpha in the folding of the monomeric Cepsilon3 domain of IgE. Soluble FcepsilonRIalpha is shown to display characteristic properties of a catalyst for the folding of Cepsilon3, with the rate of Cepsilon3 folding being dependent on the concentration of the receptor.
[Show abstract][Hide abstract] ABSTRACT: The following extracellular enzymes have been readily detected in the culture filtrates from Aeromonas salmonicida: amylase, phospholipase, lysophospholipase and ribonuclease. Amylase and phospholipase have been partially characterized. Evidence suggests that glycogen may be the natural substrate for amylase, and that the role of the enzyme in natural infection is to digest glycogen present in fish muscle. The secretion of amylase activity is suppressed by the addition of glucose to the growth medium. The amounts of amylase, phospholipase and protease that can be detected in culture filtrates decreases with increase in the growth temperature from 25 to 32°C. This marked decrease in secretion of hydrolytic enzymes occurs although the initial growth rates at 25 and 32°C are similar. Free and membrane associated ribosomes have been isolated from cultures grown at 25 and 32°C. At 32°C there is a smaller proportion of membrane-associated ribosomes and this is consistent with the hypothesis that extracellular enzymes from Aeromonas salmonicida are secreted on membrane-bound polysomes.
Journal of Fish Diseases 04/2006; 13(6):463 - 474. · 1.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The specificity of the major protease secreted by Aeromonas salmonicida has been explored using a number of proteins and p-nitroanilides as substrates. The 70kDa protease was found to hydrolyse two p-nitroanilides which have been reported to be specific substrates for thrombin. Kinetic parameters (kcat, and Km) were compared for the 70kDa protease and for thrombin as were the effects of a number of inhibitors. The 70kDa protease is able to degrade proteins which have a relatively open structure, for example, caseins or denatured bovine scrum albumin, to small fragments mostly of Mr<2500. However, proteins with a more compact structure are more resistant to the protease. It was concluded that the 70kDa protease shows some of the specificity features of thrombin, although it is less discriminating in its choice of both low and high Mr substrates than thrombin. In preliminary experiments, the 70 kDa protease was found, like thrombin, to decrease the clotting time of rainbow trout blood. The possible physiological significance of these results is discussed.
Journal of Fish Diseases 04/2006; 13(1):49 - 58. · 1.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The proteases secreted by four strains (MT004, 1102, 480 and 480P-) of Aeromonas salmonicida grown in liquid culture have been studied. Strains MT004, 1102 and 480 all show a similar pattern with two types of proteases produced; one of molecular weight 70 000 which is active against casein and gelatin and one (or more) of lower molecular weight (about 20 000) which is (are) active against gelatin but not casein. Strain 480P- produces only the latter type of protease(s). The protease of molecular weight 70 000 is classified as a serine-type protease, but further characterization of the features of its active site has not yet proved possible. The results are discussed in terms of the previously published but often contradictory data on the proteases of A. salmonicida.
Journal of Fish Diseases 04/2006; 12(3):223 - 232. · 1.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structures of the type II dehydroquinase (DHQase) from Helicobacter pylori in complex with three competitive inhibitors have been determined. The inhibitors are the substrate analogue 2,3-anhydroquinate (FA1), citrate, and an oxoxanthene sulfonamide derivative (AH9095). Despite the very different chemical nature of the inhibitors, in each case the primary point of interaction with the enzyme is via the residues that bind the C1 functionalities of the substrate, 3-dehydroquinate, i.e., N76, H102, I103, and H104. The DHQase/AH9095 complex crystal structure shows that sulfonamides can form a scaffold for nonsubstrate-like inhibitors and identifies a large conserved hydrophobic patch at the entrance to the active site as a locus that can be exploited in the development of new ligands.
Journal of Medicinal Chemistry 03/2006; 49(4):1282-90. · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Encapsulation of proteins in poly(lactide-co-glycolide) microspheres via emulsion is known to cause insoluble protein aggregates. Following protein emulsification and encapsulation in PLGA microspheres, we used circular dichroism to show that the recoverable soluble protein fraction also suffers subtle conformational changes. For a panel of proteins selected on the basis of molecular size and structural class, conformational stability measured by chemical denaturation was not indicative of stability during emulsion-encapsulation. Partial loss of structure was observed for alpha-helical proteins released from freeze-dried microspheres in aqueous buffer, with dramatic loss of structure for a beta-sandwich protein. The addition of sucrose (a lyoprotectant) did not prevent the loss of protein conformation upon encapsulation. Therefore, the conformational changes seen for the released soluble protein fraction originates during emulsification rather than microsphere freeze-drying. Analysis of the burst release for all proteins in buffer containing denaturant or surfactant showed that the degree of protein solubilisation was the dominant factor in determining the initial rate and extent of release. Our data for protein release into increasing concentrations of denaturing buffer suggest that the emulsion-denatured protein fraction remains insoluble; this fraction may represent the protein loss encountered upon comparison of protein encapsulated versus protein released.
Journal of Controlled Release 01/2006; 110(1):34-48. · 7.63 Impact Factor