scholarly journals The conformational changes of α2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes

1987 ◽  
Vol 243 (1) ◽  
pp. 47-54 ◽  
Author(s):  
L J Larsson ◽  
P Lindahl ◽  
C Hallén-Sandgren ◽  
I Björk
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Cross Linking ◽  
Bait Region ◽  
Close Proximity ◽  
Tryptophan Residues ◽  
Thioester Bond ◽  
Bond Region

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent ‘bait’ region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].

Molecular mechanism of two noncompetitive inhibitors of Na(+)-glucose cotransporter: comparison of DCCD and PCMB

1993 ◽  
Vol 264 (2) ◽  
pp. G300-G305
Author(s):  
B. E. Peerce
Keyword(s):  
Glucose Uptake ◽  
Conformational Change ◽  
Molecular Mechanism ◽  
Conformational Changes ◽  
Fluorescein Isothiocyanate ◽  
Tryptophan Fluorescence ◽  
Similar Concentration ◽  
Tryptophan Residues ◽  
Apparent Concentration ◽  
Noncompetitive Inhibitors

The effects of noncompetitive inhibitors of Na(+)-dependent glucose uptake, p-chloromercuribenzoate N,N'-dicyclohexylcarbodiimide (DCCD), on substrate-induced cotransporter conformational changes were examined using fluorescein isothiocyanate (FITC) and tryptophan fluorescence. p-chloromercuribenzoate (PCMB) inhibited both substrate-induced conformational changes with similar concentration required for 50% quenching/enhancement of tryptophan or FITC fluorescence. In contrast, DCCD inhibited the Na(+)-induced conformational change with an apparent concentration resulting in 50% inhibition (K0.5) of 18 microM and the glucose-induced conformational change with an apparent K0.5 of 5 microM. DCCD slightly increased the apparent K0.5 for the Na+ concentration required for Na(+)-induced conformational change. DCCD and PCMB altered tryptophan accessibility to quench reagents in all three conformations. Tryptophan residues on the PCMB-treated cotransporter were more Cs+ than I- sensitive in contrast to the unlabeled cotransporter. The PCMB-treated cotransporter had a reduced response to Na+, suggesting that the mode of PCMB inactivation of cotransporter activity resulted from conformational changes in the substrate-free cotransporter. DCCD had a smaller effect on cotransporter tryptophan quench reagent susceptibility. However, DCCD-labeled cotransporter was equally accessible to I- and Cs+, and the DCCD-labeled cotransporter did not respond to substrates. Loss of charge distribution around cotransporter tryptophans correlated with loss of substrate-induced conformational changes.

scholarly journals Conformational changes in plant Ins(1,4,5)P3 receptor on interaction with different myo-inositol trisphosphates and its effect on Ca2+ release from microsomal fraction and liposomes

1997 ◽  
Vol 321 (2) ◽  
pp. 355-360 ◽  
Author(s):  
Shashiprabha DASGUPTA ◽  
Dipak DASGUPTA ◽  
Aruna CHATTERJEE ◽  
Susweta BISWAS ◽  
Birendra B. BISWAS
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Dissociation Constants ◽  
Spectroscopic Studies ◽  
Binding Affinities ◽  
Inositol Trisphosphate Receptor ◽  
Single Temperature ◽  
Tryptophan Residues ◽  
Fluorescence Quencher ◽  
Trisphosphate Receptor

The interaction of the only reported plant inositol trisphosphate receptor with different myo-inositol trisphosphates (InsP3 species), namely Ins(1,4,5)P3, Ins(1,3,4)P3, Ins(1,5,6)P3 and Ins(2,4,5)P3, were studied to assess the extent of Ca2+ mobilization from microsomes/vacuoles as well as liposomes in vitro.Ins(1,4,5)P3 and Ins(2,4,5)P3 bind with the receptor with comparable affinities, as evidenced from their dissociation constants (Kd approx. 100 nM at 5 ŶC), whereas the interaction between Ins(1,3,4)P3/Ins(1,5,6)P3 and the receptor was not detected even with these ligands at 5 ƁM. Ins(1,3,4)P3/Ins(1,5,6)P3 isomers also do not elicit Ca2+ release from liposomes or microsomes/vacuoles. The ability of any InsP3 to bind the receptor for Ins(1,4,5)P3 is a prime requirement for Ca2+ release. However, the comparison of binding affinities at a single temperature does not help to correlate it directly with the extent of Ca2+ release from the intracellular stores, because the concentration of Ca2+ released by Ins(1,4,5)P3 as estimated over a period of 20 s is 3500ŷ200 nM/mg of protein and is about 4-fold higher than that by Ins(2,4,5)P3 under identical conditions. To understand the role of the receptor conformation in Ca2+ release by different isomers, we have probed the conformational change of the receptor when the different isomers bind to it. Accessibility of the tryptophan residues in the free and Ins(1,4,5)P3/Ins(2,4,5)P3-bound receptor was monitored by a neutral fluorescence quencher, acrylamide. The resulting SternŐVolmer-type quenching plots of the internal fluorescence indicate a change in the conformation of the receptor on binding to Ins(1,4,5)P3 and Ins(2,4,5)P3. It is also detected when far-UV CD spectra (205Ő250 nm) of the free and ligand [Ins(1,4,5)P3/Ins(2,4,5)P3]-bound receptor are compared. The results from CD spectroscopic studies further indicate that the conformational changes induced by the two isomers are different in nature. When thermodynamic parameters, such as enthalpy (ΔH), entropy (ΔS) and free energy (ΔG), for the formation of the two InsP3Őreceptor complexes are compared, a major difference in the extent of changes in enthalpy and entropy is noted. All these findings taken together support the proposition that it is the overall interaction leading to the requisite conformational change in the receptor that determines the potency of the InsP3 isomers in their abilities of Ca2+ mobilization from the intracellular stores or reconstituted liposomes.

scholarly journals Interaction with Lipid II Induces Conformational Changes in Bovicin HC5 Structure

2012 ◽  
Vol 56 (9) ◽  
pp. 4586-4593 ◽  
Author(s):  
Aline Dias Paiva ◽  
Nicole Irving ◽  
Eefjan Breukink ◽  
Hilário Cuquetto Mantovani
Keyword(s):  
Conformational Changes ◽  
Acyl Chain ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Blue Shift ◽  
Tryptophan Residue ◽  
Membrane Systems ◽  
Content Type ◽  
Lipid Ii ◽  
Bovicin Hc5

ABSTRACTBovicin HC5 is a lantibiotic produced byStreptococcus bovisHC5 that targets the cell wall precursor lipid II. An understanding of the modes of action against target bacteria can help broadening the clinical applicability of lantibiotics in human and veterinary medicine. In this study, the interaction of bovicin HC5 with lipid II was examined using tryptophan fluorescence and circular dichroism spectroscopy with model membrane systems that do or do not allow pore formation by bovicin HC5. In the presence of lipid II, a blue-shift of 12 nm could be observed for the fluorescence emission maximum of the tryptophan residue for all of the membrane systems tested. This change in fluorescence emission was paralleled by a decrease in accessibility toward acrylamide and phospholipids carrying a spin-label at the acyl chain; the tryptophan residue of bovicin HC5 was located near the twelfth position of the membrane phospholipid acyl chains. Moreover, the binding of lipid II by bovicin HC5 induced remarkable conformational changes in the bovicin HC5 structure. The interaction of bovicin HC5 with lipid II was highly stable even at pH 2.0. These results indicate that bovicin HC5 interacts directly with lipid II and that the topology of this interaction changes under different conditions, which is relevant for the biological activity of the peptide. To our knowledge, bovicin HC5 is the only bacteriocin described thus far that is able to interact with its target in extreme pH values, and this fact might be related to its unique structure and stability.

scholarly journals Slow conformational changes of a Neurospora glutamate dehydrogenase studied by protein fluorescence

1974 ◽  
Vol 143 (2) ◽  
pp. 317-329 ◽  
Author(s):  
Barrie Ashby ◽  
John C. Wootton ◽  
John R. S. Fincham
Keyword(s):  
Glutamate Dehydrogenase ◽  
Conformational Change ◽  
Conformational Changes ◽  
Fluorescence Emission ◽  
Enzyme Activation ◽  
Protein Fluorescence ◽  
Fluorescence Emission Spectra ◽  
Iodide Ions ◽  
Inactive State ◽  
Ph Values

1. The NADP-dependent glutamate dehydrogenase of Neurospora crassa undergoes slow reversible structural transitions, with half-times in the order of a few minutes, between active and inactive states. The inactive state of the enzyme, which predominates at pH values below 7.0, has an intrinsic tryptophan fluorescence 25% lower than that of the active state, which predominates at pH values above 7.6. The inactive state can be activated either by an increase in pH or by addition of activators such as succinate. 2. The kinetics of the slow transitions that follow activating and inactivating rapid changes in conditions have been monitored by measurements of protein fluorescence. The results show that the slow reversible conformational change detected by the change in fluorescence is the rate-limiting process for enzyme activation and inactivation. 3. In both directions this conformational change follows apparent first-order kinetics and the rate constant is independent of protein concentration. These kinetics and published measurements of molecular weight are indicative of an isomerization process. 4. In both directions the changes show a large energy of activation and a large positive entropy of activation, consistent with a considerable disturbance of conformation in the transition state. 5. Comparisons of the fluorescence emission spectra of the active and inactive states indicate that the difference in fluorescence is produced by quenching, possibly intramolecular, in the inactive conformation. Iodide ions cause similar quenching. 6. In some mutationally altered forms of the enzyme comparable but modified conformational changes can be followed by protein fluorescence.

The Intrinsic Fluorescence of Human Fibrinogen and Its Fragments D and E

1982 ◽  
Vol 48 (01) ◽  
pp. 021-023 ◽  
Author(s):  
M A Kowalska ◽  
C S Cierniewski
Keyword(s):  
Conformational Changes ◽  
Fluorescence Intensity ◽  
Degradation Products ◽  
Tryptophan Fluorescence ◽  
Intrinsic Fluorescence ◽  
Excitation Spectra ◽  
Ph Titration ◽  
Human Fibrinogen ◽  
Tryptophan Residues ◽  
Fibrinogen Molecule

SummaryThe tryptophan fluorescence of fibrinogen and its final degradation products - fragment D and E - were compared. Fibrinogen and its derivatives exhibit identical emission and excitation spectra. Their fluorescence intensity is influenced to a different extent by pH titration and temperature.Our studies showed that tryptophan residues of core fragments D and E are much more exposed to quenching effects of acrylamide and ions than intact fibrinogen, which may be caused by conformational changes occurring over the domains during plasmin digestion of fibrinogen molecule.

scholarly journals Study of Organization and Dynamics of Multi-Tryptophan Protein Molecules Utilizing Red Edge Excitation Shift Approach

2020 ◽  
Vol 32 (10) ◽  
pp. 2416-2422
Author(s):  
Anisur R. Molla ◽  
Pritha Mandal
Keyword(s):  
Fluorescence Probe ◽  
Protein Structures ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Excitation Wavelength ◽  
Denatured State ◽  
Red Edge ◽  
Tryptophan Residues ◽  
Red Edge Excitation Shift ◽  
Red Edge Effect

A shift in the fluorescence emission maxima with gradual increase in excitation wavelength is termed as red edge excitation shift (REES). Tryptophan residues are widely utilized as intrinsic fluorescence probe to investigate the protein structures. Wavelength selective tryptophan fluorescence can explore the dynamics of surrounded water molecules, the ubiquitous biological solvent. Thus REES experiment of various protein conformational states can provide significant input to the study of protein folding pathway and it can also be useful to study interaction of proteins with others. In this review article, we shall focus on red edge effect of various multi-tryptophan proteins in their respective native, intermediate and denatured state.

Conformational changes in bovine lactoferrin induced by slow or fast temperature increases

2008 ◽  
Vol 389 (8) ◽  
Author(s):  
Waleska D. Schwarcz ◽  
Lorena Carnelocce ◽  
Jerson L. Silva ◽  
Andréa C. Oliveira ◽  
Rafael B. Gonçalves
Keyword(s):  
Secondary Structure ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Heat Stability ◽  
Bovine Lactoferrin ◽  
Spectroscopic Techniques ◽  
Bioactive Component ◽  
Physiological Properties

Abstract Lactoferrin (LF) is an iron-binding protein present in several secreted substances, such as milk, and has broad antimicrobial and physiological properties. Because high temperatures may affect protein stability and its functional properties, we investigated the effect of heat on bovine LF structure and stability. The effects of temperatures used during the pasteurization process on LF and its relationship to protein functionality were studied. Conformational changes were monitored using spectroscopic techniques, such as circular dichroism (CD) and fluorescence spectroscopy. The CD data at 70°C showed that LF's secondary structure is drastically and irreversibly affected when the temperature is gradually increased. The same effect is observed when the temperature is gradually raised from 25°C to 105°C and changes are monitored by tryptophan fluorescence emission. We also verified the effects of simulating the pasteurization process; LF remained well structured during the entire process and this result was not time-dependent. Owing to preservation of the secondary structure with changes in the tertiary structure, we thus believe that pasteurization might cause LF to change into an intermediate partially folded state. A better understanding of heat stability is important for the use of LF as a bioactive component in food.

scholarly journals Structure of protease-cleavedEscherichia coliα-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

2015 ◽  
Vol 71 (7) ◽  
pp. 1478-1486 ◽  
Author(s):  
Cameron D. Fyfe ◽  
Rhys Grinter ◽  
Inokentijs Josts ◽  
Khedidja Mosbahi ◽  
Aleksander W. Roszak ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conformational Change ◽  
Domain Architecture ◽  
Activation Mechanism ◽  
Protease Inhibition ◽  
Intrinsically Disordered ◽  
Bait Region ◽  
Thioester Bond ◽  
Interdomain Interactions ◽  
Putative Mechanism

Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleavedEscherichia coliα-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintainE. coliα-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture ofEscherichia coliα-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

scholarly journals Stoichiometry of reactions of α2-macroglobulin with trypsin and chymotrypsin

1984 ◽  
Vol 217 (1) ◽  
pp. 303-308 ◽  
Author(s):  
I Björk ◽  
L J Larsson ◽  
T Lindblom ◽  
E Raub
Keyword(s):  
Conformational Change ◽  
Polypeptide Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tryptophan Fluorescence ◽  
Chain Cleavage ◽  
Α2 Macroglobulin ◽  
Thioester Bond ◽  
The Individual ◽  
Hydrolysis Of

The stoichiometry of the individual steps, i.e. polypeptide chain cleavage, hydrolysis of the putative thioester bond and conformational change, of the reaction between alpha 2-macroglobulin and trypsin or chymotrypsin was analysed. The chain cleavage was monitored by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the thioester hydrolysis by both a spectroscopic and a fluorimetric technique and the conformational change by tryptophan fluorescence. A stoichiometry of close to 2:1 was obtained for all reactions. This finding indicates that the alpha 2-macroglobulin half-molecule is an independent functional unit of the inhibitor, within which co-operativity between the two subunits may occur.

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