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.

Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase

2000 ◽  
Vol 350 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Diego F. GÓMEZ CASATI ◽  
Miguel A. AON ◽  
Alberto A. IGLESIAS
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Fluorescence Emission ◽  
Maximal Activity ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
Adp Glucose Pyrophosphorylase ◽  
Kinetics Of ◽  
Allosteric Regulators

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

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].

scholarly journals Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles

2004 ◽  
Vol 378 (3) ◽  
pp. 1059-1066 ◽  
Author(s):  
M. Soledad CELEJ ◽  
Mariana G. D'ANDREA ◽  
Patricia T. CAMPANA ◽  
Gerardo D. FIDELIO ◽  
M. Lucia BIANCONI
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Enzyme Stability ◽  
Catalytic Efficiency ◽  
Tryptophan Fluorescence ◽  
Enzyme Activation ◽  
Hexadecyltrimethylammonium Bromide ◽  
Negative Band ◽  
Cationic Micelles ◽  
Α Helix

The catalytic behaviour of α-CT (α-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme–micelle interaction leads to an increase in both the Vmax and the affinity for the substrate p-nitrophenyl acetate, indicating higher catalytic efficiency for bound α-CT. The bell-shaped profile of α-CT activity with increasing CTABr concentrations suggests that the micelle-bound enzyme reacts with the free substrate. Although more active with CTABr micelles, the enzyme stability is essentially the same as observed in buffer only. Enzyme activation is accompanied by changes in α-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the α-CT tryptophan fluorescence spectrum, suggesting the annulment of internal quenching and a more polar location of tryptophan residues. Near-UV CD also indicated the transfer of aromatic residues to a more flexible environment. CTABr micelles also induces an increase in α-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of α-CT shows an increase in the intensity of the positive band at 198 nm and in the negative band at 222 nm, indicating an increased α-helical content. This is in agreement with FTIR studies, where an increase in the band at 1655 cm−1, corresponding to the α-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the β-sheet content in micelle-bound α-CT. Our data suggest that the higher catalytic efficiency of micelle-bound α-CT results from significant conformational changes.

Intermediate studies on refolding of arginine kinase denatured by guanidine hydrochloride

2005 ◽  
Vol 83 (2) ◽  
pp. 109-114 ◽  
Author(s):  
Hong-Min Tang ◽  
Hong Yu
Keyword(s):  
Fluorescence Intensity ◽  
Tertiary Structure ◽  
Structural Characteristics ◽  
Molten Globule ◽  
Guanidine Hydrochloride ◽  
Arginine Kinase ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Blue Shift ◽  
Molten Globule State

The refolding course and intermediate of guanidine hydrochloride (GuHCl)-denatured arginine kinase (AK) were studied in terms of enzymatic activity, intrinsic fluorescence, 1-anilino-8-naphthalenesulfonte (ANS) fluorescence, and far-UV circular dichroism (CD). During AK refolding, the fluorescence intensity increased with a significantly blue shift of the emission maximum. The molar ellipticity of CD increased to close to that of native AK, as compared with the fully unfolded AK. In the AK refolding process, 2 refolding intermediates were observed at the concentration ranges of 0.8–1.0 mol/L and 0.3–0.5 mol GuHCl/L. The peak position of the fluorescence emission and the secondary structure of these conformation states remained roughly unchanged. The tryptophan fluorescence intensity increased a little. However, the ANS fluorescence intensity significantly increased, as compared with both the native and the fully unfolded states. The first refolding intermediate at the range of 0.8–1.0 mol GuHCl/L concentration represented a typical "pre-molten globule state structure" with inactivity. The second one, at the range of 0.3–0.5 mol GuHCl/L concentration, shared many structural characteristics of native AK, including its secondary and tertiary structure, and regained its catalytic function, although its activity was lower than that of native AK. The present results suggest that during the refolding of GuHCl-denatured AK there are at least 2 refolding intermediates; as well, the results provide direct evidence for the hierarchical mechanism of protein folding.Key words: arginine kinase, guanidine-denatured, refolding, intermediate, molten globule state.

scholarly journals Conformational changes in gastric H+/K+-ATPase monitored by difference Fourier-transform infrared spectroscopy and hydrogen/deuterium exchange

2004 ◽  
Vol 382 (1) ◽  
pp. 121-129 ◽  
Author(s):  
Frantz SCHEIRLINCKX ◽  
Vincent RAUSSENS ◽  
Jean-Marie RUYSSCHAERT ◽  
Erik GOORMAGHTIGH
Keyword(s):  
Fourier Transform ◽  
Secondary Structure ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Structural Information ◽  
Deuterium Exchange ◽  
Attenuated Total Reflection ◽  
Hydrogen Deuterium Exchange ◽  
Hydrogen Deuterium ◽  
Difference Fourier

Gastric H+/K+-ATPase is a P-type ATPase responsible for acid secretion in the stomach. This protein adopts mainly two conformations called E1 and E2. Even though two high-resolution structures for a P-ATPase in these conformations are available, little structural information is available about the transition between these two conformations. In the present study, we used two experimental approaches to investigate the structural differences that occur when gastric ATPase is placed in the presence of various ligands and ligand combinations. We used attenuated total reflection–Fourier-transform IR experiments under a flowing buffer to modify the environment of the protein inside the measurement cell. The high accuracy of the results allowed us to demonstrate that the E1–E2 transition induces a net change in the secondary structure that concerns 10–15 amino acid residues of a total of 1324 in the proteins. The E2.K+ structure is characterized by a decreased β-sheet content and an increase in the disordered structure content with respect to the E1 form of the enzyme. Modifications in the absorption of the side chain of amino acids are also suggested. By using hydrogen/deuterium-exchange kinetics, we show that tertiary-structure modifications occurred in the presence of the same ligands, but these changes involved several hundreds of residues. The present study suggests that conformational changes in the catalytic cycle imply secondary-structure rearrangements of small hinge regions that have an impact on large domain re-organizations.

DENATURATION OF HUMAN SERUM ALBUMIN BY CERIUM (III) CHLORIDE

2013 ◽  
Vol 08 (01n02) ◽  
pp. 59-71
Author(s):  
G. REZAEI BEHBAHANI ◽  
M. SHALBAFAN ◽  
N. GHEIBI ◽  
L. BARZEGAR ◽  
H. REZAEI BEHBAHANI ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Fluorescence Emission ◽  
Titration Calorimetry ◽  
Tryptophan Residue ◽  
Spectroscopic Methods ◽  
Irreversible Denaturation

Cerium (III) Chloride-induced conformational changes of human serum albumin, HSA, in phosphate buffer, 10 mM at pH 7.4 was investigated, using isothermal titration calorimetry (ITC), UV and fluorescence emission spectroscopic methods. The results indicate that CeCl3, Ce3+, induces irreversible denaturation of the HSA structure. The UV absorption intensity of HSA + Ce3+ shows a slight blueshift in the absorbance wavelength with increasing Ce3+ concentration. The fluorescence intensity was increased regularly and a slight redshift was observed in the emission wavelength. The HSA + Ce3+ complex quenches the fluorescence of HSA and changes the microenvironment of tryptophan residue. The emission intensity increases suggesting the loss of the tertiary structure of HSA. The results obtained from the ITC data are in agreement with the spectroscopic methods. The strong negative cooperativity of Ce3+ binding with HSA (Table 1) recovered from the extended solvation model, indicates that HSA has been denatured as a result of its interaction with Ce3+ ions.

scholarly journals A comparison of the secondary structure of human brain mitochondrial and cytosolic ‘malic’ enzyme investigated by Fourier-transform infrared spectroscopy

1995 ◽  
Vol 309 (2) ◽  
pp. 607-611 ◽  
Author(s):  
Z Kochan ◽  
J Karbowska ◽  
G Bukato ◽  
M M Zydowo ◽  
E Bertoli ◽  
...  
Keyword(s):  
Fourier Transform ◽  
Secondary Structure ◽  
Human Brain ◽  
Ir Spectra ◽  
Conformational Changes ◽  
Malic Enzyme ◽  
Tertiary Structure ◽  
Alpha Helix ◽  
Random Structure ◽  
Mitochondrial Enzymes

The secondary structure of human brain cytosolic and mitochondrial ‘malic’ enzymes purified to homogeneity has been investigated by Fourier-transform IR spectroscopy. The absorbance IR spectra of these two isoenzymes were slightly different, but calculated secondary-structure compositions were essentially similar (38% alpha-helix, 38-39% beta-sheet, 14% beta-turn and 9-10% random structure). These proportions were not affected by succinate, a positive effector of mitochondrial ‘malic’ enzyme activity. IR spectra indicate that the tertiary structures of human brain cytosolic and mitochondrial ‘malic’ enzymes are slightly different, and addition of succinate does not cause conformational changes to the tertiary structure of the mitochondrial enzyme. Thermal-denaturation patterns of the cytosolic and mitochondrial enzymes, obtained from spectra recorded at different temperatures in the absence or presence of Mg2+, suggest that the tertiary structure of both isoenzymes is stabilized by bivalent cations and that the cytosolic enzyme possesses a more compact tertiary structure.

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.

Influence of Ascorbic Acid on the Structure and Function of Alpha-2- macroglobulin: Investigations using Spectroscopic and Thermodynamic Techniques

2020 ◽  
Vol 27 (3) ◽  
pp. 201-209
Author(s):  
Syed Saqib Ali ◽  
Mohammad Khalid Zia ◽  
Tooba Siddiqui ◽  
Haseeb Ahsan ◽  
Fahim Halim Khan
Keyword(s):  
Ascorbic Acid ◽  
Conformational Changes ◽  
Structural Changes ◽  
The Body ◽  
Titration Calorimetry ◽  
Spectroscopic Techniques ◽  
Human Beings ◽  
Plasma Ascorbic Acid ◽  
Dietary Antioxidant ◽  
And Function

Background: Ascorbic acid is a classic dietary antioxidant which plays an important role in the body of human beings. It is commonly found in various foods as well as taken as dietary supplement. Objective: The plasma ascorbic acid concentration may range from low, as in chronic or acute oxidative stress to high if delivered intravenously during cancer treatment. Sheep alpha-2- macroglobulin (α2M), a human α2M homologue is a large tetrameric glycoprotein of 630 kDa with antiproteinase activity, found in sheep’s blood. Methods: In the present study, the interaction of ascorbic acid with alpha-2-macroglobulin was explored in the presence of visible light by utilizing various spectroscopic techniques and isothermal titration calorimetry (ITC). Results: UV-vis and fluorescence spectroscopy suggests the formation of a complex between ascorbic acid and α2M apparent by increased absorbance and decreased fluorescence. Secondary structural changes in the α2M were investigated by CD and FT-IR spectroscopy. Our findings suggest the induction of subtle conformational changes in α2M induced by ascorbic acid. Thermodynamics signatures of ascorbic acid and α2M interaction indicate that the binding is an enthalpy-driven process. Conclusion: It is possible that ascorbic acid binds and compromises antiproteinase activity of α2M by inducing changes in the secondary structure of the protein.

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