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PubMed2002

Binding of ricin A-chain to negatively charged phospholipid vesicles leads to protein structural changes and destabilizes the lipid bilayer.

Ricin is a heterodimeric protein toxin in which a catalytic polypeptide (the A-chain or RTA) is linked by a disulfide bond to a cell-binding polypeptide (the B-chain or RTB). During cell entry, ricin undergoes retrograde vesicular transport to reach the endoplasmic reticulum (ER) lumen, from where RTA translocates into the cytosol, probably by masquerading as a substrate for the ER-associated protein degradation (ERAD) pathway. In partitioning studies in Triton X-114 solution, RTA is predominantly found in the detergent phase, whereas ricin holotoxin, native RTB, and several single-chain ribosome-inactivating proteins (RIPs) are in the aqueous phase. Fluorescence spectroscopy and far-UV circular dichroism (CD) demonstrated significant structural changes in RTA as a result of its interaction with liposomes containing negatively charged phospholipid (POPG). These lipid-induced structural changes markedly increased the trypsin sensitivity of RTA and, on the basis of the protein fluorescence determinations, abolished its ability to bind to adenine, the product resulting from RTA-catalyzed depurination of 28S ribosomal RNA. RTA also released trapped calcein from POPG vesicles, indicating that it destabilized the lipid bilayer. We speculate that membrane-induced partial unfolding of RTA during cell entry may facilitate its recognition as an ERAD substrate.

Fluoresceins↗

PubMed1989

Structural changes in bacteriorhodopsin during proton translocation revealed by neutron diffraction.

A neutron diffraction study of spectroscopic states for the light-energized proton pump bacteriorhodopsin (BR) is presented. The photocycle states BR-568 and M were generated at temperatures above 4 degrees C and were measured after trapping at--180 degrees C. In the BR-568 to M-state transition, which is known to be a key step in transmembrane proton pumping, reversible structural changes of the protein were detected. These structural alterations occur in the neighborhood of the cyclohexene ring and at the Schiff's base end of the chromophore retinal. They are interpreted as a 1-2 degree tilt of three or four of the transmembrane alpha-helices or as positional changes of four or five amino acids. The structural changes observed are inherent in the transport mechanism of bacteriorhodopsin.

Bacteriorhodopsins↗

PubMed1998

Temperature induced structural changes of beta-crystallin and sphingomyelin binding.

The study of the binding of alpha-crystallin to membranes is potentially important for understanding the function of alpha-crystallin in the ocular lens and the formation of cataracts. Using fluorescence probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3 -phosphoethanolamine, triethylammonium salt (NBD-PE) and (1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid, dipotassium salt (bis-ANS), the temperature dependence of the binding of alpha-crystallin to sphingomyelin liposomes, and the structural changes of alpha-crystallin and sphingomyelin induced by temperature were studied. The influence of the binding of alpha-crystallin on the mobility of the head group region of liposomes of sphingomyelin was dependent on the thermal history of alpha-crystallin. Binding of alpha-crystallin to sphingomyelin caused a decrease in the anisotropy of the fluorophore NBD-PE at or below 37 degrees C. However, when alpha-crystallin or the mixture of alpha-crystallin/sphingomyelin were preincubated near the secondary structure phase transition temperature of 60 degrees C, an increase of the anisotropy of NBD-PE (decrease of lipid head group mobility) was observed when measured at 22 degrees C or 37 degrees C. An inflection near 47 degrees C in the curve of fluorescence anisotropy of bis-ANS pre-incorporated into the alpha-crystallin corresponded to a 3 degrees or 4 degrees structural change of alpha-crystallin. alpha-Crystallin either increases or decreases the flexibility of the head group of sphingomyelin liposomes depending on its structure.

Animals↗

PubMed2002

Structural changes of pulled vesicles: a Brownian dynamics simulation.

We studied the structural changes of bilayer vesicles induced by mechanical forces using a Brownian dynamics simulation. Two nanoparticles, which interact repulsively with amphiphilic molecules, are put inside a vesicle. The position of one nanoparticle is fixed, and the other is moved by a constant force as in optical-trapping experiments. First, the pulled vesicle stretches into a pear or tube shape. Then the inner monolayer in the tube-shaped region is deformed, and a cylindrical structure is formed between two vesicles. After stretching the cylindrical region, fission occurs near the moved vesicle. Soon after this the cylindrical region shrinks. The trapping force approximately 100 pN is needed to induce the formation of the cylindrical structure and fission.

Biophysics↗

PubMed2005

Kinetics of structural changes in the relay loop and SH3 domain of myosin.

The intrinsic fluorescence of smooth muscle myosin signals conformational changes associated with different catalytic states of the ATPase cycle. To elucidate this relationship, we have examined the pre-steady-state kinetics of nucleotide binding, hydrolysis, and product release in motor domain-essential light chain mutants containing a single endogenous tryptophan, either residue 512 in the rigid relay loop or residue 29 adjacent to the SH3 domain. The intrinsic fluorescence of W512 is sensitive to both nucleotide binding and hydrolysis, and appears to report structural changes at the active site, presumably through a direct connection with switch II. The intrinsic fluorescence of W29 is sensitive to nucleotide binding but not hydrolysis, and does not appear to be tightly linked with structural changes occurring at the active site. We propose that the SH3 domain may be sensitive to conformational changes in the lever arm through contacts with the essential light chain.

Adenosine Triphosphate↗

PubMed1991

Long-term structural changes in pH-sensitive hydrogels.

The long-term swelling properties of lightly cross-linked copolymer hydrogels consisting of methyl methacrylate (MMA) and N,N-dimethylaminoethyl methacrylamide (DMAA) were studied as a function of pH at 25 degrees C and 37 degrees C. In acidic pH regions, the swelling equilibria were found to be stable over 200 d. In alkaline pH environments, however, the 'equilibrium' swelling increases slowly with time. Gas chromatography of the supernatant shows that substantial methanol is produced, along with trace amounts of N,N-dimethylethylene diamine. Thus, the primary mechanism underlying the structural changes appears to be hydrolysis of ester groups in the MMA side-chains, with a much smaller contribution due to amidolysis of the DMAA side-chains. The implications of these structural changes for the application of this hydrogel, as well as other related hydrogels as long-term implantable biomaterials, are discussed.

Gels↗

PubMed2000

Magnitude of structural changes of the T-cell receptor binding regions determine the strength of T-cell antagonism: molecular dynamics simulations of HLA-DR4 (DRB1*0405) complexed with analogue peptide.

In our model system, we generated T cell clones specific for the HLA-DR4 (DRB1*0405)-index peptide (YWALEAAAD) complex. Based on response patterns of the T cell clones, analogue peptides containing single amino acid substitutions of the index peptide were classified into three types, agonists, antagonists or null peptides (non-agonistic and non-antagonistic peptides). Subtle structural changes induced by the antagonists in the T-cell receptor (TCR) binding regions have already been explained using the root mean square (r.m.s.) deviations from the DR4-index peptide complex in the molecular dynamics (MD) trajectory. In this work, we performed additional MD simulations at 300 K with explicit solvent molecules to reveal the structural character of the HLA-DR4 complexed with the analogue peptides. We examined the r.m.s. deviations of the TCR-binding sites and the exposed areas of the bound peptides. Remarkable differences of the r.m.s. deviations among the DR4-antagonist complexes, together with our previous data, suggest that the magnitude of structural changes of TCR-binding regions would determine the strength of TCR antagonism. The simulations also indicate that TCR could discriminate null peptides from other ligands mainly through the changes of exposed side chains of the bound peptide, rather than the conformational changes of TCR-binding surfaces on HLA molecule.

Binding Sites↗

PubMed2004

Three dimensional structural studies of alpha-N-acetylgalactosaminidase (alpha-NAGA) in alpha-NAGA deficiency (Kanzaki disease): different gene mutations cause peculiar structural changes in alpha-NAGAs resulting in different substrate specificities and clinical phenotypes.

BACKGROUND: Kanzaki disease (OMIM#104170) is attributable to a deficiency in alpha-N-acetylgalactosaminidase (alpha-NAGA; E.C.3.2.1.49), which hydrolyzes GalNAcalpha1-O-Ser/Thr. Missense mutations, R329W or R329Q were identified in two Japanese Kanzaki patients. Although they are on the same codon, the clinical manifestation was more severe in R329W because an amino acid substitution led to protein instability resulting in structural change, which is greater in R329W than in R329Q. OBJECTIVE: To examine whether the different clinical phenotypes are attributable to the two mutations. METHODS: Plasma alpha-NAGA activity and urinary excreted glycopeptides were measured and three-dimensional models of human alpha-NAGA and its complexes with GalNAcalpha1-O-Ser and GalNAcalpha1-O-Thr were constructed by homology modeling. RESULTS: Residual enzyme activity was significantly higher in the R329Q- than the R329W mutant (0.022+/-0.005 versus 0.005+/-0.001 nmol/h/ml: p<0.05); the urinary ratios of GalNAcalpha1-O-Ser:GalNAcalpha1-O-Thr were 2:10 and 8:10, respectively. GalNAcalpha1-O-Ser/Thr fit tightly in a narrow space of the active site pocket of alpha-NAGA. GalNAcalpha1-O-Thr requires a larger space to associate with alpha-NAGA because of the side chain (CH3) of the threonine residue. CONCLUSION: Our findings suggest that the association of alpha-NAGA with its substrates is strongly affected by the amino acid substitution at R329 and that the association with GalNAcalpha1-O-Thr is more highly susceptible to structural changes. The residual mutant enzyme in R329W could not associate with GalNAcalpha1-O-Thr and GalNAcalpha1-O-Ser. However, the residual mutant enzyme in R329Q catalyzed GalNAcalpha1-O-Ser to some extent. Therefore, the urinary ratio of GalNAcalpha1-O-Ser:GalNAcalpha1-O-Thr was lower and the clinical phenotype was milder in the R329Q mutation. Structural analysis revealed biochemical and phenotypic differences in these Kanzaki patients with the R329Q and R329W mutation.

Antigens, Tumor-Associated, Carbohydrate↗

PubMed2002

Preformed oligomeric epidermal growth factor receptors undergo an ectodomain structure change during signaling.

Fluorescence resonance energy transfer (FRET) was used to reveal aspects of the mechanism of signal transduction by epidermal growth factor receptors (EGFR). The superpositions of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha) and an antibody fragment (29.1) to the carbohydrate extremity of the receptor's ectodomain as measured by FRET, show that 14% of EGFRs in A431 cells are oligomerized before growth factor binding. After binding growth factor and signaling, these oligomers dissociate before releasing growth factor. Time courses of the FRET-derived distances between constitutively oligomerized EGFRs during signal transduction show a transient structural change in the extracellular domain, which occurs simultaneously with the production of intracellular Ca2+ signals. The FRET measurements also show a slow increase in oligomerization of EGFR monomers after growth factor binding. The structural change found in the extracellular domain of oligomeric EGFRs is similar to that shown by others for EPO, Neu, Fas, and tumor necrosis factor receptors, and may therefore be a common property of the transduction of the receptor-mediated signals.

Calcium Signaling↗

PubMed1997

A structural change occurs upon binding of syntaxin to SNAP-25.

The highly conserved proteins syntaxin and SNAP-25 are part of a protein complex that is thought to play a key role in exocytosis of synaptic vesicles. Previous work demonstrated that syntaxin and SNAP-25 bind to each other with high affinity and that their binding regions are predicted to form coiled coils. Circular dichroism spectroscopy was used here to study the alpha-helicity of the individual proteins and to gain insight into structural changes associated with complex formation. Syntaxin displayed approximately 43% alpha-helical content. In contrast, the alpha-helical content of SNAP-25 was low under physiological conditions. Formation of the SNAP-25-syntaxin complex was associated with a dramatic increase in alpha-helicity. Interaction of a 90-residue NH2-terminal fragment of SNAP-25 comprising the minimal syntaxin binding domain lead to a similar but less pronounced increase in alpha-helicity. Single amino acid replacements in the putative hydrophobic core of this fragment with hydrophilic amino acids abolished the induced structural change and disrupted the interaction monitored by binding assays. Replacements with hydrophobic residues had no effect. Our findings are consistent with induced coiled coil formation upon binding of syntaxin and SNAP-25.

Amino Acid Sequence↗

PubMed2006

Raman evidence for specific substrate-induced structural changes in the heme pocket of human cytochrome P450 aromatase during the three consecutive oxygen activation steps.

Specific substrate-induced structural changes in the heme pocket are proposed for human cytochrome P450 aromatase (P450arom) which undergoes three consecutive oxygen activation steps. We have experimentally investigated this heme environment by resonance Raman spectra of both substrate-free and substrate-bound forms of the purified enzyme. The Fe-CO stretching mode (nu(Fe)(-)(CO)) of the CO complex and Fe(3+)-S stretching mode (nu(Fe)(-)(S)) of the oxidized form were monitored as a structural marker of the distal and proximal sides of the heme, respectively. The nu(Fe)(-)(CO) mode was upshifted from 477 to 485 and to 490 cm(-)(1) by the binding of androstenedione and 19-aldehyde-androstenedione, substrates for the first and third steps, respectively, whereas nu(Fe)(-)(CO) was not observed for P450arom with 19-hydroxyandrostenedione, a substrate for the second step, indicating that the heme distal site is very flexible and changes its structure depending on the substrate. The 19-aldehyde-androstenedione binding could reduce the electron donation from the axial thiolate, which was evident from the low-frequency shift of nu(Fe)(-)(S) by 5 cm(-)(1) compared to that of androstenedione-bound P450arom. Changes in the environment in the heme distal site and the reduced electron donation from the axial thiolate upon 19-aldehyde-androstenedione binding might stabilize the ferric peroxo species, an active intermediate for the third step, with the suppression of the formation of compound I (Fe(4+)=O porphyrin(+)(*)) that is the active species for the first and second steps. We, therefore, propose that the substrates can regulate the formation of alternative reaction intermediates by modulating the structure on both the heme distal and proximal sites in P450arom.

Androstenedione↗

PubMed2002

Clarification of the mechanism of structural change induced by reoxygenation following the induction of lipid peroxidation in Caco-2 cell monolayers.

Recently, we established a system for assessing ischemia/reperfusion injury, specifically the opening of tight junctions (TJ), caused by reoxygenation following the induction of lipid peroxidation by tertiary-butylhydroperoxide (t-BuOOH), using the human intestinal epithelial cell line Caco-2 in order to focus on the barrier function of the epithelium independent of the vascular compartment. In the present study, we attempted to identify factors involved in the structural changes induced by reoxygenation using 0.5 mM t-BuOOH in Caco-2 cell monolayers. Glutathione (GSH) and N-acetylcystein, a precursor of GSH, inhibited the opening of TJ evoked by reoxygenation following the induction of lipid peroxidation by 0.5 mM of t-BuOOH. Tiron, as a cell permeable superoxide anion scavenger and deferoxamine, an iron-chelating agent ameliorated the opening in a dose-dependent manner. Also, Tiron suppressed the apical-to-basal and basal-to-apical permeability of the increased Rhodamine123 by reoxygenation in a concentration-dependent manner. These results collectively suggest that superoxide anion and iron ions play an important role or contribute to structural changes such as the opening of TJ induced by reoxygenation following the induction of lipid peroxidation by 0.5 mM t-BuOOH.

Journal Article↗

PubMed2001

An interpretation of the structural changes responsible for the chronicity of rhinoscleroma.

OBJECTIVE: To identify the structural changes of the proliferative phase of rhinoscleroma which could be responsible for the chronicity of the disease. STUDY DESIGN: Observational research. METHODS: Samples of friable tissue taken from the nasal mucosa of nine untreated patients were processed for light and ultrastructural microscopy. RESULTS: The majority of changes contributing to the chronicity of the disease occurred in the subepithelium and followed three closely related but distinct events. In the first (infiltrative), subepithelial invasion by the Klebsiella was followed by its active multiplication and proliferation of capillaries. In the second (neutrophilic), large numbers of neutrophils were delivered into this space. Neutrophils actively phagocytized the Klebsiella but appeared to die at an accelerated rate without completing digestion of the microorganisms. In the third event (histiocytic), histiocytes entered the subepithelium and engaged in unrestrained phagocytosis of decaying neutrophils, Klebsiella, and debris. During this process, the histiocytes' phagosomes underwent massive dilation, thus becoming Mikulicz cells. Mikulicz cells were unable to consistently destroy the Klebsiella and eventually ruptured, releasing them into the interstitium. Evidence was found that an autophagic process might contribute to phagosome distention and to the rupture of the vacuolar membranes and cell wall. CONCLUSIONS: Several critical changes responsible for the chronicity of rhinoscleroma occur during the proliferative phase of the disease. The majority of these take place in the subepithelium and include: 1) factors leading to the transformation of histiocytes into Mikulicz cells, 2) the inability of these cells to consistently destroy the Klebsiella, 3) their rupture releasing viable Klebsiella, and 4) the intrinsic resistance of the pathogen.

Biopsy↗

PubMedDate not supplied

[Structural changes in the gypsy moth (Ocneria dispar L.) hemogram in polyhedrosis].

Structural changes in hemogram of Gypsy moth larvae from a population in the phase of quantity increase are studied morphometrically and cytochemically. The counts of granulocytes and prohemocytes are increased, as are the counts of hemocytes possessing phenol oxidase activity and hemocytes reducing nitroblue tetrazolium. Atypical virus morphogenesis is observed.

Animals↗

PubMed2006

[Structure changes of lead silicate glasses induced by UV laser irradiation].

The structure changes of lead silicate glasses induced by UV laser irradiation were studied by UV-visible spectra and electron spin resonance (ESR). The Urbach energies of lead silicate glasses exposed to the 266 nm UV laser increased. This implied that the disorder of the lead silicate glasses was increased. The electron spin resonance experiments showed that the oxygen deficiency centers in lead silicate glass did not change to paramagnetic defects after exposure to 266 nm laser beam, and the absorption peak near 235 nm for lead silicate glass film did not change either. But this absorption peak was bleached after exposure to the 248 nm UV laser.

English Abstract↗

PubMed1988

Long-term antihypertensive treatment may induce normalization of left ventricular mass before complete regression of vascular structural changes: consequences for cardiac function at rest and during stress.

In 14 essential hypertensive patients, aged 26-59 years, blood pressure, left ventricular mass index (LVMI), systolic function (M-mode echo, two-dimensionally guided), post-ischaemic 'maximal' forearm blood flow (strain gauge venous occlusion plethysmography), plasma renin activity, plasma catecholamines and aldosterone were measured before and after 6 and 12 months of treatment (eight patients were given captopril, 100 mg/day, + hydrochlorothiazide 25 mg/day in five patients, and six patients were given nitrendipine, 20 mg/day, + atenolol 50 mg/day in four patients). Minimal vascular resistance (mean blood pressure/peak forearm blood flow) was taken as an index of arterial structural changes. After 6 months of treatment significant reductions in blood pressure (P less than 0.001), LVMI (P less than 0.001) and minimal vascular resistance were observed. After 12 months of treatment blood pressure, LVMI and minimal vascular resistance were further reduced. The LVMI was normalized in nine cases and the minimal vascular resistance in two cases only. Aldosterone and plasma catecholamines did not change, whereas plasma renin activity was increased during captopril only. Before and during treatment the left-ventricular shortening fraction in relation to end-systolic stress in each patient at rest, and at peak of handgrip and cold pressor tests, fell within the 95% confidence limits of correlation obtained in normals. Thus, in essential hypertensives long-term treatment can induce normalization of LVMI before complete regression of arterial structural changes in the forearm. Left ventricular systolic function is preserved after normalization of LVMI, both at rest and during stress.

Adult↗

PubMed2000

Structural changes in porcine bioprosthetic valves of a left ventricular assist system in human patients.

BACKGROUND AND AIM OF THE STUDY: Porcine, specially manufactured bioprosthetic valves regulate blood flow from the left ventricle to pump sac (inflow valve) and from the pump to the aorta (outflow valve) in a wearable, electrically powered left ventricular support system (LVAS, Novacor). The increased need for long-term circulatory assistance requires information on the evolution of these valves when exposed to specific hemodynamic conditions and inflammatory reactions in the device. The study aim was to examine structural changes in valves from explanted LVASs. METHODS: Thirteen patients (11 males, two females; mean age 42 years (range: 17-64 years) were supported for a mean of 285 days (range: 37-1,293 days) with LVAS. Histologic sections from explanted inflow and outflow valves were studied immunohistochemically using peroxidase-labeled antibodies and avidin-biotinylated peroxidase complex for detection. RESULTS: In the macroscopically normal inflow valves (11/13), the outflow surface (facing the pump) was covered with a discontinuous deposit of fibrin, macrophages and granulocyte elastase. Fibrinogen, IgG, complement proteins C1q and C3 had infiltrated the extracellular matrix (ECM) between 37 and 1,293 days. The crevices were enlarged during circulatory support, and fibrinogen/fibrin insudations were detected in the spongiosa. The collagen layers in the fibrosa were disrupted after 293 days, and eroded on the inflow surface in the ventricularis after 1,293 days. In a deteriorated valve from a patient with endocarditis, Gram-positive bacteria and metalloproteinases were concentrated in the ECM. In the macroscopically normal (11/13) outflow valves, fibrin and complement proteins had penetrated the ECM from the inflow side (facing the pump), while macrophages and granulocytes were localized mainly on the outflow surface. IgG and complement proteins were detected on and beneath the cusp surface up to 200 days and covered the disrupted ECM as implant time progressed. CONCLUSIONS: Structural changes appear to progress more rapidly in the inflow than in the outflow of bioprosthetic valves. This difference indicates that the effects of biological factors are modulated by mechanical stress.

Adolescent↗

PubMed1993

Fourier transform infrared difference spectroscopy of the nicotinic acetylcholine receptor: evidence for specific protein structural changes upon desensitization.

We have previously reported a new method based on Fourier transform infrared spectroscopy for probing conformational changes that occur upon the binding of ligands to the nicotinic acetylcholine receptor (nAChR) [Baenziger, J. E., Miller, K. W. & Rothschild, K. J. (1992) Biophys. J. 61, 983-992; Baenziger, J. E., Miller, K. W., McCarthy, M. P. & Rothschild, K. J. (1992) Biophys. J. 62, 64-66]. Spectra are recorded using attenuated total reflection both in the presence and absence of agonists. The resulting nAChR "resting-to-desensitized" difference spectra reveal small highly reproducible infrared bands which can arise from vibrations of the agonist and structural changes in the nAChR membrane during the conversion of the receptor from the resting to desensitized state. In this work we have used a combination of different agonists and an antagonist along with isotopic labeling to assign bands in these spectra. nAChR membranes pretreated with the competitive antagonist alpha-bungarotoxin exhibit no bands above the noise level (approximately 10(-5) au) demonstrating that vibrations of the unbound agonist do not contribute to the normal difference spectrum. In contrast, bands in the resting-to-desensitized difference spectra are identified which can be assigned to the bound agonist, providing a means to probe its interaction and orientation in the binding site. Additional difference bands are due to secondary structural changes of the protein, perturbation of tyrosine(s), and changes in carboxyl groups possibly from Asp and/or Glu residues. Remarkably, some of these spectral changes are similar to those detected during the bleaching of the photoreceptor membrane protein rhodopsin.

Animals↗