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PubMed1991

Fourier transform infrared spectroscopic study of Ca2+ and membrane-induced secondary structural changes in bovine prothrombin and prothrombin fragment 1.

Fourier transform infrared (FTIR) spectroscopy was used to monitor secondary structural changes associated with binding of bovine prothrombin and prothrombin fragment 1 to acidic lipid membranes. Prothrombin and prothrombin fragment 1 were examined under four different conditions: in the presence of (a) Na2EDTA, (b) 5 mM CaCl2, and in the presence of CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either (c) bovine brain phosphatidyl-serine (bovPS) or (d) 1,2-dioleoyl-phosphatidylglycerol (DOPG). The widely reported Ca(2+)-induced conformational change in bovine prothrombin fragment 1 was properly detected by our procedures, although Ca(2+)-induced changes in whole prothrombin spectra were too small to be reliably interpreted. Binding of prothrombin in the presence of Ca2+ to procoagulant POPC/bovPS small unilamellar vesicles produced an increase in ordered secondary structures (2% and 3% increases in alpha-helix and beta-sheet, respectively) and a decrease of random structure (5%) as revealed by spectral analysis on both the original and Fourier-self-deconvolved data and by difference spectroscopy with the undeconvolved spectra. Binding to POPC/DOPG membranes, which are less active as procoagulant membranes, produced no detectable changes in secondary structure. In addition, no change in prothrombin fragment 1 secondary structure was detectable upon binding to either POPC/bovPS or POPC/DOPG membranes. This indicates that a membrane-induced conformational change occurs in prothrombin in the nonmembrane-binding portion of the molecule, part of which is activated to form thrombin, rather than in the membrane-binding fragment 1 region. The possible significance of this conformational change is discussed in terms of differences between the procoagulant activities of different acidic lipid membranes.

Animals↗

PubMed2002

Local structural changes caused by peptidyl-prolyl cis/trans isomerization in the native state of proteins.

Peptidyl-prolyl cis/trans isomerization, observed in the native state of an increasing number of proteins, is of considerable biological significance. The first evidence for an asymmetric transmission along the polypeptide chain of the structural effects of prolyl isomerization is now derived from the statistics of the C(alpha)/C(alpha)-atom distance distributions in the crystal structures of 848 non-homologous proteins. More detailed information on how isomerization affects segments adjacent to proline is obtained from crystal structures of proteins, that are more than 95% homologous, and that exhibit two different states of isomerization at a particular prolyl bond. The resulting 64 cases, which represent 3.8% of the database used, form pairs of coordinates which were analyzed for the existence of isomer-specific intramolecular nonbonded C(alpha)/C(alpha)-atom distances around the critical proline, and for the positional preferences for particular amino acids in the isomeric sequence segment. The probability that a native protein exhibits both prolyl isomers in the crystalline state increases in particular with a Pro at the third position N-terminal to the isomeric bond (-3 position), and with Ser, Gly and Asp at the position preceding the isomeric bond (-1 position). Structural alignment of matched pairs of isomeric proteins generates three classes with respect to position-specific distribution of C(alpha)-atom displacements around an isomeric proline imide bond. In the majority of cases the distribution of these intermolecular isomer-specific C(alpha)-atom distances shows a symmetric behavior for the N-terminal and C-terminal segment flanking the proline residue, and the magnitude did not exceed 1.3+/-0.6 A including the C(alpha) atoms in proximity to the prolyl bond. However, in the remaining 12 protein pairs the structural changes are unidirectional relative to the isomerizing bond whereby the magnitude of the isomer-specific effect exceeds 3.0+/-2.0 A even at positions remote to proline. Interestingly, the magnitude of the intramolecular isomer-specific C(alpha) atom displacements reveals a lever-arm amplification of the isomerization-mediated structural changes in a protein backbone. The observed backbone effects provide a structural basis for isomer-specific reactions of proline-containing polypeptides, and thus may play a role in biological recognition and regulation.

Crystallography, X-Ray↗

PubMed2003

Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry.

An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.

Amino Acid Sequence↗

PubMedDate not supplied

[Kinetics of electron transport, proton transfer and photophosphorylation in chloroplasts and their relation to temperature-induced structural changes in the thylakoid membrane].

Effects of various temperatures on the rates of electron transport between two photosystems, the light-induced uptake of protons, kinetics of proton efflux from the chloroplasts in the dark and photophosphorylation were studied in isolated chloroplasts. There are correlations between the physical state of thylakoid membrane and the rates of electron- and proton transport processes. The temperature dependence of "structural" parameter (fluidity of lipids in membrane) as well as the rates of electron- and proton transport processes reveal the breaks under the same temperatures. Stimulation of photophosphorylation by temperature increasing correlates with the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the protein part of CF0-CF1 complex. The rate of photophosphorylation also correlates with the physical state of membrane lipids. Thermoinduced "melting" of the thylakoid membrane inhibits the ATP formation because of a decrease in photosystem 2 photochemical activity and stimulation of membrane conductivity for protons.

Chloroplasts↗

PubMed1997

Structural changes in native membrane proteins monitored at subnanometer resolution with the atomic force microscope: a review.

Three membrane proteins, OmpF porin from Escherichia coli, bacteriorhodopsin from Halobacterium salinarium, and the hexagonally packed intermediate (HPI) layer from Deinoccocus radiodurans, were investigated with the atomic force microscope in buffer solution. A resolution of up to 0.8 nm allowed structural differences of individual proteins to be detected. OmpF porin exhibits different static conformations on the outer surface, which possibly represent the two conductive states of the ion channels. Reversible structural changes in the cytoplasmic surface of purple membrane have been induced by changing the force applied to the scanning stylus: doughnut-shaped bacteriorhodopsin trimers transformed into a structure with three pronounced protrusions when the force was reduced from 300 to 100 pN. Furthermore, individual pores of the inner surface of the HPI layer were observed to switch from an "open" to a "closed" state. Together, the structural changes in proteins monitored under physiological conditions suggest that direct observation of function-related conformational changes of biomolecules with the atomic force microscope is feasible.

Bacterial Outer Membrane Proteins↗

PubMed2001

Elongation factor G-induced structural change in helix 34 of 16S rRNA related to translocation on the ribosome.

During the translocation step of the elongation cycle, two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. The movement is catalyzed by the binding of elongation factor G (EF-G) and driven by GTP hydrolysis. Here we study structural changes of the ribosome related to EF-G binding and translocation by monitoring the accessibility of ribosomal RNA (rRNA) for chemical modification by dimethyl sulfate or cleavage by hydroxyl radicals generated by Fe(II)-EDTA. In the state of the ribosome that is formed upon binding of EF-G but before the movement of the tRNAs takes place, residues 1054,1196, and 1201 in helix 34 in 16S rRNA are strongly protected. The protections depend on EF-G binding, but do not require GTP hydrolysis, and are lost upon translocation. Mutants of EF-G, which are active in ribosome binding and GTP hydrolysis but impaired in translocation, do not bring about the protections. According to cryo-electron microscopy (Stark et al., Cell, 2000, 100:301-309), there is no contact of EF-G with the protected residues of helix 34 in the pretranslocation state, suggesting that the observed protections are due to an induced conformational change. Thus, the present results indicate that EF-G binding to the pretranslocation ribosome induces a structural change of the head of the 30S subunit that is essential for subsequent tRNA-mRNA movement in translocation.

Guanosine Triphosphate↗

PubMed1994

Compactness of protein molten globules: temperature-induced structural changes of the apomyoglobin folding intermediate.

Apomyoglobin undergoes a two-step unfolding transition when the pH is lowered from 6 to 2. The partly folded intermediate (I) state at pH 4 and low ionic strength has properties of a molten globule. We have studied structural features of this state, its compactness, content of secondary structure, and specific packing of aromatic side chains, using dynamic light scattering, and small-angle X-ray scattering and far- and near-ultraviolet circular dichroism spectroscopy. Particular attention was paid to temperature-dependent structural changes. The results are discussed with reference to the native-like (N) state and the highly unfolded (U) state. It turned out that the I-state is most compact near 30 degrees C, having a Stokes radius 20% larger and a radius of gyration 30% larger than those of the N-state. Both cooling and heating relative to 30 degrees C led to an expansion of the molecule, but the structural changes at low and high temperatures were of a different kind. At temperatures above 40 degrees C non co-operative melting of structural elements was observed, while the secondary structure was essentially retained on cooling. The results are discussed in context with theoretical predictions of the compactness and the stability of apomyoglobin by Alonso et al. [Alonso, D. O. V., Dill, K. A., and Stigter, D. (1991) Biopolymers 31:1631-1649]. Comparing the I-state of apomyoglobin with the molten globules of alpha-lactalbumin and cytochrome c, we found that the compactness of the molten globule states of the three proteins decreases in the order alpha-lactalbumin > apocytochrome c > apomyoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Structural changes in lungs of magnesium-deficient weanling rats dying spontaneously or after spontaneous recovery from the seizure-shock episode. Possible methods for sudden infant death syndromes.

A light and electron microscopic study of Mg-deficient weanling rats showed structural changes of the lungs associated with the audiogenic seizure-shock episode, and with sudden, spontaneous death or spontaneous recovery after the shock episode. Pathogen-free weanling males were fed a Mg-deficient (Mg-0) or Mg-sufficient (Mg-100) diet and were raised in a gnotobiotic environment. Mg-100 rats (n = 16), unstressed or stressed with noise or strychnine, showed normal lungs. Mg-0 rats (n = 20) experienced audiogenic seizure-shock, followed by hyperventilation with tonic-clonic hyperextension of the back and extremities. The lungs of Mg-0 rats sacrificed during shock showed marked hemorrhage, including petechiae; edema; and atelectasis. Eight that died after a post-shock period of hyperventilation and hyperextension of the spine showed partial recovery of the pulmonary lesion; they showed well-expanded lungs, pleural petechiae, persistent congestion, with mild to moderate pathology. Mg-0 rats killed for study 2 days after the seizure-shock episode showed few small areas of residual lung pathology. Ultrastructural changes after Mg-O shock included aggregated platelets, leukocytes, and occasional reticulocytes in congested capillaries. Surfactant was disrupted during Mg-0 seizure-shock, but a layer closely applied to the surface of the epithelium was evident 2 days after shock. Mg-0 rats dying spontaneously showed nonspecific structural changes of the lung similar to changes reported in the sudden infant death syndrome (SIDS).

Acoustic Stimulation↗

PubMed2000

Structural changes in cytochrome c oxidase induced by cytochrome c binding. A resonance raman study.

Electrostatically stabilized complexes of fully oxidized cytochrome c oxidase from Paracoccus denitrificans and horse heart cytochrome c were studied by resonance Raman spectroscopy. The experiments were carried out with the wild-type oxidase and a variant in which a negatively charged amino acid in the binding domain (D257) is replaced by an asparagine. It is shown that cytochrome c induces structural changes at heme a and heme a(3) which are reminiscent to those found in mammalian cytochrome c oxidase-cytochrome c complex. The spectral changes are attributed to subtle changes in the heme-protein interactions implying that there is a structural communication from the binding domain even to the remote catalytic center. Only for the heme a modes minor spectral differences were found in the response of the wild-type and the D257N variant oxidase upon cytochrome c binding indicating that electrostatic interactions of aspartate 257 are not crucial for the perturbation of the catalytic site structure in the complex. On the other hand, in none of the complexes, structural changes were detected in the bound cytochrome c. These findings are in contrast to previous results obtained with beef heart cytochrome c oxidase which triggers the formation of a new conformational state of cytochrome c assumed to be involved in the biological electron transfer process.

Animals↗

PubMed1998

Neutrophil structural changes associated with chronic endotoxemia and lung injury.

Previous studies showed that chronic endotoxemia induces thickening of the alveolar wall of rabbits. The present study examines cellular changes associated with this process and attempts to define the role of PMN in this response. Rabbits received i.v. injections of either Escherichia coli lipopolysaccharide (LPS) or saline (control), 2-3 times weekly, for 28 weeks. Peripheral blood mature and immature polymorphonuclear leukocyte (PMN) cell counts were determined on Wright-stained blood smears. Lung histological analysis was performed by both light and electron microscopy. FITC-Maclura pomifera was used to identify type II cells and diaminobenzidine tetrahydrochloride-H2O2 was employed to localize peroxidase. The results show that the LPS-induced neutrophilia is associated with an increase in the circulating band cells which is consistent with active bone marrow release. PMN in the pulmonary microvessels display structural features characteristic of phagocytosis and active macromolecule synthesis. Endothelial cells, adjacent to these PMN, show numerous coated pits and large inclusions suggestive of endocytosis. The LPS-induced thickening of the alveolar wall is associated with leukocyte migration into the interstitial and alveolar spaces. Some interstitial PMN are fragmented and surrounded by dispersed elements of the connective tissue, while others appear activated and are closely associated with hyperactive fibroblasts and alveolar type II cells. The number of alveolar type II cells has increased twofold. These results show that chronic endotoxemia in rabbits causes structural changes in PMN, endothelium, interstitium, and epithelium. PMN structural changes are consistent with enhanced functional properties and their close association with modified regions of the lung parenchyma suggest that PMN play an important role in the process of this lung injury and repair.

Animals↗

PubMed1979

Structural changes of Toxoplasma gondii bradyzoites and cysts following therapy with sulfamethoxypyrazine-pyrimethamine: studies by light and electron microscopy. Consequences for chemotherapy.

Mastomys natalensis chronically infected with Toxoplasma gondii strain ALT over two months were treated with sulfamethoxypyrazine-pyrimethamine for 10 and 25 days. 72 hours after discontinuation of therapy the animals were sacrificed. The brains were removed and, following corresponding preparation, studied for the presence of the parasite and structural changes of cysts by light and transmission electron microscopy. More or less pronounced structural changes could be found in cyst walls, bradyzoites, and in particular in the endodyogeny stages. The degree of damage proved to be proportional to the intensity of the bradyzoite metabolism. The combination of drugs used was capable of passing the cyst membrane as long as the bradyzoites maintained their metabolism irrespective of its intensity. In cysts with a largely dormant metabolism that had been subject to therapy, no micromorphological differences of the ultrastructure could be recognized when compared with untreated controls of identical age; these cysts could not be influenced by treatment.

Animals↗

PubMed1993

Colicin Ia inserts into negatively charged membranes at low pH with a tertiary but little secondary structural change.

Colicin Ia, a member of the channel-forming family of colicins, inserts into model membranes in a pH- and lipid-dependent fashion. This insertion occurs with single-hit kinetics, requires negatively charged lipids in the target membrane, and increases in rate as the pH is reduced below 5.2. The low-pH requirement does not act by inducing a secondary structural change in colicin Ia, which remains 66% +/- 4% alpha-helical between pHs 7.3 and 3.1 as determined by circular dichroism. The secondary structure also remains unchanged between pHs 7.3 and 4.2 in the hydrophobic environment provided by the detergent octyl beta-D-glucopyranoside (beta-OG). However, at pH 3.1 in the presence of beta-OG, an 11% +/- 3% decrease in the alpha-helical content is observed. Further, beta-OG induces a change in tryptophan fluorescence and an altered pattern of proteolytic digestion, indicative of a tertiary structural changes. This suggests that colicin Ia undergoes a tertiary but little or no secondary structural change in its transition from a soluble to a transmembrane protein.

Chromatography, Gel↗

PubMed1995

Water structural changes at the proton uptake site (the Thr46-Asp96 domain) in the L intermediate of bacteriorhodopsin.

Fourier transform infrared spectra of the L intermediate of light-adapted bacteriorhodopsin were examined for recombinant proteins with amino acid substitutions at Thr46 and Asp96. Two O-H stretching vibrational bands of water, at 3607 and 3577 cm-1, change into stronger H-bonding states in L of the wild type. Thr46-->Val substitution abolished these bands in spite of the fact that [3-18O]threonine-labeling did not shift them, indicating that they correspond to coordination of the water with Thr46. The two water bands were restored, although with changed frequencies, by an additional Asp96-->Asn substitution in Thr46-->Val/Asp96-->Asn. A single Asp96-->Asn substitution abolished the 3607 cm-1 band. Thus, Asp96 also takes part in structural changes in water. The perturbations of these water molecules in the L intermediate displayed a weak correlation with the ratio of intensity change in the two vibrational bands of the Schiff base mode at 1312 and 1301 cm-1 and the rate for the deprotonation of the Schiff base at the L-to-M reaction of the photocycle. We find, therefore, that the water molecules in the cytoplasmic Asp96-Thr46 domain, which comprises the site of proton uptake after formation of the M intermediate, undergo structural changes in the L intermediate already. These changes are transmitted to the extracellular domain and affect interaction of the Schiff base with Asp85, that is far removed from this region.

Aspartic Acid↗

PubMed1980

Proton magnetic resonance study of p-mercuribenzoate binding and structural changes in methemoglobin.

Interaction of human adult methemoglobin with p-mercuribenzoate (pMB) was examined at 21 degrees C by monitoring the hyperfine-shifted proton nuclear magnetic resonance (NMR) spectra of several high- and low-spin derivatives. The NMR spectra show that the heme methyl proton resonances from the beta subunits in methemoglobin were selectively affected by the binding of pMB regardless of whether the heme iron was saturated with high-spin or low-spin ligand. This observation suggests that the binding of pMB to methemoglobin induces a localized tertiary structural change around the beta heme, leaving the alpha heme unaffected. The structural change of the beta subunit was correlated with an increase in the high-spin character of the beta heme iron. A model study of the azide-methemoglobin complex suggested that the increase of the high-spin character of the beta heme iron is due to a conformational change of the proximal histidine which weakens the interaction between the heme iron and the proximal base. A similar and more pronounced spectral change due to binding of pMB was observed for the isolated beta subunit. The NMR spectral change in the isolated beta subunit also suggests that the binding of pMB to methemoglobin induces a localized conformational change within the beta subunit.

Adult↗

PubMed1984

Electron-transfer kinetics in photosynthetic reaction centers cooled to cryogenic temperatures in the charge-separated state: evidence for light-induced structural changes.

We have compared the electron-transfer kinetics in reaction centers (RCs) cooled in the dark with those cooled under illumination (i.e., in the charge-separated state). Large differences between the two cases were observed. We interpreted these findings in terms of light-induced structural changes. The kinetics of charge recombination D+QA-----DQA in RCs containing one quinone were modeled in terms of a distribution of donor-acceptor electron-transfer distances. For RCs cooled under illumination the distribution broadened and shifted to larger distances compared to the distribution for RCs cooled in the dark. The model accounts for the nonexponential decay observed at low temperatures [McElroy, J. D., Mauzerall, D. C., & Feher, G. (1974) Biochim. Biophys. Acta 333, 261-277; Morrison, L.E., & Loach, P.A. (1978) Photochem. Photobiol. 27, 751-757]. A possible physiological role of the structural changes is an enhanced charge stabilization. For RCs with two quinones, the recombination kinetics D+QAQB-----DQAQB were found to be strongly temperature dependent. This was interpreted in terms of temperature-dependent transitions between structural states [Agmon, N., & Hopfield, J.J. (1983) J. Chem. Phys. 78, 6947-6959]. This interpretation requires that these transitions occur at cryogenic temperatures on a time scale t greater than or approximately 10(3) s. The electron transfer from QA- to QB was found to not take place in RCs cooled in the dark (tau ABdark greater than 10(-1) s). In RCs cooled under illumination, we found tau ABlight less than 10(-3) s. We suggest the possibility that the drastic decrease in tau AB observed in RCs cooled under illumination is due to the trapping of a proton near QB-.

Bacterial Proteins↗

PubMed1984

Local structural changes in tropomyosin detected by a trypsin-probe method.

Structural changes in tropomyosin from rabbit skeletal muscle were studied by the tryptic digestion method, which is an application of the quantitative enzyme-probe method recently developed by Ueno and Harrington [Ueno, H., & Harrington, W.F. (1984) J. Mol. Biol. 173, 35-61]. Effects of ionic strength, temperature, and an interchain disulfide bond at Cys-190 on the structure of tropomyosin were examined. A region of high susceptibility to trypsin was found to be localized in the middle portion of the molecule, and its susceptibility increased on lowering ionic strength and/or raising temperature. With the introduction of a disulfide bond at Cys-190, cleavage on the N-terminal side of Cys-190 was accelerated. The results suggest that skeletal muscle tropomyosin is flexible in the middle of the molecule in contrast to the flanking N- and C-terminal trypsin-resistant segments.

Animals↗

PubMed1997

Structural changes induced by ultrasound during aging of the boehmite phase.

Structural changes induced by ultrasound during the aging of the aluminum monohydroxide (boehmite) were studied by means of X-ray diffraction (XRD) and nitrogen adsorption. The BET surface area and the pore volume of the ultrasound stimulated hydroxide (HU) are about 40% less than those of the non-stimulated one (HS). The mean pore size practically does not change, while the mean crystallite size (L) is about 25% greater in the HU system. The increase of L alone is not enough to account for the surface area diminution, suggesting that the sonication also induces compaction by elimination of some porosity. The sonication of the precursor hydroxide does not seem to play an apparent role in the structural properties of the resulting calcinated gamma-alumina.

Journal Article↗

PubMed2003

In vivo selective inhibition of mitogen-activated protein kinase kinase 1/2 in rabbit experimental osteoarthritis is associated with a reduction in the development of structural changes.

OBJECTIVE: The primary aim of this study was to investigate, using an experimental rabbit model of osteoarthritis (OA), the effect of a selective mitogen-activated protein kinase kinase 1/2 (MEK-1/2) inhibitor, PD 198306, on the development of structural changes. Additional aims were to assess the effects of the inhibitor on levels of phosphorylated extracellular signal-regulated kinase 1/2 (phospho-ERK-1/2) and matrix metalloproteinase 1 (MMP-1; collagenase 1) in OA chondrocytes. METHODS: After surgical sectioning of the anterior cruciate ligament of the right knee joint, rabbits with OA were separated into 3 experimental groups: oral treatment with placebo or with PD 198306 at a therapeutic concentration of 10 mg/kg/day or 30 mg/kg/day. Each treatment started immediately after surgery. The animals were killed 8 weeks after surgery. Macroscopic and histologic studies were performed on the cartilage and synovial membrane. The levels of phospho-ERK-1/2 and MMP-1 in OA cartilage chondrocytes were evaluated by immunohistochemistry. Normal, untreated rabbits were used as controls. RESULTS: OA rabbits treated with the highest dosage of MEK-1/2 inhibitor showed decreases in the surface area (size) of cartilage macroscopic lesions (P < 0.002) and in osteophyte width on the lateral condyles (P = 0.05). Histologically, the severity of synovial inflammation (villous hyperplasia) was also reduced (P < 0.02). In cartilage from placebo-treated OA rabbits, a significantly higher percentage of chondrocytes in the superficial layer stained positive for phospho-ERK-1/2 and MMP-1 compared with normal controls. Rabbits treated with the highest dosage of PD 198306 demonstrated a significant and dose-dependent reduction in the level of phospho-ERK-1/2 and a lower level of MMP-1. CONCLUSION: This study demonstrates that, in vivo, PD 198306, a selective inhibitor of MEK-1/2, can partially decrease the development of some of the structural changes in experimental OA. This effect was associated with a reduction in the level of phospho-ERK-1/2 in OA chondrocytes, which probably explains the action of the drug.

Animals↗