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PubMed1989

ATP-induced structural change in myosin subfragment-1 revealed by the location of protease cleavage sites on the primary structure.

To understand the nature of the ATP-induced structural change in myosin subfragment-1, rabbit and chicken skeletal subfragments-1s were cleaved by various proteolytic enzymes in the absence, and in the presence, of ATP and the exact locations of the cleavage sites that were affected by ATP were determined from the amino end analysis of fragments by the use of a protein sequencer. It was found that subtilisin cleaved a site between Gln27 and Asn28 of rabbit subfragment-1 and between Gln28 and Asn29 of chicken subfragment-1 only in the presence of ATP. Thermolysin cleaved a site between Pro31 and Phe32 of chicken subfragment-1 in the presence of ATP, but the same site of rabbit subfragment-1 was not cleaved. The location of these sites is quite similar to the ATP-induced chymotryptic cleavage site of chicken gizzard heavy meromyosin, between Trp29 and Ser30 as reported by others. It is suggested, therefore, that the structure and the ATP-induced structural change in the regions are similar in these subfragment-1s. ATP also changes the cleavage rate of the 26K-50K junction by many proteases. Exact cleavage sites were determined and the relationship between their location and the suppression or the enhancement by ATP of the cleavage was studied. It was found that the cleavage sites were restricted to a quite narrow region and only the cleavage by thermolysin that attacked the middle of the region was enhanced by ATP. The distribution of the cleavage sites and the effect of ATP suggest that ATP induces drastic structural change at the middle of the 26K-50K junction region. The region attacked easily by many proteases coincided very well with a hydrophilic region indicated by the hydropathy index. The region probably protrudes outside and is, therefore, easily attacked by many proteases.

Adenosine Triphosphate↗
PubMed1991

19F NMR studies of the D-galactose chemosensory receptor. 1. Sugar binding yields a global structural change.

The Escherichia coli D-galactose and D-glucose receptor is an aqueous sugar-binding protein and the first component in the distinct chemosensory and transport pathways for these sugars. Activation of the receptor occurs when the sugar binds and induces a conformational change, which in turn enables docking to specific membrane proteins. Only the structure of the activated receptor containing bound D-glucose is known. To investigate the sugar-induced structural change, we have used 19F NMR to probe 12 sites widely distributed in the receptor molecule. Five sites are tryptophan positions probed by incorporation of 5-fluorotryptophan; the resulting 19F NMR resonances were assigned by site-directed mutagenesis. The other seven sites are phenylalanine positions probed by incorporation of 3-fluorophenylalanine. Sugar binding to the substrate binding cleft was observed to trigger a global structural change detected via 19F NMR frequency shifts at 10 of the 12 labeled sites. Two of the altered sites lie in the substrate binding cleft in van der Waals contact with the bound sugar molecule. The other eight altered sites, specifically two tryptophans and six phenylalanines distributed equally between the two receptor domains, are distant from the cleft and therefore experience allosteric structural changes upon sugar binding. The results are consistent with a model in which multiple secondary structural elements, known to extend between the substrate cleft and the protein surface, undergo shifts in their average positions upon sugar binding to the cleft. Such structural coupling provides a mechanism by which sugar binding to the substrate cleft can cause structural changes at one or more docking sites on the receptor surface.

Carbohydrate Metabolism↗
PubMed1977

Structural changes and fluctuations of proteins. I. A statistical thermodynamic model.

A general theory of the structural changes and fluctuations of proteins has been proposed based on statistical thermodynamic considerations at the chain level. The "structure" of protein was assumed to be characterized by the state of secondary bonds between unique pairs of specific sites on peptide chains. Every secondary bond changes between the bonded and unbonded states by thermal agitation and the "structure" is continuously fluctuating. The free energy of the "structural state" that is defined by the fraction of secondary bonds in the bonded state has been expressed by the bond energy, the cooperative interaction between bonds, the mixing entropy of bonds, and the entropy of polypeptide chains. The most probable "structural state" can be simply determined by graphical analysis and the effect of temperature or solvent composition on it is discussed. The temperature dependence of the free energy, the probability distribution of structural states and the specific heat have been calculted for two examples of structural change. The theory predicts two different types of structural changes from the ordered to disorderd state, a "structured transition" and a "gradual structural change" with rising temperature. In the "structural transition", the probability distribution has two maxima in the temperature range of transition. In the "gradual structural change", the probabilty distribution has only one maximum during the change. A considerable fraction of secondary bonds is in the unbounded state and is always fluctuating even in the ordered state at room temperature. Such structural flucutations in a single protein molecule have been discussed quantitatively. The theory is extended to include small molecules which bind to the protein molecule and affect the structural state. The changes of structural state caused by specific and non-specific binding and allosteric effects are explained in a unified manner.

Chemical Phenomena↗
PubMed1990

Salt-dependent structure change and ion binding in cytochrome c studied by two-dimensional proton NMR.

To search for salt-dependent structure changes that might help to explain physicochemical differences observed in previous solution studies, two-dimensional proton NMR spectra of reduced and oxidized cytochrome c were recorded at relatively high and low salt concentrations. The results rule out substantial ionic strength dependent structure change in either redox form over the salt concentrations tested (5 mM phosphate to 5 mM phosphate plus 200 mM NaCl, at pH 7). Chemical shift changes were found for several residues within a limited segment of the oxidized protein, most prominently in the sequence Lys-86, Lys-87, Lys-88, Thr-89. A salt-dependent binding of phosphate anion(s) at this site, as observed earlier by others, is indicated. The binding of one or two phosphates at the cytochrome c surface can explain earlier small-angle X-ray scattering observations of an increase in the calculated radius of gyration of the oxidized protein at the same low-salt condition used here. Other observations, by ultraviolet resonance Raman and 1D NMR spectroscopies, of salt-dependent changes could not be corroborated, but may depend on the still lower salt used and the absence of phosphate. The results obtained support the view that the absence of sizeable redox-dependent structure change observed in X-ray and NMR studies at varying salt conditions is characteristic of the protein at all salt conditions above the low millimolar range. Physicochemical differences between oxidized and reduced cytochrome c apparently represent differences in stability without patent structure change.

Amino Acid Sequence↗
PubMed1989

Fourier transform infrared evidence for proline structural changes during the bacteriorhodopsin photocycle.

Structural changes involving bacteriohodopsin proline residues have been investigated by Fourier transform infrared difference spectroscopy. Bacteriohodopsin (bR)-producing Halobacteria halobium were grown on a stringent medium containing either ring-perdeuterated proline or 15N-labeled proline. Comparison of the difference spectra obtained from the photoreactions of these labeled bR samples with those for unlabeled bR has led to the assignment of peaks due to proline vibrations. [proline-N15]bR exhibited a 15-cm-1 isotopic downshift of peaks in the 1420- to 1440-cm-1 region of the bR----K and bR----M difference spectra as well as a similar downshift of peaks found in the absolute absorption spectrum of bR. In contrast, [proline-D7]bR did not cause shifts in this region of the difference spectra. These results indicate that one or more prolines undergo a structural rearrangement during the bR photocycle involving the Xaa-Pro C--N peptide bond. This change may be directly coupled to the light-induced isomerization of the retinal chromophore from all-trans-retinal to 13-cis-retinal.

Bacteriorhodopsins↗
PubMed1990

Target-dependent structural changes accompanying long-term synaptic facilitation in Aplysia neurons.

The mechanisms underlying structural changes that accompany learning and memory have been difficult to investigate in the intact nervous system. In order to make these changes more accessible for experimental analysis, dissociated cell culture and low-light-level video microscopy were used to examine Aplysia sensory neurons in the presence or absence of their target cells. Repeated applications of serotonin, a facilitating transmitter important in behavioral dishabituation and sensitization, produced growth of the sensory neurons that paralleled the long-term enhancement of synaptic strength. This growth required the presence of the postsynaptic motor neuron. Thus, both the structural changes and the synaptic facilitation of Aplysia sensorimotor synapses accompanying long-term behavioral sensitization can be produced in vitro by applying a single facilitating transmitter repeatedly. These structural changes depend on an interaction of the presynaptic neuron with an appropriate postsynaptic target.

Animals↗
PubMed1989

Structural changes vary along individual arterioles in deoxycorticosterone acetate hypertensive rats.

The variability of structural changes along individual arterioles in deoxycorticosterone acetate (DOCA) hypertensive rats was measured by coefficients of variation of: (a) serial lumen diameters along microfil casts of individual mesenteric arterioles; and (b) wall and lumen indices in serial histological cross-sections along individual renal arterioles. The mean lumen diameter of third-order mesenteric arterioles decreased with increasing duration of hypertension. There was increased variability of lumen diameter along lengths of DOCA arterioles, the coefficient of variation at 10 weeks DOCA treatment being 20.2 +/- 0.6% compared to 9.2 +/- 0.2% in controls (P less than 0.001). Medial area to internal elastic lamina (IEL) radius ratio of renal arterioles was increased in DOCA rats compared with control rats (P less than 0.025) and its variability along individual arterioles expressed as the coefficient of variation was 31.70 +/- 3.87% in DOCA rats compared with 15.29 +/- 1.72% in controls (P less than 0.005). The observed increase in variability of lumen diameter and medial area along short lengths of individual arterioles in DOCA hypertensive rats indicates that hypertensive structural changes are probably not directly related to local blood pressure. We suggest that irregular functional vasoconstriction in hypertensive rats could account for this distribution of structural changes.

Animals↗
PubMed1985

UV-induced structural changes in chromatin.

UV-induced structural alterations of chromatin were studied by means of CD, electron microscopic, and gel electrophoretic measurements. The results indicate that chromatin undergoes serious structural changes after irradiation even at very low fluences. In the low fluence range the structural transitions from the higher ordered chromatin structure to the unfolded state occur without detectable changes in the content of histone H1 and of the core histones. Histone H1 disappears only at fluences above 10 kJ/m2. Furthermore, DNA in chromatin is much more sensitive against UV-irradiation and shows a higher degree of strand scission relative to free DNA. While fragmentation in free DNA occurs at fluences above 15 kJ/m2, it occurs even at 5.5 kJ/m2 in the case of chromatin. The biological meaning of the observed UV-induced structural alterations of chromatin is discussed.

Animals↗
PubMed1987

[Functional disorders and structural changes in the liver in Amanita phalloides poisoning].

The liver functional disorders and structural changes were studied in 17 patients (children 6, adults 11, 4 to 70 years of age) with phalloidine intoxication. The clinical course of this severe and lethal exogenic intoxication, its characteristic clinical and laboratory features are discussed in relation to the morphological changes of the liver. The structural changes are similar in all patients independently of their age and the time of death. They are presented by fatty degeneration, acute toxic liver dystrophy and centrilobular necroses. These changes are produced by the specific action of the phalloidine toxins on the liver.

Adolescent↗
PubMed1980

Structural changes in (Na+ + K+)-ATPase accompanying detergent inactivation.

Structural changes in the purified (Na+ + K+)-ATPase accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to beta-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of (Na+ + K+)-ATPase and p-nitrophenylphosphate activity by digitonin (which occurs during the rapid phase of inactivation) is unlikey to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either (Na+ + K+)-ATPase or p-nitrophenylphosphatase activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid.

Animals↗
PubMed1990

Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W.

High-strength bioactive glass-ceramic A-W was soaked in various acellular aqueous solutions different in ion concentrations and pH. After soaking for 7 and 30 days, surface structural changes of the glass-ceramic were investigated by means of Fourier transform infrared reflection spectroscopy, thin-film x-ray diffraction, and scanning electronmicroscopic observations, in comparison with in vivo surface structural changes. So-called Tris buffer solution, pure water buffered with trishydroxymethyl-aminomethane, which had been used by various workers as a "simulated body fluid," did not reproduce the in vivo surface structural changes, i.e., apatite formation on the surface. A solution, ion concentrations and pH of which are almost equal to those of the human blood plasma--i.e., Na+ 142.0, K+ 5.0, Mg2+ 1.5, Ca2+ 2.5, Cl- 148.8, HCO3- 4.2 and PO4(2-) 1.0 mM and buffered at pH 7.25 with the trishydroxymethyl-aminomethane--most precisely reproduced in vivo surface structure change. This shows that careful selection of simulated body fluid is required for in vitro experiments. The results also support the concept that the apatite phase on the surface of glass-ceramic A-W is formed by a chemical reaction of the glass-ceramic with the Ca2+, HPO4(2-), and OH- ions in the body fluid.

Biocompatible Materials↗
PubMed1989

Structural changes in glomeruli and proteinuria in streptozotocin diabetic rats.

In streptozotocin induced diabetes in rats, excretion of urinary protein fractions were studied in relation to structural changes in the renal glomeruli, using light and transmission electron microscopy. After six weeks of induced diabetes only beta 1 and beta 2 plasma globulins were significantly elevated. The amount of excreted proteins and degree of glomerular changes were not proportional. In the initial stages (1-2 weeks) glomerular structural changes were very mild and were accompanied by significantly elevated proteinuria. This progressed (4-8 weeks) to moderate to prominent structural changes with intermittent proteinuria except for the fractions beta 1 & beta 2 which were elevated throughout the duration of the experiment. The amount of proteinuria was not proportional to changes in the plasma protein levels. The following conclusions may be made: 1) The mild early glomerular abnormalities seem to be mainly due to acute metabolic disturbances. 2) An early indication of diabetic nephropathy is provided not only by albuminuria, but may also be an elevated excretion of beta-globulin fractions. 3) Decrease of albuminuria in the later stages of diabetes may be related to the deposition of albumin as a basement membrane-like material in the mesangium.

Animals↗
PubMed1991

19F NMR studies of the D-galactose chemosensory receptor. 2. Ca(II) binding yields a local structural change.

The Escherichia coli D-galactose and D-glucose receptor possesses a Ca(II)-binding site closely related in structure and metal-binding characteristics to the eukaryotic EF-hand sites. Only the structure of the Ca(II)-occupied site is known. To investigate the structural change triggered by Ca(II) and Sr(II) binding, we have used 19F NMR to probe five 5-fluorotryptophan (5F-Trp) and seven 3-fluorophenylalanine (3F-Phe) positions in the structure, extending the approach described in the preceding article. Of particular interest were two 5F-Trp residues near the N terminus of the Ca(II) site at positions 127 and 133. Substitution of the larger Sr(II) for Ca(II) triggered 19F NMR frequency shifts of the 5F-Trp127 and -133 resonances, indicating a detectable structural change in the Ca(II) site. In contrast, the three 5F-Trp resonances from distant regions of the structure exhibited no detectable frequency shifts. When the metal was removed from the Ca(II) site, the 5F-Trp127 and -133 frequencies shifted to a new value similar to that observed for free 5F-Trp in aqueous solvent, and this new frequency was a function of the H2O to D2O ratio, indicating that the residues had become solvent exposed. Metal removal yielded small or undetectable frequency shifts for the three distant 5F-Trp resonances and for four of the five resolved 3F-Phe resonances. The allosteric coupling of the metal and sugar binding sites was observed to be slight: depletion of metal ions was observed to reduce the D-galactose affinity of the receptor by 2-fold. Together the results indicate that the structural changes in the Ca(II) site are primarily localized in the region of the site. Removal of the metal ion from the site exposes the nearby 5F-Trp127 and -133 residues to the solvent, suggesting that the empty site has a more open structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1988

[Effect of structural changes in thyroxine-binding globulin on its biological activity and immunochemical properties].

Using spectroscopic, electrophoretic and microcalorimetric techniques, the changes in the spatial structure of human thyroxine-binding globulin (TBG) induced by exposure of protein solutions to high temperatures (45-90 degrees C) and low pH (2.5-6.0) were studied. Simultaneously the biological activity and immunoreactivity of TBG samples were measured. The structural changes were manifested at 52 degrees C or at pH 4.0 and were then aggravated with a rise in temperature or a decrease of pH. The circular dichroism spectra showed that the molecular ellipticity had a maximum decrease (by 10%) at 218-222 nm. In fluorescence spectra excitable at 280 nm the band half-width increased by 4-6 nm; their intensity decreased by 30-40%, whereas the position of the maxima did not change significantly. After addition of an equimolar amount of thyroxine to inactivated TBG the protein fluorescence was quenched by 25-40%. The electrophoregrams of treated preparations contained additional protein bands possessing no biological activity, whose mobility was less than that of native TBG. Microcalorimetric assays of native TBG revealed a thermoabsorption peak with a maximum at 62.5 degrees C and a half-width of 7.1 degrees C. The thermodynamic parameters of melting of TBG spatial structure were consistent with a model of a two-domain structure of the molecule. The biological activity and immunoreactivity of TBG showed a coordinated decrease with a rise in the degree of protein denaturation, However, the formation of TBG complex with antibodies did not screen the thyroxine-binding center of TBG and did not alter its affinity. Possible mechanisms of structural transition of TBG and its effect on the biological properties of TBG are discussed.

Binding Sites, Antibody↗
PubMed1976

[Study of structural changes of contractile muscle proteins with the aid of polarization ultraviolet fluorescence microscopy. 1. Conformational changes of F-actin in the muscle fiber caused by ATP and its analogs].

Increase of anisotropy of F-actin fluorescence of balanus and rabbit muscle fibers under the influence of ATP, AMP and pyrophosphate in EGTA presence was detected by means of the polarized ultraviolet (UV) fluorescent microscopy methods. The fluorescence anisotropy changes are assumed to be associated with the conformational changes in the actin. ATP cause more noticeable changes of actin structure, than pyrophosphate and AMP. The conformational changes in the actin of balanus and rabbit muscle fibres were similar. ATP and its analogs induced also decrease of UV fluorescence anisotropy of A-band which appears to be associated with conformational changes in myosin. It was siggested that the changes in fluorescence of anisotropy of A-bands are due to structural changes in both HMM and LMM parts of myosin molecule.

Actins↗
PubMed1976

Rate of quaternary structure change in hemoglobin measured by modulated excitation.

Using a novel technique of modulated photo-dissociation of carbon monoxide from hemoglobin, we have obtained the rates for conversion between the two quaternary states, R, and T, at 3-fold ligation. Our measurements at pH 7 and 22 degrees give rates of 780 +/- 40 sec-1 for going from R to T, and 2500 +/- 200 sec-1 from T to R. This yields an equilibrium constant of 0.31 +/- 0.04, which is in good agreement with previous estimates. The degree of agreement between this equilibrium constant and that predicted from the allosteric model provides a new, quantitative test of the allosteric description. A sequential model for the change in structure was found incompatible with the data, even if kinetic subunit inequivalence was assumed. The technique described here is quite general and can be used as long as the system under investigation can be repetitively excited in a regime in which it responds linearly to the excitation.

Allosteric Regulation↗