Biomedical subjects

R Rigler

Publications and source records attributed to R Rigler.

At least 73 records · Page 4Linked to original sources

PubMed1990

Side chain mobility in bovine calbindin D9k. Rotational motion of Tyr13.

The local motion of Tyr13 in wild type and mutant calbindin (Mr 8500, 75 amino acids) was investigated by time-resolved fluorescence spectroscopy performed at the MAX synchrotron in Lund, Sweden. Two-dimensional fluorescence spectroscopy (excitation-emission mapping) was used to characterize the emission of Tyr13 against the background of phenylalanine residues in the presence and absence of Ca2+. Local restricted motion of Tyr13 is observed in wild-type calbindin with only minor differences between the Ca2(+)-saturated and Ca2(+)-free forms. In a mutant, where Pro20 is exchanged for Gly and Ala14 and Asn21 are deleted, the local mobility of Tyr13 is enhanced close to values characteristic for free rotational diffusion. An increase of the overall rotational motion in this mutant form by a factor of two and the enhanced local mobility of Tyr13 indicate local and global conformational changes that also affect the Ca2(+)-binding properties. Tyr13 occurs in two isomeric species differing in lifetime of the excited state; the major species is populated to 85-90%.

Animals↗

PubMed1990

A 1H NMR study of the solution conformation of the neuropeptide galanin.

The conformations of the neuropeptide galanin in water and trifluoroethanol solutions have been examined by 1H NMR spectroscopy. Analysis of two-dimensional NMR experiments enabled the assignment of virtually all the 1H resonances of galanin in trifluoroethanol solution and many of the 1H resonances in aqueous solution. Interpretation of the NMR data in structural terms suggests that in trifluoroethanol galanin is predominantly helical while in water it does not adopt a fixed conformation.

Amino Acid Sequence↗

PubMed1990

Biophysical models of ciliary activity: Gaussian frequency distributions.

A model of a freely rotating extended scatterer is proposed to describe light scattering from beating cilia. Gaussian rotation frequency distributions, characterized by a mean angular frequency and a standard deviation, are introduced in order to simulate intensity autocorrelation functions and to fit the model to experimental data. Thus the ciliary beats are characterized by a mean beat frequency and a standard deviation of the beat frequency distribution. The standard deviation influences the damping of the intensity autocorrelation function of light scattered from cilia. The calculated intensity autocorrelation function shows a more prominent oscillating behaviour the smaller the standard deviation of the beat frequency. The validity of the model is supported by experimental data in two ways: 1) The model fits very well to experimental data in computer evaluations, 2) Neither the model nor information obtained from measurements are dependent on the measuring angle.

Animals↗

PubMed1990

Studies on somatostatin with time-resolved spectroscopy and molecular dynamics simulations.

Somatostatin was studied by time-resolved fluorescence spectroscopy and by molecular dynamics simulations. The results obtained indicate the existence of mainly one conformation in DMSO, and the existence of several conformations in other solvents. Molecular dynamics simulations showed that the most important region for activity (residue 7-9) is also the most flexible region in the peptide.

Computer Simulation↗

PubMed1989

Structure and dynamics of a fluorescent DNA oligomer containing the EcoRI recognition sequence: fluorescence, molecular dynamics, and NMR studies.

The self-complementary DNA decamer duplex d(CTGAATTCAG)2 and its modified counterpart d(CTGA[2AP]TTCAG)2, where the innermost adenine (6-aminopurine) has been replaced with the fluorescent analogue 2-aminopurine (2AP), have been studied by fluorescence and NMR spectroscopy and simulated by molecular dynamics. Both decamers are recognized and cleaved by the EcoRI restriction endonuclease. 2D NMR results show that both decamers have a standard B-type conformation below 20 degrees C, though a disturbance exists to the 5' side of the 2AP site which may originate from increased local mobility. The fluorescence and fluorescence anisotropy decays of both decamers, as well as the one containing 2AP in only one chain, were studied as a function of temperature. The data show that the 2AP base exists in a temperature-dependent distribution of states and shows rapid motions, suggesting interconversion among these states on a time scale of about 10(-10) s. The integrated fluorescence of the decamer with 2AP in both chains shows a large increase around the helix melting temperature whereas the decamer with one 2AP shows only a mild increase, showing that the mixed helix has a different structural transition as sensed by the 2AP base. The data suggest a model of conformational states which have distinct fluorescence decay times. The various states may differ in the degree of base stacking. Fluctuations in the degree of stacking of the A or 2AP base are supported by molecular dynamics simulations, which additionally show that the 2AP-T or A-T base pair hydrogen bonds remain intact during these large motions.

DNA↗

PubMed1989

Picosecond tryptophan fluorescence of thioredoxin: evidence for discrete species in slow exchange.

The steady-state tryptophan fluorescence and time-resolved tryptophan fluorescence of Escherichia coli thioredoxin, calf thymus thioredoxin, and yeast thioredoxin have been studied. In all proteins, the tryptophan residues undergo strong static and dynamic quenching, probably due to charge-transfer interactions with the nearby sulfur atoms of the active cysteines. The use of a high-resolution photon counting instrument, with a time response of 60 ps full width at half-maximum, allowed the detection of fluorescence lifetimes ranging from a few tens of picoseconds to 10 ns. The data were analyzed both by classical nonlinear least squares and by a new method of entropy maximization (MEM) for the recovery of lifetime distributions. Simulations representative of the experimental data were used to test the MEM analysis. Strong support was obtained in this way for a small number of averaged discrete species in the fluorescence decays. Wavelength studies show that each of these components spreads over closely spaced excited states, while the temperature studies indicate that they do not exchange significantly on the nanosecond time scale. The oxidized form of thioredoxin is characterized by a high content of a very short lifetime below 70 ps, the amplitude of which is sharply decreased upon reduction. On the other hand, the fluorescence anisotropy decays indicate that reduction causes an increase of the very fast tryptophan rotations in an otherwise relatively rigid structure. While the calf thymus and E. coli proteins have mostly similar dynamical fluorescence properties, the yeast thioredoxin differs in many respects.

Animals↗

PubMed1989

Molecular dynamics simulations of ribonuclease T1: comparison of the free enzyme and the 2' GMP-enzyme complex.

Molecular dynamics simulations were performed on free RNase T1 and the 2'GMP-RNase T1 complex in vacuum and with water in the active site along with crystallographically identified waters, allowing analysis of both active site and overall structural and dynamics changes due to the presence of 2'GMP. Differences in the active site include a closing in the presence of 2'GMP, which is accompanied by a decrease in mobility of active site residues. The functional relevance of the active site fluctuations is discussed. 2'GMP alters the motion of Tyr-45, suggesting a role for that residue in providing a hydrophobic environment for the protein-nucleic acid interactions responsible for the specificity of RNase T1. The presence of 2'GMP causes a structural change of the C-terminus of the alpha-helix, indicating the transmission of structural changes from the active site through the protein matrix. Overall fluctuations of both the free and 2'GMP enzyme forms are in good agreement with X-ray temperature factors. The motion of Trp-59 is influenced by 2'GMP, indicating differences in enzyme dynamics away from the active site, with the calculated changes following those previously seen in time-resolved fluorescence experiments.

Binding Sites↗

PubMed1989

Laser light scattering spectroscopy: a new method to measure tracheobronchial mucociliary activity.

Laser light scattering spectroscopy is based on the evaluation of the frequency shift of coherent light scattered by moving particles. This makes it particularly suitable for use in light guiding systems. In this study laser light scattering spectroscopy was assessed for its ability to provide information on the motility of respiratory cilia. In vitro and in vivo measurements were undertaken with animal tracheal mucosa. The intensity fluctuations of laser light scattered from moving cilia were analysed in terms of their autocorrelation functions to provide information on the frequency and synchrony of beating cilia. In vitro measurements were performed on fresh bovine trachea to estimate a safe laser power level for mucosal exposure and to test the method by defining the temperature dependence of the ciliary beat frequency. Power densities not exceeding 0.3 kW/cm2 were found to be the upper limit for long term exposure of the mucosa in vitro. Ciliary beat frequency showed a pronounced temperature dependence, ranging from 5.8 to 28.3 Hz over the temperature range 20-43.5 degrees C. A maximum frequency was found at 41.5 degrees C. In vivo measurements of ciliary activity were performed in six pigs by means of optical fibres for light transmission combined with fibreoptic bronchoscopy. A ciliary beat frequency of 5 Hz was obtained; heart and breathing frequencies could be separated and identified. These results suggest that laser light scattering spectroscopy might provide a convenient method of studying the mucociliary system of the lower airways.

Animals↗

PubMed1988

Diffusion of fluorescein-labelled molecules in suspensions of erythrocyte ghosts.

The diffusion of fluorescein isothiocyanate-labelled dextran molecules in suspensions of centrifugally, tightly packed, erythrocyte ghosts was measured by fluorescence recovery after photobleaching. In comparison with diffusion in aqueous solution, the diffusion coefficients for probe molecules of varying size were about two orders of magnitude smaller. It was established that the dextran molecules remained in the space between the ghosts. Since crosslinking membrane surface carbohydrates with antibodies further inhibits diffusion, it is assumed that interactions between surface carbohydrates and the probe molecules are the cause of slow diffusion. Two alternative models are discussed.

Antibodies↗

PubMed1988

Molecular dynamics simulations of ribonuclease T1: analysis of the effect of solvent on the structure, fluctuations, and active site of the free enzyme.

Molecular dynamics simulations were performed on ribonuclease T1 (RNase T1; EC 3.1.27.3) to determine a structure for the free enzyme. Simulations starting with the X-ray coordinates for the 2'GMP-RNase T1 complex were done in vacuo and with an 18-A water ball around the active site using stochastic boundary conditions to understand the influence of water on both the structure and fluctuations of the enzyme. Removal of 2'GMP caused structural changes in the loop regions, including those directly interacting with the bound inhibitor in the crystal structure, while regions of secondary structure were less affected. The presence of solvent in the simulation damped the structural changes observed, which may be related to the use of full charges in both simulations. Fluctuations were also affected by the water, which generally increased both at the surface and in the interior of the protein. The active site in vacuo collapsed upon itself, forming a number of protein-protein hydrogen bonds leading to larger structural changes and lowered fluctuations while the presence of water kept the active site open, minimized structural changes, and increased fluctuations. Such fluctuations in the active site may be important for the binding of inhibitors or substrates to the enzyme. Lastly, results from the water simulation allow the prediction of a motion for a hypothetical tryptophan at position 45, which can ultimately be tested experimentally via time-resolved fluorescence using a site-specific mutant of the enzyme.

Aspergillus oryzae↗

PubMed1988

Molecular dynamics simulations of ribonuclease T1. Effect of solvent on the interaction with 2'GMP.

Molecular dynamics simulations in vacuum and with a water sphere around the active site were performed on the 2'GMP-RNase T1 complex. The presence of water led to the maintenance of the 2'-GMP-RNase T1 interactions as compared to the X-ray structure, including the hydrogen bonds implicated in the enzyme-inhibitor recognition process. The sidechain of His92 in the molecular dynamics water simulation, however, hydrogen bonds directly to the phosphate of 2'GMP in contrast to the X-ray structure but in support of the role of that residue in the enzyme's catalytic mechanism. Fluctuations of active-site residues are not strongly influenced by water, possibly owing to the exclusion of water by the bound 2'GMP, which did show an increase in mobility. Analysis of the 2'GMP-RNase T1 interactions versus time reveal an equilibrium fluctuation in the presence of water, leading to a less favorable 2'GMP-RNase T1 interaction energy, suggesting a possible relationship between picosecond fluctuations and inhibitor dissociation occurring in the millisecond time domain.

Endoribonucleases↗

PubMed1988

Structural fluctuations of a helical polypeptide traversing a lipid bilayer.

Time-resolved fluorescence anisotropy (FA) measurements are reported for five helical bilayer-spanning henicosapeptides, each containing one tryptophan at sequence position 1, 6, 11, 16, or 21. The FA decay reflects two molecular processes in all cases: local internal fluctuations of the tryptophan side chain with a relaxation time of 200-500 ps, and motions of the whole polypeptide molecule with a relaxation time of 9-10 ns. The amplitudes of the fast fluctuation are largest at the helix ends and decrease toward the center of the helix. A similar observation was made for the helical polypeptides dissolved in organic solvents showing that the mobility gradient along the polypeptide sequence is an inherent property of the polypeptide helix and not due to the lipid bilayer. However, the amplitudes of the fast fluctuations can be modulated by the physical state of the lipid bilayer. With decreasing temperature, the internal mobility of the tryptophan in all positions decreases with an abrupt change at the lipid-phase transition. Concomitant molecular dynamics calculations indicate a correlation between the fast FA decay and conformational fluctuations within the helix. According to the simulation, the conformation of the polypeptide is on average predominantly helical, but actually the molecule can fluctuate between a variety of different substructures. The conformational fluctuations are largest at the helix ends and provide the free space required for rotation of the indole ring around the tryptophan side chain bonds.

Amino Acid Sequence↗

PubMed1988

Continuous wave (CW) dye laser vs. CW argon laser treatment of port wine stain (PWS).

Pulsed dye laser treatment has shown specific vessel damage and thrombosis and good clinical bleaching of PWS at lower energy densities than continuous wave (CW) argon laser treatment. Based on a pilot study with CW dye laser treatment of PWS, we wanted to investigate whether CW dye laser would be more beneficial in PWS treatment than argon laser treatment. The hypothesis was tested in 20 randomized PWS patients, but no difference in treatment results was detected between the two treatment modalities.

Adolescent↗

PubMed1987

Base pair opening dynamics of a 2-aminopurine substituted Eco RI restriction sequence and its unsubstituted counterpart in oligonucleotides.

Studies of 1H NMR selective saturation recovery were performed to determine the imino proton exchange with solvent water of the base pairs in the Eco RI endonuclease recognition sequence GAATTC, placed at the center of self-complementary decamer and dodecamer oligonucleotides. In one oligonucleotide the innermost adenine was replaced by the fluorescent base analogue 2-aminopurine (2AP). From the measurements at different concentrations of TRIS buffer acting as proton exchange catalyst, base pair lifetimes were evaluated. The results at 25 degrees show that the AT base pairs have lifetimes of the order of a few ms, whereas the surrounding GC base pairs in a dodecamer have lifetimes of about 100 ms. The (2AP)T base pair has a shorter lifetime than the corresponding AT base pair. The temperature dependent optical absorption, and for the 2AP containing oligonucleotide fluorescence, were used to study the single strand-duplex equilibrium of the decamers. The results indicate that NMR and the optical techniques, although applied at very different concentrations, monitor the same conformational transition of the oligonucleotide.

2-Aminopurine↗

PubMed1987

Fibrinogen and the early stages of polymerization to fibrin as studied by dynamic laser light scattering.

Human fibrinogen and the polymerization of fibrin after activation by the enzymes, thrombin and Batroxobin, were studied by means of dynamic laser light scattering (DLS). The apparent diffusion constant, D, for fibrinogen was measured and has a value of (1.80 +/- 0.42) X 10(-7) cm2 X s-1. D was found to contain contributions from the translational diffusion constant (Dt) as well as from the rotational diffusion constant (Dr). A comparison between experimental and calculated values of Dr and Dt suggests that fibrinogen in the absence of added Ca2+ expresses a certain degree of flexibility, while it is straightened in the presence of added Ca2+. The time dependence of D showed periodic oscillations, while the average D values decreased with time. Thrombin and Batroxobin caused similar behaviour of D. The period length was related to the enzyme concentration, clotting time (Ct) and the rate of release of fibrinopeptide A (FPA). No periodic oscillations were observed in experiments where the enzyme was replaced by saline, or in experiments using a dysfunctional fibrinogen (fibrinogen Aarhus) which displayed slow rates of FPA-release and polymerization. We propose that the periodic oscillations in a system far from equilibrium may be explained by conformational changes occurring in the fibrinogen molecule during enzyme activation and polymerization.

Batroxobin↗

PubMed1987

Protein dynamics. A time-resolved fluorescence, energetic and molecular dynamics study of ribonuclease T1.

Studies using time-resolved fluorescence depolarization were performed on the internal motion of Trp 59 of ribonuclease T1 (EC 3.1.27.3) in the free enzyme, 2'-GMP-enzyme complex and 3'-GMP-enzyme complex. The Trp 59 motion was also studied in the free enzyme using molecular dynamics simulations. Energetic analysis of activation barriers to the Trp 59 motion was performed using both the transition state theory and Kramers' theory. The activation parameters showed a dependence on solvent viscosity indicating the transition state approach in aqueous solution to be inadequate. When taking solvent viscosity contributions into account agreement between the transition state and Kramers' theories was obtained. The results indicate the three enzyme forms to have different conformations with the free enzyme and 3'-GMP-enzyme complex being similar. Comparison of the experimental and theoretical results showed a good agreement on the Trp 59 motion in the free enzyme. Trp 59 appears to vibrate rapidly, with a relaxation time of the order of 1 ps, within free space in the protein matrix and to have a slower motion, with a relaxation time of the order of 100 ps, which is related to breathing of the surrounding protein matrix. Molecular dynamics results indicate high mobility in regions of the enzyme involved in the interaction with the guanine base of the inhibitor or substrate while much lower mobility occurred in residues involved in the catalytic mechanism of ribonuclease T1.

Aspergillus oryzae↗

PubMed1986

Conformational dynamics of the anticodon loop in yeast tRNAPhe as sensed by the fluorescence of wybutine.

Conformational and dynamic properties of the anticodon loop of yeast tRNAPhe were investigated by analyzing the time resolved fluorescence of wybutine serving as a local structural probe adjacent to the anticodon GmAA on its 3' side. The influence of Mg2+, important for stabilizing the tertiary structure of tRNA, and of the complementary anticodon s2UUC of E. coli tRNA2Glu were investigated. Fluorescence lifetimes and anisotropies were measured with ps time resolution using time correlated single photon counting and a mode locked synchronously pumped and frequency doubled dye laser as excitation source. From the analysis of lifetimes (tau) and rotational relaxation times (tau R) we conclude that wybutine occurs in various structural states: one stacked conformation where the base has no free mobility and the only rotational motion reflects the mobility of the whole tRNA molecule (tau = 6 ns, tau R = 19 ns), an unstacked conformation where the base can freely rotate (tau = 100 ps, tau R = 370 ps) and an intermediary state (tau = 2 ns, tau R = 1.6 ns). Under biological conditions, i.e. in the presence of Mg2+ and neutral salts, wybutine is found in a stacked and immobile state which is consistent with the crystallographic picture. In the presence of the complementary codon however, as exemplified by the E. coli-tRNA2Glu anticodon, our analysis indicates that the codon-anticodon complex exists in an equilibrium of structural states with different rotational mobility of wybutine. The conformation with wybutine freely mobile is the predominant one and suggests that this conformation of the codon-anticodon structure differs from the canonical 3'-5' stack.

Anticodon↗

PubMed1984

Rotational and translational swimming of human spermatozoa: a dynamic laser light scattering study.

The rotational swimming motion of human spermatozoa is evaluated from measurements of depolarized dynamic laser light scattering at zero angle. The analysis is based on a Maxwellian angular velocity distribution and yields a rotational frequency of about 4 Hz that is ascribed to the rotation of the sperm head. From comparison with the translational swimming motion, a propelling efficiency of about 10 micron per turn is deduced. This parameter describes the linkage between the rotational and translational swimming motion and is likely to be discriminatory in the analysis of physiological and pathological sperm motions.

Cell Movement↗