| 11. |
- Friedman, Ran, et al.
(författare)
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The role of small intraprotein cavities in the catalytic cycle of bacteriorhodopsin
- 2003
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 85:2, s. 886-896
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Tidskriftsartikel (refereegranskat)abstract
- The last phase of the proton transfer cycle of bacteriorhodopsin calls for a passage of a proton from D38 to D96.This reaction utilizes a narrow shaft ;10-A˚ long that connects the two carboxylates that cross through a very hydrophobicdomain. As the shaft is too narrow to be permanently hydrated, there are two alternatives for the proton propagation into thechannel. The proton may propagate through the shaft without solvation at the expense of a high electrostatic barrier;alternatively, the shaft will expand to accommodate some water molecules, thus lowering the Born energy for the insertion ofthe charge into the protein (B. Scha¨ tzler, N. A. Dencher, J. Tittor, D. Oesterhelt, S. Yaniv-Checover, E. Nachliel, and G. Gutman,2003, Biophys. J. 84:671–686). A comparative study of nine published crystal-structures of bacteriorhodopsin identified, next tothe shaft, microcavities in the protein whose position and surrounding atoms are common to the reported structures. Some ofthe cavities either shrink or expand during the photocycle. It is argued that the plasticity of the cavities provides a working spaceneeded for the transient solvation of the shaft, thus reducing the activation energy necessary for the solvation of the shaft. Thissuggestion is corroborated by the recent observations of Klink et al. (B. U. Klink, R. Winter, M. Engelhard, and I. Chizhov, 2002,Biophys. J. 83:3490–3498) that the late phases of the photocycle (t $ 1 ms) are strongly inhibited by external pressure.
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| 12. |
- Hofsäss, Christofer, et al.
(författare)
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Molecular dynamics simulations of phospholipid bilayers with cholesterol
- 2003
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 84:4, s. 2192-206
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Tidskriftsartikel (refereegranskat)abstract
- To investigate the microscopic interactions between cholesterol and lipids in biological membranes, we have performed a series of molecular dynamics simulations of large membranes with different levels of cholesterol content. The simulations extend to 10 ns, and were performed with hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers. The bilayers contain 1024 lipids of which 0-40% were cholesterol and the rest DPPC. The effects of cholesterol on the structure and mesoscopic dynamics of the bilayer were monitored as a function of cholesterol concentration. The main effects observed are a significant ordering of the DPPC chains (as monitored by NMR type order parameters), a reduced fraction of gauche bonds, a reduced surface area per lipid, less undulations--corresponding to an increased bending modulus for the membrane, smaller area fluctuations, and a reduced lateral diffusion of DPPC-lipids as well as cholesterols.
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| 13. |
- Karolin, J, et al.
(författare)
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Donor-donor energy migration for determining intramolecular distances in proteins : I. Application of a model to the latent plasminogen activator inhibitor-1 (PAI-1).
- 1998
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 74:1, s. 11-21
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Tidskriftsartikel (refereegranskat)abstract
- A new fluorescence spectroscopic method is presented for determining intramolecular and intermolecular distances in proteins and protein complexes, respectively. The method circumvents the general problem of achieving specific labeling with two different chromophoric molecules, as needed for the conventional donor-acceptor transfer experiments. For this, mutant forms of proteins that contain one or two unique cysteine residues can be constructed for specific labeling with one or two identical fluorescent probes, so-called donors (d). Fluorescence depolarization experiments on double-labeled Cys mutant monitor both reorientational motions of the d molecules, as well as the rate of intramolecular energy migration. In this report a model that accounts for these contributions to the fluorescence anisotropy is presented and experimentally tested. Mutants of a protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1), containing one or two cysteine residues, were labeled with sulfhydryl specific derivatives of 4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacence (BODIPY). From the rate of energy migration, the intramolecular distance between the d groups was calculated by using the Forster mechanism and by accounting for the influence of local anisotropic orientation of the d molecules. The calculated intramolecular distances were compared with those obtained from the crystal structure of PAI-1 in its latent form. To test the stability of parameters extracted from experiments, synthetic data were generated and reanalyzed.
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| 14. |
- Leitz, Guenther, et al.
(författare)
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Stress response in Caenorhabditis elegans caused by optical tweezers : wavelength, power, and time dependence
- 2002
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 82:4, s. 2224-2231
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Tidskriftsartikel (refereegranskat)abstract
- Optical tweezers have emerged as a powerful technique for micromanipulation of living cells. Although the technique often has been claimed to be nonintrusive, evidence has appeared that this is not always the case. This work presents evidence that near-infrared continuous-wave laser light from optical tweezers can produce stress in Caenorhabditis elegans. A transgenic strain of C. elegans, carrying an integrated heat-shock-responsive reporter gene, has been exposed to laser light under a variety of illumination conditions. It was found that gene expression was most often induced by light of 760 nm, and least by 810 nm. The stress response increased with laser power and irradiation time. At 810 nm, significant gene expression could be observed at 360 mW of illumination, which is more than one order of magnitude above that normally used in optical tweezers. In the 700-760-nm range, the results show that the stress response is caused by photochemical processes, whereas at 810 nm, it mainly has a photothermal origin. These results give further evidence that the 700-760-nm wavelength region is unsuitable for optical tweezers and suggest that work at 810 nm at normal laser powers does not cause stress at the cellular level.
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| 15. |
- Lindahl, E, 1972-, et al.
(författare)
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Mesoscopic undulations and thickness fluctuations in lipid bilayers from molecular dynamics simulations.
- 2000
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 79:1, s. 426-33
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Tidskriftsartikel (refereegranskat)abstract
- Molecular dynamics simulations of fully hydrated Dipalmitoylphosphatidylcholine bilayers, extending temporal and spatial scales by almost one order of magnitude, are presented. The present work reaches system sizes of 1024 lipids and times 10-60 ns. The simulations uncover significant dynamics and fluctuations on scales of several nanoseconds, and enable direct observation and spectral decomposition of both undulatory and thickness fluctuation modes. Although the former modes are strongly damped, the latter exhibit signs of oscillatory behavior. From this, it has been possible to calculate mesoscopic continuum properties in good agreement with experimental values. A bending modulus of 4 x 10(-20) J, bilayer area compressibility of 250-300 mN/m, and mode relaxation times in the nanosecond range are obtained. The theory of undulatory motions is revised and further extended to cover thickness fluctuations. Finally, it is proposed that thickness fluctuations is the explanation to the observed system-size dependence of equilibrium-projected area per lipid.
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| 16. |
- Månsson, Alf
(författare)
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Changes in force and stiffness during stretch of skeletal muscle fibers, effects of hypertonicity
- 1989
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 56:2, s. 429-433
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Tidskriftsartikel (refereegranskat)abstract
- Slow stretch ramps (velocity: 0.17 fiber lengths s-1) were imposed during fused tetanic contractions of intact muscle fibers of the frog (1.4–3.0 degrees C; sarcomere length: 2.12–2.21 microns). Instantaneous force-extension relations were derived both under isometric conditions and during slow stretch by applying fast (0.2 ms) length steps to the fiber. An increase in tonicity (98 mM sucrose added to control Ringer solution) led to significant reduction of the maximum isometric tension but at the same time to marked increase in the force enhancement during slow stretch. The maximum force level reached during the stretch was affected very little. Experiments on relaxed fibers showed that recruitment of passive parallel elastic components were of no relevance for these effects. Hypertonicity slightly increased the instantaneous stiffness of the active fiber both in the presence and in the absence of stretch. The total extension of the undamped fiber elasticity was considerably reduced by increased tonicity under isometric conditions but was only slightly affected during slow stretch. The change in length of the undamped cross-bride elasticity upon stretch was thus greater in the hypertonic than in the normotonic solution suggesting a greater increase in force per cross-bridge in the hypertonic medium. The contractile effects are consistent with the assumptions that hypertonicity reduces the capability of the individual cross-bridge to produce active force and, furthermore, that hypertonicity has only minor effects on the number of attached cross-bridges and the maximum load-bearing capacity of the individual bridge.
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| 17. |
- Ratilainen, Tommi, 1970, et al.
(författare)
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A simple model for gene targeting
- 2001
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 81:5, s. 2876-2885
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Tidskriftsartikel (refereegranskat)abstract
- Sequence-specific binding to genomic-size DNA sequences by artificial agents is of major interest for the development of gene-targeting strategies, gene-diagnostic applications, and biotechnical tools. The binding of one such agent, peptide nucleic acid (PNA), to a randomized human genome has been modeled with statistical mass action calculations. With the length of the PNA probe, the average per-base binding constant k(0), and the binding affinity loss of a mismatched base pair as main parameters, the specificity was gauged as a "therapeutic ratio" G = maximum safe [PNA](tot)/minimal efficient [PNA](tot). This general, though simple, model suggests that, above a certain threshold length of the PNA, the microscopic binding constant k(0) is the primary determinant for optimal discrimination, and that only a narrow range of rather low k(0) values gives a high therapeutic ratio G. For diagnostic purposes, the value of k(0) could readily be modulated by changing the temperature, due to the substantial DeltaH(o) associated with the binding equilibrium. Applied to gene therapy, our results stress the need for appropriate control of the binding constant and added amount of the gene-targeting agent, to meet the varying conditions (ionic strength, presence of competing DNA-binding molecules) found in the cell.
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| 18. |
- Song, Z., et al.
(författare)
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Conformational transitions of the phosphodiester backbone in native DNA: two-dimensional magic-angle-spinning 31P-NMR of DNA fibers
- 1997
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 73:3, s. 1539-1552
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Tidskriftsartikel (refereegranskat)abstract
- Solid-state 31P-NMR is used to investigate the orientation of the phosphodiester backbone in NaDNA-, LiDNA-, MgDNA-, and NaDNA-netropsin fibers. The results for A- and B-DNA agree with previous interpretations. We verify that the binding of netropsin to NaDNA stabilizes the B form, and find that in NaDNA, most of the phosphate groups adopt a conformation typical of the A form, although there are minor components with phosphate orientations close to the B form. For LiDNA and MgDNA samples, on the other hand, we find phosphate conformations that are in variance with previous models. These samples display x-ray diffraction patterns that correspond to C-DNA. However, we find two distinct phosphate orientations in these samples, one resembling that in B-DNA, and one displaying a twist of the PO4 groups about the O3-P-O4 bisectors. The latter conformation is not in accordance with previous models of C-DNA structure.
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| 19. |
- Thomson, Neil H., et al.
(författare)
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Oriented, active Escherichia coli RNA polymerase : an atomic force microscope study
- 1999
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 76:2, s. 1024-1033
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Tidskriftsartikel (refereegranskat)abstract
- RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM). Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different Ω-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion. AFM images showed that these two alkanethiols phase-segregate. Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell. The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts. These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.
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| 20. |
- Westemark, Pål, et al.
(författare)
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A model of phosphofructokinase and glycolytic oscillations in the pancreatic beta-cell
- 2003
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 85:1, s. 126--139
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Tidskriftsartikel (refereegranskat)abstract
- We have constructed a model of the upper part of the glycolysis in the pancreatic beta-cell. The model comprises the enzymatic reactions from glucokinase to glyceralclehyde-3-phosphate dehydrogenase (GAPD). Our results show, for a substantial part of the parameter space, an oscillatory behavior of the glycolysis for a large range of glucose concentrations. We show how the occurrence of oscillations depends on glucokinase, aldolase and/or GAPD activities, and how the oscillation period depends on the phosphofructokinase activity. We propose that the ratio of glucokinase and aldolase and/or GAPD activities are adequate as characteristics of the glucose responsiveness, rather than only the glucokinase activity. We also propose that the rapid equilibrium between different oligomeric forms of phosphofructokinase may reduce the oscillation period sensitivity to phosphofructokinase activity. Methodologically, we show that a satisfying description of phosphofructokinase kinetics can be achieved using the irreversible Hill equation with allosteric modifiers. We emphasize the use of parameter ranges rather than fixed values, and the use of operationally well-defined parameters in order for this methodology to be feasible. The theoretical results presented in this study apply to the study of insulin secretion mechanisms, since glycolytic oscillations have been proposed as a cause of oscillations in the ATP/ADP ratio which is linked to insulin secretion.
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