| 1. |
- Arner, Anders, et al.
(författare)
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Calcium transients and the effect of a photolytically released calcium chelator during electrically induced contractions in rabbit rectococcygeus smooth muscle
- 1998
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Ingår i: Biophysical Journal. - 1542-0086 .- 0006-3495. ; 75:4, s. 1895-1903
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Tidskriftsartikel (refereegranskat)abstract
- Intracellular Ca2+ was determined with the fura-2 technique during electrically induced contractions in the rabbit rectococcygeus smooth muscle at 22 degreesC. The muscles were electrically activated to give short, reproducible contractions. Intracellular [Ca2+] increased during activation; the increase in [Ca2+] preceded force development by approximately 2 s. After cessation of stimulation Ca2+ fell, preceding the fall in force by approximately 4 s. The fluorescence properties of fura-2 were determined with time-resolved spectroscopy using synchrotron light at the MAX-storage ring, Lund, Sweden. The fluorescence decay of free fura-2 was best described by two exponential decays (time constants approximately 0.5 and 1.5 ns) at low Ca2+ (pCa 9). At high Ca2+ (pCa 4.5), fluorescence decay became slower and could be fitted by one exponential decay (1.9 ns). Time-resolved anisotropy of free fura-2 was characteristic of free rotational motion (correlation time 0.3 ns). Motion of fura-2 could be markedly inhibited by high concentrations of creatine kinase. Time-resolved spectroscopy measurements of muscle fibers loaded with fura-2 showed that the fluorescence lifetime of the probe was longer, suggesting an influence of the chemical environment. Anisotropy measurements revealed, however, that the probe was mobile in the cells. The Ca2+-dependence of contraction and relaxation was studied using a photolabile calcium chelator, diazo-2, which could be loaded into the muscle cells in a similar manner as fura-2. Photolysis of diazo-2 leads to an increase in its Ca2+-affinity and a fall in free Ca2+. When muscles that had been loaded with diazo-2 were illuminated with UV light flashes during the rising phase of contraction, the rate of contraction became slower, suggesting a close relation between intracellular Ca2+ and the cross-bridge interaction. In contrast, photolysis during relaxation did not influence the rate of force decay, suggesting that relaxation of these contractions is not determined by the rate of Ca2+ removal or due to an increased Ca2+ sensitivity, but instead is limited by other processes such as deactivation by dephosphorylation or detachment of tension-bearing cross-bridges, possibly regulated by thin filament systems.
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| 2. |
- Barg, Sebastian, et al.
(författare)
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Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells
- 2001
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Ingår i: Biophysical Journal. - 1542-0086 .- 0006-3495. ; 81:6, s. 3308-3323
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Tidskriftsartikel (refereegranskat)abstract
- The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.
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| 3. |
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| 4. |
- André, Ingemar, et al.
(författare)
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Salt enhances calmodulin-target interaction
- 2006
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 90:8, s. 2903-2910
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Tidskriftsartikel (refereegranskat)abstract
- Calmodulin (CaM) operates as a Ca2+ sensor and is known to interact with and regulate hundreds of proteins involved in a great many aspects of cellular function. It is of considerable interest to understand the balance of forces in complex formation of CaM with its target proteins. Here we have studied the importance of electrostatic interactions in the complex between CaM and a peptide derived from smooth-muscle myosin light-chain kinase by experimental methods and Monte Carlo simulations of electrostatic interactions. We show by Monte Carlo simulations that, in agreement with experimental data, the binding affinity between CaM and highly charged peptides is surprisingly insensitive to changes in the net charge of both the protein and peptide. We observe an increase in the binding affinity between oppositely charged partners with increasing salt concentration from zero to 100 mM, showing that formation of globular CaM-kinase type complexes is facilitated at physiological ionic strength. We conclude that ionic interactions in complex formation are optimized at pH and saline similar to the cell environment, which probably overrules the electrostatic repulsion between the negatively charged Ca2+-binding domains of CaM. We propose a conceivable rationalization of CaM electrostatics associated with interdomain repulsion.
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| 5. |
- André, Ingemar, et al.
(författare)
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The role of electrostatic interactions in calmodulin-peptide complex formation
- 2004
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 87:3, s. 1929-1938
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Tidskriftsartikel (refereegranskat)abstract
- The complex between calmodulin and the calmodulin-binding portion of smMLCKp has been studied. Electrostatic interactions have been anticipated to be important in this system where a strongly negative protein binds a peptide with high positive charge. Electrostatic interactions were probed by varying the pH in the range from 4 to 11 and by charge deletions in CaM and smMLCKp. The change in net charge of CaM from similar to-5 at pH 4.5 to -15 at pH 7.5 leaves the binding constant virtually unchanged. The affinity was also unaffected by mutations in CaM and charge substitutions in the peptide. The insensitivity of the binding constant to pH may seem surprising, but it is a consequence of the high charge on both protein and peptide. At low pH it is further attenuated by a charge regulation mechanism. That is, the protein releases a number of protons when binding the positively charged peptide. We speculate that the role of electrostatic interactions is to discriminate against unbound proteins rather than to increase the affinity for any particular target protein.
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| 6. |
- Balogh, Johanna, et al.
(författare)
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Desmin filaments influence myofilament spacing and lateral compliance of slow skeletal muscle fibres.
- 2005
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 88:2, s. 1156-1165
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Tidskriftsartikel (refereegranskat)abstract
- Intermediate filaments composed of desmin interlink Z-disks and sarcolemma in skeletal muscle. Depletion of desmin results in lower active stress of smooth, cardiac, and skeletal muscles. Structural functions of intermediate filaments in fast (psoas) and slow (soleus) skeletal muscle were examined using x-ray diffraction on permeabilized muscle from desmin-deficient mice (Des–/–) and controls (Des+/+). To examine lateral compliance of sarcomeres and cells, filament distances and fiber width were measured during osmotic compression with dextran. Equatorial spacing (x-ray diffraction) of contractile filaments was wider in soleus Des–/– muscle compared to Des+/+, showing that desmin is important for maintaining lattice structure. Osmotic lattice compression was similar in Des–/– and Des+/+. In width measurements of single fibers and bundles, Des–/– soleus were more compressed by dextran compared to Des+/+, showing that intermediate filaments contribute to whole-cell compliance. For psoas fibers, both filament distance and cell compliance were similar in Des–/– and Des+/+. We conclude that desmin is important for stabilizing sarcomeres and maintaining cell compliance in slow skeletal muscle. Wider filament spacing in Des–/– soleus cannot, however, explain the lower active stress, but might influence resistance to stretch, possibly minimizing stretch-induced cell injury.
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| 7. |
- Behrens, Manja, et al.
(författare)
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The Shapes of Z-alpha(1)-Antitrypsin Polymers in Solution Support the C-Terminal Domain-Swap Mechanism of Polymerization
- 2014
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 107:8, s. 1905-1912
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Tidskriftsartikel (refereegranskat)abstract
- Emphysema and liver cirrhosis can be caused by the Z mutation (Glu342Lys) in the serine protease inhibitor alpha 1-antitrypsin (alpha 1AT), which is found in more than 4% of the Northern European population. Homozygotes experience deficiency in the lung concomitantly with a massive accumulation of polymers within hepatocytes, causing their destruction. Recently, it was proposed that Z-alpha 1AT polymerizes by a C-terminal domain swap. In this study, small-angle x-ray scattering (SAXS) was used to characterize Z-alpha 1AT polymers in solution. The data show that the Z-alpha 1AT trimer, tetramer, and pentamer all form ring-like structures in strong support of a common domain-swap polymerization mechanism that can lead to self-terminating polymers.
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| 8. |
- Bernado, Pau, et al.
(författare)
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Structure and Dynamics of Ribosomal Protein L12: An Ensemble Model Based on SAXS and NMR Relaxation
- 2010
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 98:10, s. 2374-2382
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomal protein L12 is a two-domain protein that forms dimers mediated by its N-terminal domains. A 20-residue linker separates the N- and C-terminal domains. This linker results in a three-lobe topology with significant flexibility, known to be critical for efficient translation. Here we present an ensemble model of spatial distributions and correlation times for the domain reorientations of L12 that reconciles experimental data from small-angle x-ray scattering and nuclear magnetic resonance. We generated an ensemble of L12 conformations in which the structure of each domain is fixed but the domain orientations are variable. The ensemble reproduces the small-angle x-ray scattering data and the optimized correlation times of its reorientational eigenmodes fit the N-15 relaxation data. The ensemble model reveals intrinsic conformational properties of L12 that help explain its function on the ribosome. The two C-terminal domains sample a large volume and extend further away from the ribosome anchor than expected for a random-chain linker, indicating that the flexible linker has residual order. Furthermore, the distances between each C-terminal domain and the anchor are anticorrelated, indicating that one of them is more retracted on average. We speculate that these properties promote the function of L12 to recruit translation factors and control their activity on the ribosome.
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| 9. |
- Bhattacherjee, Arnab, et al.
(författare)
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Coupled Folding-Binding in a Hydrophobic/Polar Protein Model: Impact of Synergistic Folding and Disordered Flanks
- 2012
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 102:3, s. 569-578
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Tidskriftsartikel (refereegranskat)abstract
- Coupled folding-binding is central to the function of many intrinsically disordered proteins, yet not fully understood. With a continuous three-letter protein model, we explore the free-energy landscape of pairs of interacting sequences and how it is impacted by 1), variations in the binding mechanism; and 2), the addition of disordered flanks to the binding region. In particular, we focus on two sequences, one with 16 and one with 35 amino acids, which make a stable dimeric three-helix bundle at low temperatures. Three distinct binding mechanisms are realized by altering the stabilities of the individual monomers: docking, coupled folding-binding of a single α-helix, and synergistic folding and binding. Compared to docking, the free-energy barrier for binding is reduced when the single α-helix is allowed to fold upon binding, but only marginally. A greater reduction is found for synergistic folding, which in addition results in a binding transition state characterized by very few interchain contacts. Disordered flanking chain segments attached to the α-helix sequence can, despite a negligible impact on the dimer stability, lead to a downhill free-energy surface in which the barrier for binding is eliminated.
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| 10. |
- Björklund, Sebastian, et al.
(författare)
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Skin membrane electrical impedance properties under the influence of a varying water gradient
- 2013
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Ingår i: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 104:12, s. 2639-2650
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Tidskriftsartikel (refereegranskat)abstract
- The stratum corneum (SC) is an effective permeability barrier. One strategy to increase drug delivery across skin is to increase the hydration. A detailed description of how hydration affects skin permeability requires characterization of both macroscopic and molecular properties and how they respond to hydration. We explore this issue by performing impedance experiments on excised skin membranes in the frequency range 1 Hz to 0.2 MHz under the influence of a varying gradient in water activity (aw). Hydration/dehydration induces reversible changes of membrane resistance and effective capacitance. On average, the membrane resistance is 14 times lower and the effective capacitance is 1.5 times higher when the outermost SC membrane is exposed to hydrating conditions (aw ¼ 0.992), as compared to the case of more dehydrating conditions (aw ¼ 0.826). Molecular insight into the hydration effects on the SC components is provided by natural-abundance 13C polarization transfer solidstate NMR and x-ray diffraction under similar hydration conditions. Hydration has a significant effect on the dynamics of the keratin filament terminals and increases the interchain spacing of the filaments. The SC lipids are organized into lamellar structures with ~ 12.6 nm spacing and hexagonal hydrocarbon chain packing with mainly all-trans configuration of the acyl chains, irrespective of hydration state. Subtle changes in the dynamics of the lipids due to mobilization and incorporation of cholesterol and long-chain lipid species into the fluid lipid fraction is suggested to occur upon hydration, which can explain the changes of the impedance response. The results presented here provide information that is useful in explaining the effect of hydration on skin permeability.
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