| 1. |
- 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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| 2. |
- 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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| 3. |
- Dell'Orco, D, et al.
(författare)
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Electrostatic contributions to the kinetics and thermodynamics of protein assembly
- 2005
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 88:3, s. 1991-2002
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Tidskriftsartikel (refereegranskat)abstract
- The role of electrostatic interactions in the assembly of a native protein structure was studied using fragment complementation. Contributions of salt, pH, or surface charges to the kinetics and equilibrium of calbindin D-9k reconstitution was measured in the presence of Ca2+ using surface plasmon resonance and isothermal titration calorimetry. Whereas surface charge substitutions primarily affect the dissociation rate constant, the association rates are correlated with subdomain net charge in a way expected for Coulomb interactions. The affinity is reduced in all mutants, with the largest effect (260-fold) observed for the double mutant K25E+K29E. At low net charge, detailed charge distribution is important, and charges remote from the partner EF-hand have less influence than close ones. The effects of salt and pH on the reconstitution are smaller than mutational effects. The interaction between the wild-type EF-hands occurs with high affinity (K-A = 1.3 x 10(10) M-1; K-D = 80 pM). The enthalpy of association is overall favorable and there appears to be a very large favorable entropic contribution from the desolvation of hydrophobic surfaces that become buried in the complex. Electrostatic interactions contribute significantly to the affinity between the subdomains, but other factors, such as hydrophobic interactions, dominate.
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| 4. |
- Hellstrand, Erik, et al.
(författare)
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Retardation of Abeta fibril formation by phospholipid vesicles depends on membrane phase behavior.
- 2010
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 98:10, s. 2206-2214
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Tidskriftsartikel (refereegranskat)abstract
- An increasing amount of evidence suggests that in several amyloid diseases, the fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. We have studied Alzheimer's disease related amyloid beta peptide (Abeta). Recombinant Abeta(M1-40) and Abeta(M1-42) produced in Escherichia coli, allows us to carry out large scale kinetics assays with good statistics. The amyloid formation process is followed in means of thioflavin T fluorescence at relatively low (down to 380 nM) peptide concentration approaching the physiological range. The lipid membranes are introduced in the system as large and small unilamellar vesicles. The aggregation lagtime increases in the presence of lipid vesicles for all situations investigated and the phase behavior of the membrane in the vesicles has a large effect on the aggregation kinetics. By comparing vesicles with different membrane phase behavior we see that the solid gel phase dipalmitoylphosphatidylcholine bilayers cause the largest retardation of Abeta fibril formation. The membrane-induced retardation reaches saturation and is present when the vesicles are added during the lag time up to the nucleation point. No significant difference is detected in lag time when increasing amount of negative charge is incorporated into the membrane.
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| 5. |
- Herling, Therese W., et al.
(författare)
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A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions
- 2016
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 110:9, s. 1957-1966
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Tidskriftsartikel (refereegranskat)abstract
- The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions. We apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, we explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. We further explore the effect of calmodulin on creatine kinase activity and show that it is increased by the interaction between the two proteins. These findings demonstrate the potential of quantitative microfluidic techniques to characterize binding equilibria between biomolecules under native solution conditions.
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| 6. |
- Lindman, Stina, et al.
(författare)
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pK(a) values for side-chain carboxyl groups of a PGB1 variant explain salt and pH-dependent stability
- 2007
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 92:1, s. 257-266
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Tidskriftsartikel (refereegranskat)abstract
- Determination of pK(a) values of titrating residues in proteins provides a direct means of studying electrostatic coupling as well as pH-dependent stability. The B1 domain of protein G provides an excellent model system for such investigations. In this work, we analyze the observed pK(a) values of all carboxyl groups in a variant of PGB1 (T2Q, N8D, N37D) at low and high ionic strength as determined using H-1-C-13 heteronuclear NMR in a structural context. The pK(a) values are used to calculate the pH-dependent stability in low and high salt and to investigate electrostatic coupling in the system. The observed pK(a) values can explain the pH dependence of protein stability but require pKa shifts relative to model values in the unfolded state, consistent with persistent residual structure in the denatured state. In particular, we find that most of the deviations from the expected random coil values can be explained by a significantly upshifted pK(a) value. We show also that C-13 backbone carbonyl data can be used to study electrostatic coupling in proteins and provide specific information on hydrogen bonding and electrostatic potential at nontitrating sites.
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| 7. |
- Lindman, Stina, et al.
(författare)
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pK(a) Values for the Unfolded State under Native Conditions Explain the pH-Dependent Stability of PGB1.
- 2010
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 99:10, s. 3365-3373
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Tidskriftsartikel (refereegranskat)abstract
- Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pK(a) values. However, pK(a) values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pK(a) values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pK(a) values were determined for carboxyl groups by monitoring their pH-dependent (13)C chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pK(a) values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pK(a) values of the folded and unfolded states and compared to experimental stability data. The use of experimental pK(a) values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone.
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| 8. |
- Lindman, Stina, et al.
(författare)
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Salting the charged surface: pH and salt dependence of protein G B1 stability
- 2006
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 90:8, s. 2911-2921
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Tidskriftsartikel (refereegranskat)abstract
- This study shows signicant effects of protein surface charges on stability and these effects are not eliminated by salt screening. The stability for a variant of protein G B1 domain was studied in the pH-range of 1.5-11 at low, 0.15 M, and 2 M salt. The variant has three mutations, T2Q, N8D, and N37D, to guarantee an intact covalent chain at all pH values. The stability of the protein shows distinct pH dependence with the highest stability close to the isoelectric point. The stability is pH-dependent at all three NaCl concentrations, indicating that interactions involving charged residues are important at all three conditions. We find that 2 M salt stabilizes the protein at low pH (protein net charge is +6 and total number of charges is 6) but not at high pH (net charge is <=-6 and total number of charges is >= 18). Furthermore, 0.15 M salt slightly decreases the stability of the protein over the pH range. The results show that a net charge of the protein is destabilizing and indicate that proteins contain charges for reasons other than improved stability. Salt seems to reduce the electrostatic contributions to stability under conditions with few total charges, but cannot eliminate electrostatic effects in highly charged systems.
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