Function Of Total Ligand Concentration Research Articles

Obtaining Quantitative Parameters of DNA-Ligand Cooperative Binding from Persistence Length Measurements

Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We have measured the persistence length of DNA molecule for cationic and neutral beta-cyclodextrin binding, using optical tweezers. We propose a methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation. We obtain an expression for the effective persistence length as a function of total ligand concentration, which fits very well our data of the DNA-cationic beta-cyclodextrin and the DNA-HU protein data available in the literature. The fit returns the values of the local persistence lengths, the dissociation constant and the degree of cooperativity for both sets of data. In both cases the persistence length behaves non-monotonically as a function of total ligand concentration. We discuss some physical mechanisms for these binding processes and their interplay with DNA flexibility.ReferencesM. S. Rocha et al., J. Chem. Phys. 127 (10), 105108 (2007);J. van Noort et al., PNAS 101 (18), 6969 (2004);D. Sagi et al., J. Mol. Biol., 341, 419 (2004).Sponsors: CNPq, Fapemig, Capes, Pronex-Facepe and INCT-FCx

Physical Basis behind Achondroplasia, the Most Common Form of Human Dwarfism

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physical-chemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia.

Mg2+ and Ca2+ Differentially Regulate DNA Binding and Dimerization of DREAM

DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca(2+) binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca(2+) binding to the various mutants revealed that, in the absence of Mg(2+), Ca(2+) binds independently and sequentially to EF-3 (DeltaH = -2.4 kcal/mol), EF-4 (DeltaH = +5.2 kcal/mol), and EF-2 (DeltaH = +1 kcal/mol). By contrast, only two Ca(2+) bind to DREAM in the presence of physiological levels of Mg(2+) for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg(2+). ITC measurements demonstrate that one Mg(2+) binds enthalpically with high affinity (K(d) = 13 mum and DeltaH = -0.79 kcal/mol) and two or more Mg(2+) bind entropically in the millimolar range. Size-exclusion chromatography studies revealed that Mg(2+) stabilizes DREAM as a monomer, whereas Ca(2+) induces protein dimerization. Electrophoretic mobility shift assays indicated that Mg(2+) is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca(2+) levels, suggesting that binding of a single Ca(2+) at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca(2+) and Mg(2+) induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg(2+) binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca(2+)-induced protein dimerization disrupts DNA binding.

Application of the oscillator strength of “hypersensitive” transitions to the investigation of complex equilibria of lanthanide ions—I: Method of the evaluation of stability constants

A method of evaluation of stability constants for the lanthanide complexes from the changes of oscillator strengths of “hypersensitive” f- f transitions as a function of total ligand concentration is proposed. The method is discussed for neodymium acetate and glycolate and holmium lactate complexes. Stability constants calculated for mononuclear species are compared with the potentiometric data of Sonesson. This method is also useful for the calculation of thermodynamic parameters using measurements of the dependence of the oscillator strength on temperature. The method is limited to lanthanide ions with f 2- f 6 and f 8- f 12 electronic configurations.