Effect of thiols on beta 2-adrenoceptors in human mononuclear leucocytes

The effect of the disulfide reducing agent dithiothreitol (DTT) and other thiols on binding of the beta-adrenoceptor antagonist (-)-125iodocyanopindolol (125ICYP) to human mononuclear leucocytes (MNL) was investigated. Saturation experiments and dissociation kinetics revealed two classes of specific 125ICYP binding sites, one of high and the other of low affinity, respectively. In intact MNL DTT caused a decrease in specific binding. This was due almost selectively to a decrease in the affinity of high affinity binding sites, which decreased gradually in a concentration-dependent manner to the affinity of low affinity binding sites. In MNL membranes DTT decreased not only the affinity but also the number of high affinity binding sites. The DTT effect was completely reversible by simple reoxidation on air. The structural isomers (+/-)-DTT. (-)-DTT and dithioerythritol revealed identical effects on specific binding, whereas the monothiols mercaptoethanol and alpha-monothioglycerol, having a lower redox potential, were considerably less effective. In the same concentration range that influenced specific binding. DTT stimulated intracellular cAMP production. These results suggest functionally important disulfide bridges which regulate the affinity of beta-adrenoceptor binding sites in human MNL. They stabilize the receptor in a high affinity state; their reduction causes the conversion of the high affinity state into a low affinity state in a process associated with stimulation of adenylate cyclase. Available evidence indicates that a similar transformation is made by beta-adrenoceptor agonists. Consequently low affinity 125ICYP binding sites preexistent in untreated cells could represent a reduced receptor state resulting from agonist-receptor interaction in vivo

Improving binding affinity through cyclization

Cancer chemotherapy results in systematic damage as the drugs used are also toxic to benign tissue. Sensitizing a cancer cell to therapy by interfering with the DNA repair mechanisms would decrease overall toxicity, as the necessary dosage of chemotherapy drugs would be lowered. The Hartman lab developed a peptide (8.6) that binds with a KD of 1 μM to the C-terminal domain of breast cancer associated protein (BRCA1), blocking homologous recombination. The crystal structure of the peptide shows the tyrosine and threonine residues are close together, suggesting that by cyclizing these positions, the peptide may already be constrained into its bound conformation. A series of dibromomethylnaphthalene linkers of various length were synthesized and cyclized through alkylation of the cysteine residues on peptide 8.6. The binding of the cyclic peptides with the BRCA1 (BRCT)2 domain will be compared to peptide 8.6 through the use of fluorescence polarization.https://scholarscompass.vcu.edu/uresposters/1248/thumbnail.jp

Steric regulation of tandem calponin homology domain actin-binding affinity.

Tandem calponin homology (CH1-CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1-CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for F-actin. To investigate features that affect CH1-CH2 affinity for F-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1-CH2 interface, replacing the linker domain, and adding a polyethylene glycol (PEG) polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1-CH2 affinity is both increased and decreased by modifications that change the effective "openness" of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution "openness," suggesting additional influences on affinity. Interestingly, we also observe nonuniform subcellular localization of utrophin CH1-CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1-CH2 binding properties

Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum

125I-Calmodulin (125I-CaM) binding to sarcoplasmic reticulum (SR) membranes isolated from skeletal muscle cells was investigated, and the CaM receptors associated with the membrane were identified by using the photoaffinity cross-linker methyl-4-azidobenzimidate or the chemical cross-linker dithiobis-N-hydroxysuccinimidyl propionate. Exogenous CaM binds to CaM-depleted membranes in a Ca2+- or Mg2+-dependent way. When both cations are added together to the reaction medium, the stimulatory effects appear to be additive, suggesting that Ca2+ and Mg2+ act by two distinct mechanisms. The Ca2+/Mg2+-dependent binding of CaM is specific since it is inhibited by unlabeled CaM or by trifluoperazine. Furthermore, it is saturable and shows one class of high affinity binding sites with a KD of about 52 nM and a beta max of about 5 pmol/mg of protein. The sensitivity of Ca2+ is expressed in two steps reaching half-saturation at free Ca2+ concentrations of about 1.6 x 10(-7) and 3 x 10(-5) M, respectively. On the other hand, the sensitivity to Mg2+ is expressed in one step with a half-saturation Mg2+ concentration of about 2 x 10(-3) M. Electrophoretic analysis in a polyacrylamide gradient and subsequent autoradiography demonstrated a major CaM-binding protein of about 60 kDa and five minor CaM receptors of about 148, 125, 41, 33, and 23 kDa, respectively. The major labeled protein (60 kDa) probably represents the CaM-dependent component involved in Ca2+ release from SR, whereas the others represent a previously unrecognized class of CaM receptors in skeletal SR

Anion transport inhibitor binding to band 3 in red blood cell membranes.

The inhibitor of anion exchange 4,4'-dibenzoamido-2,2'-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site

Site-Directed Mutagenesis and Site-Specific Binding Analysis of Calmodulin (CaM)

Calcium signaling is a major regulatory system in cells and a crucial part of cell biology. An important element in the decoding of intracellular calcium concentration into downstream processes is the ubiquitous and highly conserved calcium binding protein calmodulin (CaM) which can bind to and modulate the function of hundreds of different target proteins, regulating such processes as synaptic plasticity, gene expression and electrical signaling. The biophysical characterization of binding affinity and cooperative interactions between each of calmodulin’s four EF-hand calcium binding sites is essential for understanding calcium signaling. Highly conserved amino acid sequence differences in the ion binding loops of the EF-hands give each site unique affinity for calcium. EF-hands are almost always found in pairs, where binding to one of the sites affects the affinity of the paired site. We have used spectroscopy to measure site-specific binding in each of the paired binding sites in the CaM N-lobe, along with site-directed mutagenesis, to study the contributions of individual amino acids to the ion binding affinity in the mutated site (cis effects) and in the neighboring site (trans effects). Of the twelve amino acids in the binding loops, five are different between Site 1 and Site 2. We constructed proteins with substituted individual residues from Site 1 to Site 2. CaM with the full Site 1 sequence in both Site 1 and Site 2 shows significant changes in affinity and binding characteristics in both sites. To investigate the contributions of the individual amino acid differences, we made intermediate mutants containing individual amino acid changes in Site 2. The cis-effects of the intermediate mutations on the mutated site, Site 2, seem to be independent and additive, whereas the trans-effects on the non-mutated Site 1 showed unexpected dependence on combinations of amino acid changes in Site 2.Neuroscienc

Structural Analysis and Stochastic Modelling Suggest a Mechanism for Calmodulin Trapping by CaMKII

Activation of CaMKII by calmodulin and the subsequent maintenance of constitutive activity through autophosphorylation at threonine residue 286 (Thr286) are thought to play a major role in synaptic plasticity. One of the effects of autophosphorylation at Thr286 is to increase the apparent affinity of CaMKII for calmodulin, a phenomenon known as “calmodulin trapping”. It has previously been suggested that two binding sites for calmodulin exist on CaMKII, with high and low affinities, respectively. We built structural models of calmodulin bound to both of these sites. Molecular dynamics simulation showed that while binding of calmodulin to the supposed low-affinity binding site on CaMKII is compatible with closing (and hence, inactivation) of the kinase, and could even favour it, binding to the high-affinity site is not. Stochastic simulations of a biochemical model showed that the existence of two such binding sites, one of them accessible only in the active, open conformation, would be sufficient to explain calmodulin trapping by CaMKII. We can explain the effect of CaMKII autophosphorylation at Thr286 on calmodulin trapping: It stabilises the active state and therefore makes the high-affinity binding site accessible. Crucially, a model with only one binding site where calmodulin binding and CaMKII inactivation are strictly mutually exclusive cannot reproduce calmodulin trapping. One of the predictions of our study is that calmodulin binding in itself is not sufficient for CaMKII activation, although high-affinity binding of calmodulin is