An extended Monod-Wyman-Changeaux-model expressed in terms of the Herzfeld-Stanley formalism applied to oxygen and carbonmonoxide binding curves of hemoglobin trout IV

An extended Monod, Wyman, Changeaux (MWC)-model, the mathematical basis of which had been formulated by Herzfeld and Stanley (J. Mol. Blol. 82:231. 1974.) was used to fit oxygen and CO-binding curves of hemoglobin trout IV measured at different pH-values between pH = 8.0 and 6.0. From this calculation one obtains that even the fully liganded molecule exhibits a R----T quaternary transition upon approaching the acid pH-region. In the case of O2-binding, the cooperativity becomes negative below pH = 6.5. This can be related to the difference between the equilibrium constants of proton binding to the alpha- and beta-subunits. Furthermore, it can be shown that the interaction between the quaternary T----R- and the tertiary t----r-transitions is different for the alpha- and beta-subunits

Ab initio study of alanine polypeptide chains twisting

We have investigated the potential energy surfaces for alanine chains consisting of three and six amino acids. For these molecules we have calculated potential energy surfaces as a function of the Ramachandran angles Phi and Psi, which are widely used for the characterization of the polypeptide chains. These particular degrees of freedom are essential for the characterization of proteins folding process. Calculations have been carried out within ab initio theoretical framework based on the density functional theory and accounting for all the electrons in the system. We have determined stable conformations and calculated the energy barriers for transitions between them. Using a thermodynamic approach, we have estimated the times of characteristic transitions between these conformations. The results of our calculations have been compared with those obtained by other theoretical methods and with the available experimental data extracted from the Protein Data Base. This comparison demonstrates a reasonable correspondence of the most prominent minima on the calculated potential energy surfaces to the experimentally measured angles Phi and Psi for alanine chains appearing in native proteins. We have also investigated the influence of the secondary structure of polypeptide chains on the formation of the potential energy landscape. This analysis has been performed for the sheet and the helix conformations of chains of six amino acids.Comment: 24 pages, 10 figure

A Kinetic Model of Trp-Cage Folding from Multiple Biased Molecular Dynamics Simulations

Trp-cage is a designed 20-residue polypeptide that, in spite of its size, shares several features with larger globular proteins. Although the system has been intensively investigated experimentally and theoretically, its folding mechanism is not yet fully understood. Indeed, some experiments suggest a two-state behavior, while others point to the presence of intermediates. In this work we show that the results of a bias-exchange metadynamics simulation can be used for constructing a detailed thermodynamic and kinetic model of the system. The model, although constructed from a biased simulation, has a quality similar to those extracted from the analysis of long unbiased molecular dynamics trajectories. This is demonstrated by a careful benchmark of the approach on a smaller system, the solvated Ace-Ala3-Nme peptide. For the Trp-cage folding, the model predicts that the relaxation time of 3100 ns observed experimentally is due to the presence of a compact molten globule-like conformation. This state has an occupancy of only 3% at 300 K, but acts as a kinetic trap. Instead, non-compact structures relax to the folded state on the sub-microsecond timescale. The model also predicts the presence of a state at of 4.4 Å from the NMR structure in which the Trp strongly interacts with Pro12. This state can explain the abnormal temperature dependence of the and chemical shifts. The structures of the two most stable misfolded intermediates are in agreement with NMR experiments on the unfolded protein. Our work shows that, using biased molecular dynamics trajectories, it is possible to construct a model describing in detail the Trp-cage folding kinetics and thermodynamics in agreement with experimental data

Analysis of Fc(epsilon)RI-mediated mast cell stimulation by surface-carried antigens.

Clustering of the type I receptor for IgE (Fc[epsilon]RI) on mast cells initiates a cascade of biochemical processes that result in secretion of inflammatory mediators. To determine the Fc(epsilon)RI proximity, cluster size, and mobility requirements for initiating the Fc(epsilon)RI cascade, a novel experimental protocol has been developed in which mast cells are reacted with glass surfaces carrying different densities of both antigen and bound IgE, and the cell's secretory response to these stimuli is measured. The results have been analyzed in terms of a model based on the following assumptions: 1) the glass surface antigen distribution and consequently that of the bound IgE are random; 2) Fc(epsilon)RI binding to these surface-bound IgEs immobilizes the former and saturates the latter; 3) the cell surface is formally divided into small elements, which function as a secretory stimulus unit when occupied by two or more immobilized IgE-Fc(epsilon)RI complexes; 4) alternatively, similar stimulatory units can be formed by binding of surface-carried IgE dimers to two Fc(epsilon)RI. This model yielded a satisfactory and self-consistent fitting of all of the different experimental data sets. Hence the present results establish the essential role of Fc(epsilon)RI immobilization for initiating its signaling cascade. Moreover, it provides independent support for the notion that as few as two Fc(epsilon)RIs immobilized at van der Waals contact constitute an "elementary stimulatory unit" leading to mast cell (RBL-2H3 line) secretory response

pH-induced conformational changes of the Fe(2+)-N epsilon (His F8) linkage in deoxyhemoglobin trout IV detected by the Raman active Fe(2+)-N epsilon (His F8) stretching mode.

To investigate heme-protein coupling via the Fe(2+)-N epsilon (His F8) linkage we have measured the profile of the Raman band due to the Fe(2+)-N epsilon (His F8) stretching mode (nu Fe-His) of deoxyHb-trout IV and deoxyHbA at various pH between 6.0 and 9.0. Our data establish that the band of this mode is composed of five different sublines. In deoxyHb-trout IV, three of these sublines were assigned to distinct conformations of the alpha-subunit (omega alpha 1 = 202 cm-1, omega alpha 2 = 211 cm-1, omega alpha 3 = 217 cm-1) and the other two to distinct conformations of the beta-subunit (omega beta 1 = 223 cm-1 and omega beta 2 = 228 cm-1). Human deoxyHbA exhibits two alpha-chain sublines at omega alpha 1 = 203 cm-1, omega alpha 2 = 212 cm-1 and two beta-chain sublines at omega beta 1 = 217 cm-1 and omega beta 2 = 225 cm-1. These results reveal that each subunit exists in different conformations. The intensities of the nu Fe-His sublines in deoxyHb-trout IV exhibit a significant pH dependence, whereas the intensities of the corresponding sublines in the deoxyHbA spectrum are independent on pH. This finding suggests that the structural basis of the Bohr effect is different in deoxyHbA and deoxyHb-trout IV. To analyse the pH dependence of the deoxyHb-trout IV sublines we have applied a titration model describing the intensity of each nu Fe-His subline as an incoherent superposition of the intensities from sub-sublines with the same frequency but differing intrinsic intensities due to the different protonation states of the respective subunit. The molar fractions of these protonation states are determined by the corresponding Bohr groups (i.e., pK alpha 1 = pK alpha 2 = 8.5, pK beta 1 = 7.5, pK beta 2 = 7.4) and pH. Hence, the intensities of these sublines reflect the pH dependence of the molar fractions of the involved protonation states. Fitting this model to the pH-dependent line intensities yields a good reproduction of the experimental data. To elucidate the structural basis of the observed results we have employed models proposed by Bangchoroenpaurpong, O., K. T. Schomaker, and P. M. Champion. (1984. J. Am. Chem. Soc. 106:5688-5698) and Friedman, J. M., B. F. Campbell, and R. W. Noble. (1990. Biophys. Chem. 37:43-59) which describe the coupling between the sigma *orbitals of the Fe2+-NJ(His F8) bond and the phi * orbitals of the pyrrole nitrogens in terms of the tilt angle theta between its Fe2+-N,(HisF8)-bond and the heme normal and the azimuthal angle phi between the Fe2+-N.(His F8) projection on the heme and the N1-N3 axis.Our results indicate that each subconformation reflected by different frequencies of the VFe His-subline is related to different tilt angles theta, whereas the pH-induced intensity variations of each VFe His subline of the deoxy Hb trout IV spectrum are caused by changes of the azimuthal angle phi

Correspondence of the pK values of oxyHb-titration states detected by resonance Raman scattering to kinetic data of ligand dissociation and association.

The dispersion of the depolarization ratio of oxidation and spinmarker lines of oxyhemoglobin at low C1- concentration (less than 0.08 M) have been examined for different pH values in the acid and alkaline region. Interpreting the depolarization ratio dispersion curves by fifth order Loudon theory of the polarizibility tensor, we obtain tensor parameters depending linearly on symmetry classified distortions of the functional hemegroup. The pH dependence of these parameters are explained by assuming the influence of three titrable groups with pK = 7.8, 6.6, and 5.8 on the heme. Using these pK values, we are able to interpret the pH dependence of CO(O2)-dissociation and CO-association of the fourth hemoglobin subunit. We conclude from our measurements that the change of the Tyr HC2 beta-configuration induces heme-apoprotein interaction via the Tyr HC2 beta-Val FG5 beta H-bond, which are transduced to the heme via central and peripheral coupling

Dimerization kinetics of the IgE-class antibodies by divalent haptens. I. The Fab-hapten interactions.

The binding of divalent haptens to IgE-class antibodies leads predominantly to their oligomerization into open and closed dimers. Kinetics of the open dimer formation was investigated by fluorescence titrations of Fab fragments of monoclonal DNP-specific IgE antibodies with divalent haptens having different spacer length (gamma = 14-130 A). Binding was monitored by quenching of intrinsic tryptophan emission of the Fab. Addition of divalent haptens with short spacers (gamma = 14-21 A) to the Fabs at rates larger than a distinct threshold value caused a significant decrease of Fab-binding site occupation in the initial phase of the titration. This finding was interpreted to reflect a nonequilibrium state of hapten-Fab-binding. Such nonequilibrium titrations were analyzed by inserting a kinetic model into a theory of antibody aggregation as presented by Dembo and Golstein (Histamine release due to bivalent penicilloyl haptens. 1978. J. Immunol. 121, 345). Fitting of this model to the fluorescence titrations yielded dissociation rate constants of 7.8 x 10(-3) s-1 and 6 x 10(-3) s-1 for the Fab dimers formed by the flexible divalent haptens N alpha, N epsilon-di(dinitrophenyl)-L-lysine (gamma = 16 A) and bis(N beta-2,4-dinitrophenyl-alanyl)-meso-diamino-succinate (gamma = 21 A). Making the simplifying assumption that a single step binding equilibrium prevails, the corresponding dimer formation rate constants were calculated to be 1.9 x 10(5) M-1 s-1 and 1.1 x 10(4) M-1 s-1, respectively. In contrast, all haptens with spacers longer than 40 A (i.e., bis(N alpha-2,4-dinitrophenyl-tri-D-alanyl)-1,7-diamino-heptane, and di(N epsilon-2,4-dinitrophenyl)-6-aminohexanoate-aspartyl-(prolyl)n-L-l ysyl (n = 24, 27, 33) exhibit a relative fast dimerization rate of the Fab fragments (greater than 7 x 10(6) M-1 s-1). These observations were interpreted as being caused by orientational constraints set by the limited solid angle of the reaction between the macromolecular reactants. Thus, ligands having better access to the binding site would react faster