Gusztáv Schay

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R- and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl–, 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (αFeO2)2-(βZn)2 as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70–200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl–. We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states.

Conformational Dynamics of Titin PEVK Explored with FRET Spectroscopy

The proline-, glutamate-, valine-, and lysine-rich (PEVK) domain of the giant muscle protein titin is thought to be an intrinsically unstructured random-coil segment. Various observations suggest, however, that the domain may not be completely devoid of internal interactions and structural features. To test the validity of random polymer models for PEVK, we determined the mean end-to-end distances of an 11- and a 21-residue synthetic PEVK peptide, calculated from the efficiency of the fluorescence resonance energy transfer (FRET) between an N-terminal intrinsic tryptophan donor and a synthetically added C-terminal IAEDANS acceptor obtained in steady-state and time-resolved experiments. We find that the contour-length scaling of mean end-to-end distance deviates from predictions of a purely statistical polymer chain. Furthermore, the addition of guanidine hydrochloride decreased, whereas the addition of salt increased the FRET efficiency, pointing at the disruption of structure-stabilizing interactions. Increasing temperature between 10 and 50°C increased the normalized FRET efficiency in both peptides but with different trajectories, indicating that their elasticity and conformational stability are different. Simulations suggest that whereas the short PEVK peptide displays an overall random structure, the long PEVK peptide retains residual, loose helical configurations. Transitions in the local structure and dynamics of the PEVK domain may play a role in the modulation of passive muscle mechanics.

Millisecond time-scale protein dynamics exists prior to the activation of the bulk solvent matrix

Conformational dynamics of proteins is of fundamental importance in their physiological functions. The exact mechanisms and determinants of protein motions, including the regulatory interplay between protein and solvent motions, are not yet fully understood. In the present work, the thermal activation of phosphorescence quenching was measured in oxygen-saturated aqueous protein solutions to explore protein dynamics in the millisecond range. The sample was brought to cryogenic temperatures in a fast cooling process to avoid the bulk crystallization of ice. The phosphorescence quenching effect was followed by the phosphorescence lifetime of either Zn-protoporphyrin substituting the heme in the β-subunits of human hemoglobin (Zn-HbA) or tryptophan residues of Zn-HbA and human myoglobin (Mb), measured in thermal equilibrium at temperatures varied from 8 to 273 K. The quenching effect was attributed primarily to the activation of collisions with O(2) molecules made possible by the activated millisecond time-scale dynamics of the matrix around the chromophores. We find that, in the studied temperature range, the activation of protein global dynamics facilitating oxygen diffusion takes place at clearly separated lower temperatures and independently from bulk solvent dynamics and that the energy and entropy differences between the studied frozen and thermally activated states are specific for the protein.

Without Binding ATP, Human Rad51 Does Not Form Helical Filaments on ssDNA.

Construction of the presynaptic filament (PSF) of proper helical structure by Rad51 recombinases is a prerequisite of the progress of homologous recombination repair. We studied the contribution of ATP-binding to this structure of wt human Rad51 (hRad51). We exploited the protein-dissociation effect of high hydrostatic pressure to determine the free energy of dissociation of the protomer interfaces in hRad51 oligomer states and used electron microscopy to obtain topological parameters. Without cofactors ATP and Ca(2+) and template DNA, hRad51 did not exist in monomer form, but it formed rodlike long filaments without helical order. ΔG(diss) indicated a strong inherent tendency of aggregation. Binding solely ssDNA left the filament unstructured with slightly increased ΔG(diss). Adding only ATP and Ca(2+) to the buffer disintegrated the self-associated rods into rings and short helices of further increased ΔG(diss). Rad51 binding to ssDNA only with ATP and Ca bound could lead to ordered helical filament formation of proper pitch size with interface contacts of K(d) ∼ 2 × 10(-11) M, indicating a structure of outstanding stability. ATP/Ca binding increased the ΔG(diss) of protomer contacts in the filament by 16 kJ/mol. The results emphasize that ATP-binding in the PSF of hRad51 has an essential, yet purely structural, role.

Role of Domain Interactions in the Collective Motion of Phosphoglycerate Kinase

Protein function is governed by the underlying conformational dynamics of the molecule. The experimental and theoretical work leading to contemporary understanding of enzyme dynamics was mostly restricted to the large-scale movements of single-domain proteins. Collective movements resulting from a regulatory interplay between protein domains is often crucial for enzymatic activity. It is not clear, however, how our knowledge could be extended to describe collective near-equilibrium motions of multidomain enzymes. We examined the effect of domain interactions on the low temperature near equilibrium dynamics of the native state, using phosphoglycerate kinase as model protein. We measured thermal activation of tryptophan phosphorescence quenching to explore millisecond-range protein motions. The two protein domains of phosphoglycerate kinase correspond to two dynamic units, but interdomain interactions link the motion of the two domains. The effect of the interdomain interactions on the activation of motions in the individual domains is asymmetric. As the temperature of the frozen protein is increased from the cryogenic, motions of the N domain are activated first. This is a partial activation, however, and the full dynamics of the domain becomes activated only after the activation of the C domain.

Dissimilar flexibility of α and β subunits of human adult hemoglobin influences the protein dynamics and its alteration induced by allosteric effectors

The general question by what mechanism an “effector” molecule and the hemes of hemoglobin interact over widely separated intramolecular distances to change the oxygen affinity has been extensively investigated, and still has remained of central interest. In the present work we were interested in clarifying the general role of the protein matrix and its dynamics in the regulation of human adult hemoglobin (HbA). We used a spectroscopy approach that yields the compressibility (κ) of the protein matrix around the hemes of the subunits in HbA and studied how the binding of heterotropic allosteric effectors modify this parameter. κ is directly related to the variance of volume fluctuation, therefore it characterizes the molecular dynamics of the protein structure. For the experiments the heme groups either in the α or in the β subunits of HbA were replaced by fluorescent Zn-protoporphyrinIX, and series of fluorescence line narrowed spectra were measured at varied pressures. The evaluation of the spectra yielded the compressibility that showed significant dynamic asymmetry between the subunits: κ of the α subunit was 0.17±0.05/GPa, while for the β subunit it was much higher, 0.36±0.07/GPa. The heterotropic effectors, chloride ions, inositol hexaphosphate and bezafibrate did not cause significant changes in κ of the α subunits, while in the β subunits the effectors lead to a significant reduction down to 0.15±0.04/GPa. We relate our results to structural data, to results of recent functional studies and to those of molecular dynamics simulations, and find good agreements. The observed asymmetry in the flexibility suggests a distinct role of the subunits in the regulation of Hb that results in the observed changes of the oxygen binding capability.