Ernesto E. Di Iorio

A Novel Hamiltonian Replica Exchange MD Protocol to Enhance Protein Conformational Space Sampling

Limited searching in the conformational space is one of the major obstacles for investigating protein dynamics by numerical approaches. For this reason, classical all-atom molecular dynamics (MD) simulations of proteins tend to be confined to local energy minima, particularly when the bulk solvent is treated explicitly. To overcome this problem, we have developed a novel replica exchange protocol that uses modified force-field parameters to treat interparticle nonbonded potentials within the protein and between protein and solvent atoms, leaving unperturbed those relative to solvent-solvent interactions. We have tested the new protocol on the 18-residue-long tip of the P domain of calreticulin in an explicit solvent. With only eight replicas, we have been able to considerably enhance the conformational space sampled during a 100 ns simulation, compared to as many parallel classical molecular dynamics simulations of the same length or to a single one lasting 450 ns. A direct comparison between the various simulations has been possible thanks to the implementation of the weighted histogram analysis method, by which conformations simulated with modified force-field parameters can be assigned different weights. Interatom, inter-residue distances in the structural ensembles obtained with our novel replica exchange approach and by classical MD simulations compare equally well with those derived from NMR data. Rare events, such as unfolding and refolding, occur with reasonable statistical frequency. Visiting of conformations characterized by very small Boltzmann weights is also possible. Despite their low probability, such regions of the conformational space may play an important role in the search for local potential-energy minima and in dynamically controlled functions.

Molecular Dynamics Simulations of Human Glutathione Transferase P1-1: Analysis of the Induced-Fit Mechanism by GSH Binding

We report here a 1‐ns molecular dynamics simulation on the ligand‐free monomer of human glutathione transferase P1‐1 in bulk water. The average conformation obtained from the last 500 ps of simulation is taken as a model for the apo‐structure of this protein and compared to the available crystallographic data. Remarkable changes in the tertiary structure take place during the simulation and are ascribed to the removal of the ligand. They support an induced fit mechanism occurring upon glutathione binding, whose major features can be described in detail. A portion of helix 2 (residues 42–50), which participates in the formation of the active site, undergoes the most prominent conformational changes. Other protein segments, such as the C‐terminal loop and helix 4, also show relevant structural rearrangements. All these transitions cause a significant shielding from the solvent of the hydrophobic binding site of the co‐substrate, whose exposed surface goes from 4.6 nm 2 in the holo‐structure to about 3.1 nm 2 in the apo‐conformation. The results of this simulation are consistent with numerous experimental observations previously obtained on GST P1‐1 and provide new insights for their explanation at the molecular level. Proteins 1999;37:1–9.

MOLECULAR DYNAMICS SIMULATIONS OF HUMAN GLUTATHIONE TRANSFERASE P1-1 : CONFORMATIONAL FLUCTUATIONS OF THE APO-STRUCTURE

We have investigated by molecular dynamics simulations the conformational fluctuations of the monomer of human apo‐glutathione transferase P1–1. After attainment of steady‐state dynamics, the structural fluctuations involve mainly the protein segments that participate also in the holo‐apo transition discussed in the accompanying article (Stella et al., 1999:37:1–9.). The most mobile region is the C‐terminal segment of helix 2. In contrast, helices 1, 6, 7, and 8 constitute a relatively rigid protein core. An “essential dynamics” analysis of the simulation shows that the largest fluctuations involve specific regions of glutathione transferases. In such regions, atomic motions are correlated. Motions of helix 2 are accounted for by the second most prominent principal component, which reveals a fluctuation between two distinct conformations. The residues that constitute the H‐site undergo a breathing motion, possibly relevant during the binding of hydrophobic cosubstrates. Based on our simulation, several experimental findings can be rationalized, including the viscosity‐dependent reactivity of Cys 47 and Cys 101 as well as the selective proteolysis of the peptide bond between Lys 44 and Ala 45. We have also modeled the structural changes that lead to the formation of an intrachain disulfide bridge between cysteines 47 and 101 and to the inactivation of the enzyme. The resulting structure maintains essentially the native fold except for helix 2, which closes the G‐site. Proteins 1999;37:10–19.

Protein Dynamics, Thermal Stability, and Free-Energy Landscapes: A Molecular Dynamics Investigation

Proteins have a complex free-energy landscape because of their rich topology and the nature of their nonbonded interaction potential. This has important consequences because the roughness of the landscape affects the ease with which a chain folds and also determines the dynamic behavior of the folded structure, thus influencing its functional and stability properties. A detailed description of the free-energy landscape is therefore of paramount importance for a quantitative understanding of the relationships between structure, dynamics, stability, and functional behavior of proteins. The free-energy landscape of a protein is a high-dimensional hypersurface, difficult to rationalize. Therefore, achieving its detailed graphical representation in a way that goes beyond the familiar funnel-like free-energy model is still a big challenge. We describe here an approach based on global structural parameters that allows a two-dimensional representation of the free-energy landscape from simulated atomic trajectories. As shown in this and in the accompanying article, our representation of the landscape, combined with other conformational analyses, provides valuable information on its roughness and on how atomic trajectories evolve with time.

Electron paramagnetic resonance properties of liver fluke (Dicrocoelium dendriticum) nitrosyl hemoglobin

The electron paramagnetic resonance properties of the nitric oxide derivative of liver fluke (Dicrocoelium dendriticum) hemoglobin (DD‐Hb) have been investigated in the pH range from 4.8 to 7.8. In the neutral and alkaline regions the spectra have a rhombic shape, with gx =2.09, gy =1.99 and gz =2.009, and a triplet hyperfine structure of 2.2 mT, due to the nitrogen of the bound NO molecule, in the center resonance. No superhyperfine lines in the gz region, related to the interaction of the iron with the proximal histidine, are detected, suggesting a large distance between the metal and the Nϵ of the imidazole. By lowering the pH the EPR spectrum undergoes a reversible change showing a 3‐line pattern in the high‐field region. Such a spectrum is fully formed at pH 4.8 and is interpreted in terms of a dissociation of the proximal histidine from the heme iron.

Erythrocytic Proteases: Preferential Degradation of Alpha Hemoglobin Chains

Proteolytic activity against native hemoglobin polypeptide chains is demonstrated, under strictly physiological conditions, in human reticulocytes of both normal subjects and individuals suffering from a variety of pathologic conditions involving erythrocytes, including beta-thalassemia. Two thirds of the activity are found in the cytoplasm and the remainder of it is associated with the reticulocyte membrane. That this proteolytic activity is due to contamination by WBC is excluded. The activity preferentially degrades the alpha-hemoglobin chains. An increase in this substrate within the erythroid cells, as observed in beta-thalassemia, does not enhance proteolysis. Protease inhibitors produce a variable decrease in proteolysis. None inhibit completely, thus showing that several enzymes, with different specificities, are involved.

Isoelectric focusing of cytochrome P450: Isolation of six phenobarbital-inducible rat liver microsomal isoenzymes

A procedure for the isolation of native proteins from membranes by isoelectric focusing is described. It was used to resolve into six components the major fraction of cytochrome P450, obtained from liver microsomes of phenobarbital-treated rats, after chromatography on DE-52 cellulose. When eluted from the gel, these proteins are in a native form as shown by (a) the light absorption spectra of the Soret region of their reduced carbonyl derivatives, all characterized by maxima around 450 nm, and (b) their enzymatic activities toward three different substrates. Characterization by a monoclonal antibody and partial sequence analysis of tryptic peptides reveal that three of the IEF-purified proteins have P450IIB1 character, whereas the other three are related to P450IIB2.

Dynamics of RNase-A and S-Protein: A Molecular Dynamics Simulation of the Transition Toward a Folding Intermediate

The description at atomic level of protein folding is an ambitious goal in biophysics, particularly because of the difficulty in obtaining structural information on unfolded states. Computer simulations can contribute in achieving this goal. Here we report the results of a 10-ns comparative simulation on bovine ribonuclease A and its S-protein, obtained by removal from the native molecule of the first 20 residues, the so-called S-peptide. The atomic trajectories have been analyzed by standard procedures and by applying concepts previously developed for disordered systems. Furthermore, we used a novel approach, described in the preceding paper, to represent graphically the energy landscape of the simulated systems. Relative to RNase-A, the S-protein, while largely maintaining its structural organization, displays an increased structural flexibility, it gains ergodicity and its core loses order, thus indicating that the removal of the S-peptide from ribonuclease A triggers the transition to a folding intermediate with reduced compactness. This finding also has biochemical relevance since the S-protein is recognized as not properly folded by the machinery responsible for the control of the folding quality in the endoplasmic reticulum.

Kinetic properties of cobalt—iron hybrid hemoglobins

The replacement of O2 with CO was studied on cobalt—iron hemoglobin hybrids. Both proto‐ and meso‐cobalt hemes were used for the reconstitution. In the oxy quaternary conformation no difference is observed between α‐ and β‐subunits when only proto hemes are present in the hybrid (k 4 = 30 s−1, k′ 4/l′ 4 = 2.5). If Co‐meso heme is present on the β‐chains the binding properties of the partner subunit are modified (k′ 4/l′ 4 = 4).

Protein dynamics An overview on flash‐photolysis over broad temperature ranges

Abstract Ligand binding kinetics to heme-proteins between 40 and 300 K point to a regulatory role of protein dynamics. A protein-specific susceptibility of the heme-iron reactivity to dynamic fluctuations emerges from the distribution of reaction enthalpies derived from flash-photolysis measurements below ca. 180 K; we quantify it in terms of ‘intramolecular viscosity’, postulating that narrow low-temperature enthalpy distributions correspond to low internal viscosity and vice versa. The thermal evolution of ligand binding kinetics suggests, with other results, an interplay between high-frequency transitions of the amino acid side chains and low-frequency collective motions as a possible regulatory mechanism of protein dynamics.Ligand binding kinetics to heme‐proteins between 40 and 300 K point to a regulatory role of protein dynamics. A protein‐specific susceptibility of the heme‐iron reactivity to dynamic fluctuations emerges from the distribution of reaction enthalpies derived from flash‐photolysis measurements below ca. 180 K; we quantify it in terms of ‘intramolecular viscosity’, postulating that narrow low‐temperature enthalpy distributions correspond to low internal viscosity and vice versa. The thermal evolution of ligand binding kinetics suggests, with other results, an interplay between high‐frequency transitions of the amino acid side chains and low‐frequency collective motions as a possible regulatory mechanism of protein dynamics.