Biomedical subjects

David A Case

Publications and source records attributed to David A Case.

At least 37 records · Page 2Linked to original sources

PubMed2004

NMR structure of a cyclic polyamide-DNA complex.

The solution structure of a cyclic polyamide ligand complexed to a DNA oligomer, derived from NMR restrained molecular mechanics, is presented. The polyamide, cyclo-gamma-ImPyPy-gamma-PyPyPy-, binds to target DNA with a nanomolar dissociation constant as characterized by quantitative footprinting previously reported. 2D (1)H NMR data were used to generate distance restraints defining the structure of this cyclic polyamide with the DNA duplex d(5'-GCCTGTTAGCG-3'):d(5'-CGCTAACAGGC-3'). Data interpretation used complete relaxation matrix analysis of the NOESY cross-peak intensities with the program MARDIGRAS. The NMR-based distance restraints (276 total) were applied in restrained molecular dynamics calculations using a solvent model, yielding structures with an rmsd for the ligand and binding site of approximately 1 A. The resulting structures indicate some distortion of the DNA in the binding site. The constraints from cyclization lead to altered stacking of the rings in the halves of the cyclic ligand relative to unlinked complexes. Despite this, the interactions with DNA are very similar to what has been found in unlinked complexes. Measurements of ligand amide and DNA imino proton exchange rates indicate very slow dissociation of the ligand and show that the DNA can undergo opening fluctuations while the ligand is bound although the presence of the ligand decreases their frequency relative to the free DNA.

Amides↗

PubMed2004

Exploring protein native states and large-scale conformational changes with a modified generalized born model.

Implicit solvation models provide, for many applications, a reasonably accurate and computationally effective way to describe the electrostatics of aqueous solvation. Here, a popular analytical Generalized Born (GB) solvation model is modified to improve its accuracy in calculating the solvent polarization part of free energy changes in large-scale conformational transitions, such as protein folding. In contrast to an earlier GB model (implemented in the AMBER-6 program), the improved version does not overstabilize the native structures relative to the finite-difference Poisson-Boltzmann continuum treatment. In addition to improving the energy balance between folded and unfolded conformers, the algorithm (available in the AMBER-7 and NAB molecular modeling packages) is shown to perform well in more than 50 ns of native-state molecular dynamics (MD) simulations of thioredoxin, protein-A, and ubiquitin, as well as in a simulation of Barnase/Barstar complex formation. For thioredoxin, various combinations of input parameters have been explored, such as the underlying gas-phase force fields and the atomic radii. The best performance is achieved with a previously proposed modification to the torsional potential in the Amber ff99 force field, which yields stable native trajectories for all of the tested proteins, with backbone root-mean-square deviations from the native structures being approximately 1.5 A after 6 ns of simulation time. The structure of Barnase/Barstar complex is regenerated, starting from an unbound state, to within 1.9 A relative to the crystal structure of the complex.

Bacterial Proteins↗

PubMed2004

Proton binding to proteins: pK(a) calculations with explicit and implicit solvent models.

Ionizable residues play important roles in protein structure and activity, and proton binding is a valuable reporter of electrostatic interactions in these systems. We use molecular dynamics free energy simulations (MDFE) to compute proton pKa shifts, relative to a model compound in solution, for three aspartate side chains in two proteins. Simulations with explicit solvent and with an implicit, dielectric continuum solvent are reported. The implicit solvent simulations use the generalized Born (GB) model, which provides an approximate, analytical solution to Poisson's equation. With explicit solvent, the direction of the pKa shifts is correct in all three cases with one force field (AMBER) and in two out of three cases with another (CHARMM). For two aspartates, the dielectric response to ionization is found to be linear, even though the separate protein and solvent responses can be nonlinear. For thioredoxin Asp26, nonlinearity arises from the presence of two substates that correspond to the two possible orientations of the protonated carboxylate. For this side chain, which is partly buried and has a large pKa upshift, very long simulations are needed to correctly sample several slow degrees of freedom that reorganize in response to the ionization. Thus, nearby Lys57 rotates to form a salt bridge and becomes buried, while three waters intercalate along the opposite edge of Asp26. Such strong and anisotropic reorganization is very difficult to predict with Poisson-Boltzmann methods that only consider electrostatic interactions and employ a single protein structure. In contrast, MDFE with a GB dielectric continuum solvent, used for the first time for pKa calculations, can describe protein reorganization accurately and gives encouraging agreement with experiment and with the explicit solvent simulations.

Animals↗

PubMed2004

High-resolution field-cycling NMR studies of a DNA octamer as a probe of phosphodiester dynamics and comparison with computer simulation.

Phosphorus-spin longitudinal relaxation rates of the DNA duplex octamer [d(GGAATTCC)](2) have been measured from 0.1 to 17.6 T by means of conventional and new field-cycling NMR methods. The high-resolution field-cycling method is identical to a conventional relaxation experiment, except that after preparation the sample is moved pneumatically from its usual position at the center of the high-resolution magnet upward to a lower field above its normal position and then returned to the center for readout after it has relaxed for the programmed relaxation delay at the low field. This is the first measurement of all longitudinal relaxation rates R(1) of a nuclear species in a macromolecule over virtually the entire accessible magnetic field range. For detailed analysis, three magnetic field regions can be delineated: (i) dipolar relaxation dominates at fields below 2 T, (ii) chemical shift anisotropy (CSA) relaxation is roughly constant from 2 to 6 T, and (iii) a square-law increasing dependence is seen at fields higher than approximately 6 T due to internal motion CSA relaxation. The analysis provides a rotational correlation time (tau(r) = 4.1 +/- 0.3 ns) for the duplex at both 1.5 and 0.25 mM concentrations (of duplex) at 22 degrees C. For comparison, extraction of tau(r) in the conventional way from the ratio of T(1)/T(2) at 14 T yields 3.2 ns. The tau(r) discrepancy disappears when we exclude the contribution of internal motion from the R(1) in the ratio. The low-field dipolar relaxation provides a weighted inverse sixth power sum of the distances from the phosphorus to the protons responsible for relaxation. This average is similar for all phosphates in the octamer and similar to that in previous B-DNA structures (its inverse sixth root is about 2.40 A for two different concentrations of octamer). The CSA relaxation at intermediate field provides an estimate of the order parameter squared, S(c)(2), for each phosphorus. S(c)(2) is about 0.7-1, clearly different for different phosphate linkages in the octamer duplex. The increasing R(1) at high fields reflects CSA relaxation due to internal motions, for which a correlation time, tau(hf), can be approximately extracted with the aid of additional measurements at 14.0 and 17.6 T. We conclude that tau(hf) values are relatively large, in the range of about 150 ps. Insight into the motions leading to this correlation time was gained by a 28 ns molecular dynamics simulation of the molecule. S(2) and tau(s) (corresponding to tau(hf)) predicted by this simulation were in good agreement with the experimental values from the field-cycling data. Both the effect of Mg(2+) on the dynamic parameters extracted from (31)P relaxation rates and the field dependence of relaxation rates for several protons of the octamer were measured. High-resolution field cycling opens up the possibility of monitoring residue-specific dipolar interactions and dynamics for the phosphorus nuclei of diverse oligonucleotides.

Cations, Divalent↗

PubMed2004

Performance comparison of generalized born and Poisson methods in the calculation of electrostatic solvation energies for protein structures.

This study compares generalized Born (GB) and Poisson (PB) methods for calculating electrostatic solvation energies of proteins. A large set of GB and PB implementations from our own laboratories as well as others is applied to a series of protein structure test sets for evaluating the performance of these methods. The test sets cover a significant range of native protein structures of varying size, fold topology, and amino acid composition as well as nonnative extended and misfolded structures that may be found during structure prediction and folding/unfolding studies. We find that the methods tested here span a wide range from highly accurate and computationally demanding PB-based methods to somewhat less accurate but more affordable GB-based approaches and a few fast, approximate PB solvers. Compared with PB solvation energies, the latest, most accurate GB implementations were found to achieve errors of 1% for relative solvation energies between different proteins and 0.4% between different conformations of the same protein. This compares to accurate PB solvers that produce results with deviations of less than 0.25% between each other for both native and nonnative structures. The performance of the best GB methods is discussed in more detail for the application for force field-based minimizations or molecular dynamics simulations.

Hydrogen Bonding↗

PubMed2004

Converging free energy estimates: MM-PB(GB)SA studies on the protein-protein complex Ras-Raf.

Estimating protein-protein interaction energies is a very challenging task for current simulation protocols. Here, absolute binding free energies are reported for the complex H-Ras/C-Raf1 using the MM-PB(GB)SA approach, testing the internal consistency and model dependence of the results. Averaging gas-phase energies (MM), solvation free energies as determined by Generalized Born models (GB/SA), and entropic contributions calculated by normal mode analysis for snapshots obtained from 10 ns explicit-solvent molecular dynamics in general results in an overestimation of the binding affinity when a solvent-accessible surface area-dependent model is used to estimate the nonpolar solvation contribution. Applying the sum of a cavity solvation free energy and explicitly modeled solute-solvent van der Waals interaction energies instead provides less negative estimates for the nonpolar solvation contribution. When the polar contribution to the solvation free energy is determined by solving the Poisson-Boltzmann equation (PB) instead, the calculated binding affinity strongly depends on the atomic radii set chosen. For three GB models investigated, different absolute deviations from PB energies were found for the unbound proteins and the complex. As an alternative to normal-mode calculations, quasiharmonic analyses have been performed to estimate entropic contributions due to changes of solute flexibility upon binding. However, such entropy estimates do not converge after 10 ns of simulation time, indicating that sampling issues may limit the applicability of this approach. Finally, binding free energies estimated from snapshots of the unbound proteins extracted from the complex trajectory result in an underestimate of binding affinity. This points to the need to exercise caution in applying the computationally cheaper "one-trajectory-alternative" to systems where there may be significant changes in flexibility and structure due to binding. The best estimate for the binding free energy of Ras-Raf obtained in this study of -8.3 kcal mol(-1) is in good agreement with the experimental result of -9.6 kcal mol(-1), however, further probing the transferability of the applied protocol that led to this result is necessary.

Calorimetry↗

PubMed2004

Constant pH molecular dynamics in generalized Born implicit solvent.

A new method is proposed for constant pH molecular dynamics (MD), employing generalized Born (GB) electrostatics. Protonation states are modeled with different charge sets, and titrating residues sample a Boltzmann distribution of protonation states as the simulation progresses, using Monte Carlo sampling based on GB-derived energies. The method is applied to four different crystal structures of hen egg-white lysozyme (HEWL). pK(a) predictions derived from the simulations have root-mean-square (RMS) error of 0.82 relative to experimental values. Similarity of results between the four crystal structures shows the method to be independent of starting crystal structure; this is in contrast to most electrostatics-only models. A strong correlation between conformation and protonation state is noted and quantitatively analyzed, emphasizing the importance of sampling protonation states in conjunction with dynamics.

Algorithms↗

PubMed2003

Reintroducing electrostatics into protein X-ray structure refinement: bulk solvent treated as a dielectric continuum.

Structural refinement of proteins involves the minimization of a target function that combines X-ray data with a set of restraints enforcing stereochemistry and packing. Electrostatic interactions are not ordinarily included in the target function, partly because they cannot be calculated reliably without a description of dielectric screening by solvent in the crystal. With the recent development of accurate implicit solvent models to describe this screening, the question arises as to whether a more detailed target function including electrostatic and solvation terms can yield more accurate structures or somewhat different structures of equivalent accuracy. The Generalized Born (GB) model is one such model that describes the solvent as a dielectric continuum, taking into account its heterogeneous distribution within the crystal. It is used here for X-ray refinements of three protein structures with experimental diffraction data to 2.4, 2.9 and 3.2 A, respectively. In each case, a higher resolution structure is available for comparison. The new target function includes stereochemical restraints, van der Waals, Coulomb and solvation interactions, along with the usual X-ray pseudo-energy term, which employs the likelihood estimator of Pannu and Read. Multiple simulated-annealing refinements were performed in torsion-angle space with a conventional target function and the new GB target function, yielding ensembles of refined structures. The new target function yields structures of similar accuracy, as measured by the free R factor, map/model correlations and deviations from the high-resolution structures. About 10% of side-chain conformations differ between the two sets of refinements, in the sense that the two ensembles of conformations do not completely overlap. Over 75% of the differences correspond to surface side chains. For one of the proteins, the GB set has a greater dispersion, indicating that for this case the conventional target function overestimates the true precision. As GB parameterization continues to improve, we expect that this approach will become increasingly useful.

Aspartate-tRNA Ligase↗

PubMed2003

Design and synthesis of highly constrained factor Xa inhibitors: amidine-substituted bis(benzoyl)--diazepan-2-ones and bis(benzylidene)-bis(gem-dimethyl)cycloketones.

Two conformationally constrained templates have been designed to provide selective inhibitors of the coagulation cascade serine protease, Factor Xa (FXa). The most active inhibitor, 2,7-bis[(Z)-p-amidinobenzylidene)]-3,3,6,6-tetramethylcycloheptanone, exhibits a K(i) of 42 nM against FXa, with strong selectivity against thrombin (1000-fold), trypsin (300-fold) and plasmin (900-fold). With only two freely rotatable bonds, molecular modeling suggests that one amidine group is positioned into the S1 pocket, forming hydrogen bonds with the side chain of Asp189, similar to other amidine-based inhibitors, with the second benzamidine positioned into the S4 pocket in a position to form strong cation-pi bonding with the S4 aryl cage. We suggest that this interaction plays an important role in the specificity of these inhibitors against other serine proteases.

Antithrombin III↗

PubMed2003

Insights into the mobility of methyl-bearing side chains in proteins from (3)J(CC) and (3)J(CN) couplings.

Side-chain dynamics in proteins can be characterized by the NMR measurement of (13)C and (2)H relaxation rates. Evaluation of the corresponding spectral densities limits the slowest motions that can be studied quantitatively to the time scale on which the overall molecular tumbling takes place. A different measure for the degree of side-chain order about the C(alpha)-C(beta) bond (chi(1) angle) can be derived from (3)J(C)(')(-)(C)(gamma) and (3)J(N)(-)(C)(gamma) couplings. These couplings can be measured at high accuracy, in particular for Thr, Ile, and Val residues. In conjunction with the known backbone structures of ubiquitin and the third IgG-binding domain of protein G, and an extensive set of (13)C-(1)H side-chain dipolar coupling measurements in oriented media, these (3)J couplings were used to parametrize empirical Karplus relationships for (3)J(C)(')(-)(C)(gamma) and (3)J(N)(-)(C)(gamma). These Karplus curves agree well with results from DFT calculations, including an unusual phase shift, which causes the maximum (3)J(CC) and (3)J(CN) couplings to occur for dihedral angles slightly smaller than 180 degrees, particularly noticeable in Thr residues. The new Karplus curves permit determination of rotamer populations for the chi(1) torsion angles. Similar rotamer populations can be derived from side-chain dipolar couplings. Conversion of these rotamer populations into generalized order parameters, S(J)(2) and S(D)(2), provides a view of side-chain dynamics that is complementary to that obtained from (13)C and (2)H relaxation. On average, results agree well with literature values for (2)H-relaxation-derived S(rel)(2) values in ubiquitin and HIV protease, but also identify a fraction of residues for which S(J,D)(2) < S(rel)(2). This indicates that some of the rotameric averaging occurs on a time scale too slow to be observable in traditional relaxation measurements.

HIV Protease↗

PubMed2003

Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes.

Absolute binding free energy calculations and free energy decompositions are presented for the protein-protein complexes H-Ras/C-Raf1 and H-Ras/RalGDS. Ras is a central switch in the regulation of cell proliferation and differentiation. In our study, we investigate the capability of the molecular mechanics (MM)-generalized Born surface area (GBSA) approach to estimate absolute binding free energies for the protein-protein complexes. Averaging gas-phase energies, solvation free energies, and entropic contributions over snapshots extracted from trajectories of the unbound proteins and the complexes, calculated binding free energies (Ras-Raf: -15.0(+/-6.3)kcal mol(-1); Ras-RalGDS: -19.5(+/-5.9)kcal mol(-1)) are in fair agreement with experimentally determined values (-9.6 kcal mol(-1); -8.4 kcal mol(-1)), if appropriate ionic strength is taken into account. Structural determinants of the binding affinity of Ras-Raf and Ras-RalGDS are identified by means of free energy decomposition. For the first time, computationally inexpensive generalized Born (GB) calculations are applied in this context to partition solvation free energies along with gas-phase energies between residues of both binding partners. For selected residues, in addition, entropic contributions are estimated by classical statistical mechanics. Comparison of the decomposition results with experimentally determined binding free energy differences for alanine mutants of interface residues yielded correlations with r(2)=0.55 and 0.46 for Ras-Raf and Ras-RalGDS, respectively. Extension of the decomposition reveals residues as far apart as 25A from the binding epitope that can contribute significantly to binding free energy. These "hotspots" are found to show large atomic fluctuations in the unbound proteins, indicating that they reside in structurally less stable regions. Furthermore, hotspot residues experience a significantly larger-than-average decrease in local fluctuations upon complex formation. Finally, by calculating a pair-wise decomposition of interactions, interaction pathways originating in the binding epitope of Raf are found that protrude through the protein structure towards the loop L1. This explains the finding of a conformational change in this region upon complex formation with Ras, and it may trigger a larger structural change in Raf, which is considered to be necessary for activation of the effector by Ras.

Biophysical Phenomena↗

PubMed2003

Structural, spectroscopic, and redox consequences of a central ligand in the FeMoco of nitrogenase: a density functional theoretical study.

Broken symmetry density functional and electrostatics calculations have been used to shed light on which of three proposed atoms, C, N, or O, is most likely to be present in the center of the FeMoco, the active site of nitrogenase. At the Mo(4+)4Fe(2+)3Fe(3+) oxidation level, a central N(3-) anion results in (1) calculated Fe-N bond distances that are in very good agreement with the recent high-resolution X-ray data of Einsle et al.; (2) a calculated redox potential of 0.19 eV versus the standard hydrogen electrode (SHE) for FeMoco(oxidized) + e(-) --> FeMoco(resting), in good agreement with the measured value of -0.042 V in Azotobacter vinelandii; and (3) average Mössbauer isomer shift values (IS(av) = 0.48 mm s(-1)) compatible with experiment (IS(av) = 0.40 mm s(-1)). At the more reduced Mo(4+)6Fe(2+)1Fe(3+) level, the calculated geometry around a central N(3-) anion still correlates well with the X-ray data, but the average Mössbauer isomer shift value (IS(av) = 0.54 mm s(-1)) and the redox potential of -2.21 eV show a much poorer agreement with experiment. These calculated structural, spectroscopic, and redox data indicate the most likely iron oxidation state for the resting FeMoco of nitrogenase to be 4Fe(2+)3Fe(3+). At this favored oxidation state, oxygen or carbon coordination leads to (1) Fe-O distances in poor agreement and Fe-C distances in good agreement with experiment and (2) calculated redox potentials of +0.97 eV for O(2-) and -1.31 eV for C(4-). The calculated structural parameters and/or redox data suggest either O(2-) or C(4-) is unlikely as a central anion.

Molybdoferredoxin↗

PubMed2003

Symmetry and bonding in metalloporphyrins. A modern implementation for the bonding analyses of five- and six-coordinate high-spin iron(III)-porphyrin complexes through density functional calculation and NMR spectroscopy.

Bonding interactions between the iron and the porphyrin macrocycle of five- and six-coordinate high-spin iron(III)-porphyrin complexes are analyzed within the framework of approximate density functional theory with the use of the quantitative energy decomposition scheme in combination with removal of the vacant pi orbitals of the porphyrin from the valence space. Although the relative extent of the iron-porphyrin interactions can be evaluated qualitatively through the spin population and orbital contribution analyses, the bond strengths corresponding to different symmetry representations can be only approximated quantitatively by the orbital interaction energies. In contrast to previous suggestions, there are only limited Fe --> P pi back-bonding interactions in high-spin iron(III)-porphyrin complexes. It is the symmetry-allowed bonding interaction between d(z)2 and a(2u) orbitals that is responsible for the positive pi spin densities at the meso-carbons of five-coordinate iron(III)-porphyrin complexes. Both five- and six-coordinate complexes show significant P --> Fe pi donation, which is further enhanced by the movement of the metal toward the in-plane position for six-coordinate complexes. These bonding characteristics correlate very well with the NMR data reported experimentally. The extraordinary bonding interaction between d(z)2 and a(2u) orbitals in five-coordinate iron(III)-porphyrin complexes offers a novel symmetry-controlled mechanism for spin transfer between the axial ligand sigma system and the porphyrin pi system and may be critical to the electron transfer pathways mediated by hemoproteins.

Ferric Compounds↗

PubMed2003

Modeling of flap endonuclease interactions with DNA substrate.

Structure-specific 5' nucleases play an important role in DNA replication and repair uniquely recognizing an overlap flap DNA substrate and processing it into a DNA nick. However, in the absence of a high-resolution structure of the enzyme/DNA complex, the mechanism underlying this recognition and substrate specificity, which is key to the enzyme's function, remains unclear. Here, we propose a three-dimensional model of the structure-specific 5' flap endonuclease from Pyrococcus furiosus in its complex with DNA. The model is based on the known X-ray structure of the enzyme and a variety of biochemical and molecular dynamics (MD) data utilized in the form of distance restraints between the enzyme and the DNA. Contacts between the 5' flap endonuclease and the sugar-phosphate backbone of the overlap flap substrate were identified using enzyme activity assays on substrates with methylphosphonate or 2'-O-methyl substitutions. The enzyme footprint extends two to four base-pairs upstream and eight to nine base-pairs downstream of the cleavage site, thus covering 10-13 base-pairs of duplex DNA. The footprint data are consistent with a model in which the substrate is bound in the DNA-binding groove such that the downstream duplex interacts with the helix-hairpin-helix motif of the enzyme. MD simulations to identify the substrate orientation in this model are consistent with the results of the enzyme activity assays on the methylphosphonate and 2'-O-methyl-modified substrates. To further refine the model, 5' flap endonuclease variants with alanine point substitutions at amino acid residues expected to contact phosphates in the substrate and one deletion mutant were tested in enzyme activity assays on the methylphosphonate-modified substrates. Changes in the enzyme footprint observed for two point mutants, R64A and R94A, and for the deletion mutant in the enzyme's beta(A)/beta(B) region, were interpreted as being the result of specific interactions in the enzyme/DNA complex and were used as distance restraints in MD simulations. The final structure suggests that the substrate's 5' flap interacts with the enzyme's helical arch and that the helix-hairpin-helix motif interacts with the template strand in the downstream duplex eight base-pairs from the cleavage site. This model suggests specific interactions between the 3' end of the upstream oligonucleotide and the enzyme. The proposed structure presents the first detailed description of substrate recognition by structure-specific 5' nucleases.

Base Sequence↗

PubMed2003

The circumsphere as a tool to assess distortion in [4Fe-4S] atom clusters.

The geometry proposition that "four points not in a plane describe one and only one sphere" provides a novel tool for analyzing protein-induced distortions in [4Fe-4S] clusters. A geometrically perfect reference structure comprises interlaced, regular tetrahedra of Fe, S, and S gamma atoms having T(d) symmetry. Three circumspheres are defined by the three sets of four atoms, the circumcenters of which are unique points within the cluster. The structure is thus re-defined by the positions of the circumcenters in xyz space and the r, theta, phi of each atom on its respective sphere. Analysis of 12 high-resolution structures of protein-bound and small molecule [4Fe-4S](SR)(4) clusters revealed: (a) the circumcenters are generally non-coincident by approximately 0.01 to approximately 0.06 A; (b) the Fe radius, r(Fe), is nominally independent of core oxidation state, having values between 1.66 to 1.69 A, whereas r(S) and r(SG), which have ranges of 2.18-2.24 A and 3.87-3.94 A, respectively, both increase by as much as approximately 3% upon reduction from the 3+ to the 1+ core valence; (c) deviation of some atoms from the theta, phi of a perfect tetrahedron can be large, approximately 10 degrees, and sets of atoms can show patterns of motion on their spheres that result from changes in Fe-S bond lengths. Density functional theory calculations suggest that the [4Fe-4S] core itself requires rather little energy to distort (approximately 2 kcal/mol), whereas significantly more energy is required to distort the Sgamma shell (~4 kcal/mol) to that of cluster I in Clostridium acidurici ferredoxin.

Algorithms↗

PubMed2003

Density functional and reduction potential calculations of Fe4S4 clusters.

Density functional theory geometry optimizations and reduction potential calculations are reported for all five known oxidation states of [Fe(4)S(4)(SCH(3))(4)](n)()(-) (n = 0, 1, 2, 3, 4) clusters that form the active sites of iron-sulfur proteins. The geometry-optimized structures tend to be slightly expanded relative to experiment, with the best comparison found in the [Fe(4)S(4)(SCH(3))(4)](2)(-) model cluster, having bond lengths 0.03 A longer on average than experimentally observed. Environmental effects are modeled with a continuum dielectric, allowing the solvent contribution to the reduction potential to be calculated. The calculated protein plus solvent effects on the reduction potentials of seven proteins (including high potential iron proteins, ferredoxins, the iron protein of nitrogenase, and the "X", "A", and "B" centers of photosystem I) are also examined. A good correlation between predicted and measured absolute reduction potentials for each oxidation state of the cluster is found, both for relative potentials within a given oxidation state and for the absolute potentials for all known couples. These calculations suggest that the number of amide dipole and hydrogen bonding interactions with the Fe(4)S(4) clusters play a key role in modulating the accessible redox couple. For the [Fe(4)S(4)](0) (all-ferrous) system, the experimentally observed S = 4 state is calculated to lie lowest in energy, and the predicted geometry and electronic properties for this state correlate well with the EXAFS and Mössbauer data. Cluster geometries are also predicted for the [Fe(4)S(4)](4+) (all-ferric) system, and the calculated reduction potential for the [Fe(4)S(4)(SCH(3))(4)](1)(-)(/0) redox couple is in good agreement with that estimated for experimental model clusters containing alkylthiolate ligands.

Electrochemistry↗

PubMed2003

Structural details, pathways, and energetics of unfolding apomyoglobin.

Protein folding is often difficult to characterize experimentally because of the transience of intermediate states, and the complexity of the protein-solvent system. Atomistic simulations, which could provide more detailed information, have had to employ highly simplified models or high temperatures, to cope with the long time scales of unfolding; direct simulation of folding is even more problematic. We report a fully atomistic simulation of the acid-induced unfolding of apomyoglobin in which the protonation of acidic side-chains to simulate low pH is sufficient to induce unfolding at room temperature with no added biasing forces or other unusual conditions; and the trajectory is validated by comparison to experimental characterization of intermediate states. Novel insights provided by their analysis include: characterization of a dry swollen globule state forming a barrier to initial unfolding or final folding; observation of cooperativity in secondary and tertiary structure formation and its explanation in terms of dielectric environments; and structural details of the intermediate and the completely unfolded states. These insights involve time scales and levels of structural detail that are presently beyond the range of experiment, but come within reach through the simulation methods described here. An implicit solvation model is used to analyze the energetics of protein folding at various pH and ionic strength values, and a reasonable estimate of folding free energy is obtained. Electrostatic interactions are found to disfavor folding.

Apoproteins↗

PubMed2003

Free energy calculations for theophylline binding to an RNA aptamer: Comparison of MM-PBSA and thermodynamic integration methods.

We have applied the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method (J. Srinivasan, T. E. Cheatham, P. Cieplak, P. A. Kollman, and D. A. Case, Journal of the American Chemical Society, 1998, Vol. 120, pp. 9401-9409) to study the interaction of an RNA aptamer with theophylline and its analogs. The MM-PBSA free energy analysis provides a reasonable absolute binding free energy for the RNA aptamer-theophylline complex formation. Energetic analysis reveals that the van der Waals interaction and the nonpolar contribution to solvation provide the basis for the favorable absolute free energy of complex. This trend is similar to other protein-ligand interactions studied previously. The MM-PBSA method also ranks the relative binding energies of five theophylline analogs approximately correctly, but not as well as the more conventional thermodynamic integration calculations, which were carried out to convert theophylline into its analogs. The comparison of MM-PBSA with TI suggests that the MM-PBSA method has some difficulties with the first-solvation-shell energetics.

Binding Sites↗