Electrostatic and non-electrostatic contributions to the binding free energies of anthracycline antibiotics to DNA.

The knowledge about molecular factors driving simple ligand-DNA interactions is still limited. The aim of the present study was to investigate the electrostatic and non-electrostatic contributions to the binding free energies of anthracycline compounds with DNA. Theoretical calculations based on continuum methods (Poisson-Boltzmann and solvent accessible surface area) were performed to estimate the binding free energies of five selected anthracycline ligands (daunomycin, adriamycin, 9-deoxyadriamycin, hydroxyrubicin, and adriamycinone) to DNA. The free energy calculations also took into account the conformational change that DNA undergoes upon ligand binding. This conformational change appeared to be very important for estimating absolute free energies of binding. Our studies revealed that the absolute values of all computed contributions to the binding free energy were quite large compared to the total free energy of binding. However, the sum of these large positive and negative values produced a small negative value of the free energy around -10 kcal/mol. This value is in good agreement with experimental data. Experimental values for relative binding free energies were also reproduced for charged ligands by our calculations. Together, it was found that the driving force for ligand-DNA complex formation is the non-polar interaction between the ligand and DNA even if the ligand is positively charged.

Electrostatic effects in homeodomain-DNA interactions.

We report here an investigation of the role of electrostatics in homeodomain-DNA interactions using techniques based around the use of the Poisson-Boltzmann equation. In the present case such a study is of particular interest, since in contrast to other proteins previously studied with this method, the homeodomain is a small, highly charged protein that forms extensive ion pairs upon binding DNA. We have investigated the salt dependence of the binding constant for specific association and for a variety of models for non-specific association. The results indicate that, in line with the models proposed by Manning and Record, the entropy of counterion release accounts for a significant fraction of the salt dependence of the binding free energy, though this is perhaps due to fortuitous cancellation of other contributing terms. The thermodynamic effects of a number of specific homeodomain mutants were also investigated, and partly rationalized in terms of favorable electrostatic interactions in the major goove of DNA. Investigation of the temperature-dependence of the free energy of association indicates that the electrostatic contributions become increasingly favorable as the temperature rises. For this particular system, however, there appears to be no significant electrostatic contribution to the delta(delta C(p)) of association. Finally, an analysis of the free energy of interaction when the homeodomain is moved ca one Debye length from the DNA suggests that pure electrostatic forces are able to steer the homeodomain into a partially correct orientation for binding to the DNA.

Unfolding and refolding of bovine beta-lactoglobulin monitored by hydrogen exchange measurements.

Bovine beta-lactoglobulin (beta-LG) is a widely studied protein belonging to the lipocalin family, whose structural characterisation has been reported by X-ray crystallography and NMR studies at physiological and acidic pH, respectively. Bovine beta-LG consists of nine antiparallel beta-sheets and a terminal alpha-helix segment. The beta-sheets form a calyx structure with a hydrophobic buried cluster conferring stability to the protein while a hydrophobic surface patch provides stabilising interactions between the barrel and the flanking terminal helix. Here, the stability and the folding properties of bovine beta-LG in the presence of a chemical denaturant is probed. The analysis of the NMR spectra recorded in aqueous solution with increasing amounts of urea revealed that the intensities of the backbone cross-peaks decrease upon increasing urea concentration, while their secondary shifts do not change significantly on going from 0 to 5 M urea, thus suggesting the presence of slow exchange between native and unfolded protein. Hydrogen exchange measurements at different urea concentrations were performed in order to evaluate the exchange rates of individual backbone amide protons. The opening reactions that determine protein exchange can be computed for the most slowly exchanging hydrogen atoms, and the measured exchange rates and the corresponding free energies can be expressed in terms of the equilibrium energetic for the global transition and the local fluctuations. Most of the residues converge to define a common isotherm identifying a unique cooperative folding unit, encompassing all the strands, except strand betaI, and the terminal region of the helix. The amides that do not join the same global unfolding isotherm are characterised by low DeltaGH20op and especially by low m values, indicating that they are already substantially exposed in the native state. A two-state unfolding model N <==> U is therefore proposed for this rather big protein, in agreement with CD data. Renaturation studies show that the unfolding mechanism is reversible up to 6 M urea and suggest a similar unfolding and refolding pathway. Present results are discussed in light of the hypothesis of an alpha-->beta transition proposed for bovine beta-LG refolding.

Bovine beta-lactoglobulin: interaction studies with palmitic acid.

Bovine beta-lactoglobulin (BLG) in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure-function relationship in this protein, the interactions between 13C enriched palmitic acid (PA) and BLG were investigated by means of one-, two-, and three-dimensional NMR spectroscopy in the pH range 8.4-2.1. The NMR spectra revealed that at neutral pH the ligand is bound within the central cavity of BLG, with the methyl end deeply buried within the protein. The analysis of 13C spectra of the holo protein revealed the presence of conformational variability of bound PA carboxyl end in the pH range 8.4-5.9, related to the Tanford transition. The release of PA starts at pH lower than 6.0, and it is nearly complete at acidic pH. This finding is relevant in relation to the widely reported hypothesis that this protein can act as a transporter through the acidic gastric tract. Ligand binding and release is shown to be completely reversible over the entire pH range examined, differently from other fatty acid binding proteins whose behavior is analyzed throughout the paper. The mode of interaction of BLG is compatible with the proposed function of facilitating the digestion of milk fat during the neonatal period of calves.

Comparative stability analysis of the thyroid transcription factor 1 and Antennapedia homeodomains: evidence for residue 54 in controlling the structural stability of the recognition helix.

The thyroid transcription factor 1 homeodomain (TTF-1 HD) shows a peculiar DNA-binding specificity which is partially dictated by several amino acids of the recognition helix. TTF-1 preferentially recognizes sequences containing the 5'-CAAG-3' core motif while most other homeodomains, such as Antennapedia (Antp), recognizes sites containing the 5'-TAAT-3' core motif. Since phenomena of 'induced fit' may occur during protein/DNA interaction, a primary role for high affinity binding and target discrimination has to be searched in the effect played by subtle structural determinants in these proteins. By using spectroscopic analysis in aqueous solution, we compared the structural stability of TTF-1 and Antp homeodomains. Although the three-dimensional structural architecture of homeodomains is conserved, some differences are detectable in terms of their structural stability. At 24 degrees C the TTF-1 HD is less structured than the Antp HD with 24 and 34% of the residues in the alpha-helical conformation, respectively. This poor folded structure reflects into different thermal and isothermal stability between the two homeodomains. TTF-1 HD exhibits a Tm of 39 degrees C and is stabilized by a delta GDH2O of +1487 cal/mol, calculated by Urea unfolding, while Antp HD exhibits a Tm of 48 degrees C and is stabilized by a delta GDH2O of +2742 cal/mol. By using mutants of both TTF-1 and Antp HDs we demonstrate that one of the major determinants in controlling the structural stability of the recognition helix is the residue at position 54. Since previous studies have shown that also residue at position 56 is involved in stabilization of the recognition helix, we conclude that the structure of this critical element is controlled by an interplay between residues at position 54 and 56 of the homeodomain.

Structural study of rat thyroid transcription factor 1 homeodomain (TTF-1 HD) by nuclear magnetic resonance.

The 500 MHz 1H NMR spectrum of a 68-residue peptide, encompassing the rat thyroid transcription factor 1 homeodomain (TTF-1 HD), was fully assigned using standard 2D NMR methodology. The secondary structure elements and their spatial organization were determined and led to a structure very similar to that previously described for other homeodomains and expected also for TTF-1 HD from homology modeling predictions. The three-dimensional arrangement of the three helix fragments of TTF-1 HD preserves the helix-turn-helix motif commonly occurring in many classes of DNA-binding proteins.

Analysis of the conformation and stability of rat TTF-1 homeodomain by circular dichroism.

The conformational stability of TTF-1HD has been determined by CD-monitored thermal denaturation and isothermal urea unfolding studies. The Gibbs free energy of stabilization found are 1.44 and 1.26 kcal.mol-1, respectively. TTF-1HD exhibits a Tm of 42 degrees C and a delta Cp of 80 cal.mol-1.K-1 indicating that TTF-1HD, when free in solution, is a mobile flexible segment folded into loose helices. Such a flexibility would be relevant for the DNA-binding function of this homeodomain. In fact, a small reduction of the alpha-helical content of TTF-1HD significantly modifies its DNA-binding activity.

Monomeric bovine beta-lactoglobulin adopts a beta-barrel fold at pH 2.

We have determined a crude structure of the apo form of bovine beta-lactoglobulin, a protein of 162 amino acid residues with a molecular mass of 18 kDa, at a low pH on the basis of data collected using only homonuclear 1H NMR spectroscopy. An ensemble of protein conformations was calculated with the distance-geometry algorithm for NMR applications (DYANA). The monomeric protein at low pH adopts a beta-barrel fold, well-superimposable on the structure determined by X-ray crystallography for the dimer at physiological pH. NMR evidence suggests the presence of disordered loop regions and terminal segments. Structural differences between the monomer at pH 2 and the dimer at pH 7, obtained by X-ray crystallography, are discussed, paying particular attention to surface electrostatic properties, in view of the high charge state of the protein at low pH.

NMR and circular dichroism studies of the lantibiotic nisin in non-aqueous environments.

The lantibiotic, nisin, which is known to interact with membranes of certain Gram-positive bacteria, was studied in three model systems which mimic a membrane-like environment, i.e. a mixture of trifluoroethanol and water, or micelles of sodium dodecyl sulfate or dodecylphosphocholine. The 1H NMR spectra of nisin in the non-aqueous environments, at 40 degrees C and pH 3.5, have been assigned completely. The CD and NMR results indicate that the conformation of nisin in the three non-aqueous environments differs from that in aqueous solution, and that the conformation in the two micellar systems is similar. The major conformational changes, relative to nisin in aqueous solution, occur in the N-terminus.

Molecular mechanics and dynamics of biomolecules using a solvent continuum model.

An easy implementation of molecular mechanics and molecular dynamics simulation using a continuum solvent model is presented that is particularly suitable for biomolecular simulations. The computation of solvation forces is made using the linear Poisson-Boltzmann equation (polar contribution) and the solvent-accessible surface area approach (nonpolar contribution). The feasibility of the methodology is demonstrated on a small protein and a small DNA hairpin. Although the parameters employed in this model must be refined to gain reliability, the performance of the method, with a standard choice of parameters, is comparable with results obtained by explicit water simulations. Copyright 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1830-1842, 2001

Limiting-laws of polyelectrolyte solutions. Ionic distribution in mixed-valency counterions systems. I: The model.

An extension of the counterion-condensation (CC) theory of linear polyelectrolytes has been developed for the case of a system containing a mixture of counterions of different valency, i and j. The main assumption in the derivation of the model is that the relative amount of the condensed counterions of the type i and j is strongly correlated and it is determined by the overall physical bounds of the system. The results predicted by the model are consistent, in the limiting cases of single species component, with those of the original CC theory. The most striking results are obtained for the cases of low charge density and excess of counterion species: in particular, an apparent positive "binding" cooperativity of divalent ions is revealed for small, increasing additions of M2+ ions to a solution containing a swamping amount of monovalent salt and a polyelectrolyte of low charge density. Apparent "competitive binding" of mono- and divalent ions derives as a bare consequence of the electrostatic interactions. Theoretical calculations of experimentally accessible quantities, namely single-(counter) ion activity coefficients, confirm the surprising predictions at low charge density, which qualitatively agree with the measured quantities.

Free energies calculated according to Manning's polyelectrolyte model compared with Poisson Boltzmann predictions.

The values of the different terms contributing to the free energy of a polyelectrolyte in ionic solution, calculated according to Manning's model are compared with those predicted by the Poisson-Boltzmann(P-B) equation solved for the cell model. On this ground, the limits of applicability of Manning's model and some of its features are discussed. The comparison confirms the usefulness of Manning's model for low charge density polyelectrolytes, while for higher charge densities some care should be used due to the breakdown of the approximations underlying the model.

Mitochondrial F(0) F(1) -ATP synthase is a molecular target of 3-iodothyronamine, an endogenous metabolite of thyroid hormone.

BACKGROUND AND PURPOSE: 3-iodothyronamine (T1AM) is a metabolite of thyroid hormone acting as a signalling molecule via non-genomic effectors and can reach intracellular targets. Because of the importance of mitochondrial F(0) F(1) -ATP synthase as a drug target, here we evaluated interactions of T1AM with this enzyme. EXPERIMENTAL APPROACH: Kinetic analyses were performed on F(0) F(1) -ATP synthase in sub-mitochondrial particles and soluble F(1) -ATPase. Activity assays and immunodetection of the inhibitor protein IF(1) were used and combined with molecular docking analyses. Effects of T1AM on H9c2 cardiomyocytes were measured by in situ respirometric analysis. KEY RESULTS: T1AM was a non-competitive inhibitor of F(0) F(1) -ATP synthase whose binding was mutually exclusive with that of the inhibitors IF(1) and aurovertin B. Both kinetic and docking analyses were consistent with two different binding sites for T1AM. At low nanomolar concentrations, T1AM bound to a high-affinity region most likely located within the IF(1) binding site, causing IF(1) release. At higher concentrations, T1AM bound to a low affinity-region probably located within the aurovertin binding cavity and inhibited enzyme activity. Low nanomolar concentrations of T1AM increased ADP-stimulated mitochondrial respiration in cardiomyocytes, indicating activation of F(0) F(1) -ATP synthase consistent with displacement of endogenous IF(1,) , reinforcing the in vitro results. CONCLUSIONS AND IMPLICATIONS: Effects of T1AM on F(0) F(1) -ATP synthase were twofold: IF(1) displacement and enzyme inhibition. By targeting F(0) F(1) -ATP synthase within mitochondria, T1AM might affect cell bioenergetics with a positive effect on mitochondrial energy production at low, endogenous, concentrations. T1AM putative binding locations overlapping with IF(1) and aurovertin binding sites are described.

Thermodynamic and electrostatic properties of ternary Oxytricha nova TEBP-DNA complex.

Telomeres constitute the nucleoprotein ends of eukaryotic chromosomes which are essential for their proper function. Telomere end binding protein (TEBP) from Oxytricha nova was among the first telomeric proteins, which were well characterized biologically. TEBP consists of two protein subunits (alpha, beta) and forms a ternary complex with single stranded telomeric DNA containing tandem repeats TTTTGGGG. This work presents the characterization of the thermodynamic and electrostatic properties of this complex by computational chemistry methods (continuum Poisson-Boltzmann and solvent accessible surface calculations). Our calculations give a new insight into molecular properties of studied system. Based on the thermodynamic analysis we provide a rationale for the experimental observation that alpha and ssDNA forms a binary complex and the beta subunit joins alpha:ssDNA complex only after the latter is formed. Calculations of distribution of the molecular electrostatic potential for protein subunits alone and for all possible binary complexes revealed the important role of the "guiding funnel" potential generated by alpha:ssDNA complex. This potential may help the beta subunit to dock to the already formed alpha:DNA intermediate in highly steric and electrostatic favorable manner. Our pK(a) calculations of TEBP are able to explain the experimental mobility shifts of the complex in electrophoretic non-denaturating gels.

Singular value decomposition analysis of protein sequence alignment score data.

One of the standard tools for the analysis of data arranged in matrix form is singular value decomposition (SVD). Few applications to genomic data have been reported to date mainly for the analysis of gene expression microarray data. We review SVD properties, examine mathematical terms and assumptions implicit in the SVD formalism, and show that SVD can be applied to the analysis of matrices representing pairwise alignment scores between large sets of protein sequences. In particular, we illustrate SVD capabilities for data dimension reduction and for clustering protein sequences. A comparison is performed between SVD-generated clusters of proteins and annotation reported in the SWISS-PROT Database for a set of protein sequences forming the calycin superfamily, entailing all entries corresponding to the lipocalin, cytosolic fatty acid-binding protein, and avidin-streptavidin Prosite patterns.

The Poisson-Boltzmann equation for biomolecular electrostatics: a tool for structural biology.

Electrostatics plays a fundamental role in virtually all processes involving biomolecules in solution. The Poisson-Boltzmann equation constitutes one of the most fundamental approaches to treat electrostatic effects in solution. The theoretical basis of the Poisson-Boltzmann equation is reviewed and a wide range of applications is presented, including the computation of the electrostatic potential at the solvent-accessible molecular surface, the computation of encounter rates between molecules in solution, the computation of the free energy of association and its salt dependence, the study of pKa shifts and the combination with classical molecular mechanics and dynamics. Theoretical results may be used for rationalizing or predicting experimental results, or for suggesting working hypotheses. An ever-increasing body of successful applications proves that the Poisson-Boltzmann equation is a useful tool for structural biology and complementary to other established experimental and theoretical methodologies.

Biomolecular electrostatics with the linearized Poisson-Boltzmann equation.

Electrostatics plays a key role in many biological processes. The Poisson-Boltzmann equation (PBE) and its linearized form (LPBE) allow prediction of electrostatic effects for biomolecular systems. The discrepancies between the solutions of the PBE and those of the LPBE are well known for systems with a simple geometry, but much less for biomolecular systems. Results for high charge density systems show that there are limitations to the applicability of the LPBE at low ionic strength and, to a lesser extent, at higher ionic strength. For systems with a simple geometry, the onset of nonlinear effects has been shown to be governed by the ratio of the electric field over the Debye screening constant. This ratio is used in the present work to correct the LPBE results to reproduce fairly accurately those obtained from the PBE for systems with a simple geometry. Since the correction does not involve any geometrical parameter, it can be easily applied to real biomolecular systems. The error on the potential for the LPBE (compared to the PBE) spans few kT/q for the systems studied here and is greatly reduced by the correction. This allows for a more accurate evaluation of the electrostatic free energy of the systems.

Modeling of polypeptide chains as C alpha chains, C alpha chains with C beta, and C alpha chains with ellipsoidal lateral chains.

In an effort to reduce the number of degrees of freedom necessary to describe a polypeptide chain we analyze the statistical behavior of polypeptide chains when represented as C alpha chains, C alpha chains with C beta atoms attached, and C alpha chains with rotational ellipsoids as models of side chains. A statistical analysis on a restricted data set of 75 unrelated protein structures is performed. The comparison of the database distributions with those obtained by model calculation on very short polypeptide stretches allows the dissection of local versus nonlocal features of the distributions. The database distribution of the bend angles of polypeptide chains of pseudo bonded C alpha atoms spans a restricted range of values and shows a bimodal structure. On the other hand, the torsion angles of the C alpha chain may assume almost all possible values. The distribution is bimodal, but with a much broader probability distribution than for bend angles. The C alpha - C beta vectors may be taken as representative of the orientation of the lateral chain, as the direction of the bond is close to the direction of the vector joining C alpha to the ad hoc defined center of the "steric mass" of the side chain. Interestingly, both the bend angle defined by C alpha i-C alpha i+1-C beta i+1 and the torsional angle offset of the pseudo-dihedral C alpha i-C alpha i+1-C alpha i+2-C beta i+2 with respect to C alpha i-C alpha i+1-C alpha i+2-C alpha i+3 span a limited range of values. The latter results show that it is possible to give a more realistic representation of polypeptide chains without introducing additional degrees of freedom, i.e., by just adding to the C alpha chain a C beta with given side-chain properties. However, a more realistic description of side chains may be attained by modeling side chains as rotational ellipsoids that have roughly the same orientation and steric hindrance. To this end, we define the steric mass of an atom as proportional to its van der Waals volume and we calculate the side-chain inertia ellipsoid assuming that the steric mass of each atom is uniformly distributed within its van der Waals volume. Finally, we define the rotational ellipsoid representing the side chain as the uniform density ellipsoid possessing the same rotationally averaged inertia tensor of the side chain. The statistics of ellipsoid parameters support the possibility of representing a side chain via an ellipsoid, independently of the local conformation. To make this description useful for molecular modeling we describe ellipsoid-ellipsoid interactions via a Lennard-Jones potential that preserves the repulsive core of the interacting ellipsoids and takes into account their mutual orientation. Tests are performed for two different forms of the interaction potential on a set of high-resolution protein structures. Results are encouraging, in view of the drastic simplifications that were introduced.

Improved processing of selective NMR spectra of biopolymers by separation of noise and signal subspaces through singular-value decomposition.

A novel sensitivity-enhancement procedure is introduced for 2D NMR data matrices. It is based on the separation of the signal-and-noise subspaces by means of singular-value decomposition of the experimental 2D array. Although no theoretical limitation exists for a general application of the method, the reliability of the results increases considerably with reduced data sets such as those of selective measurements. Advantageous applications can be envisaged for the quantitation of NMR parameters in biopolymers whose linewidths are often large enough to undermine severely the sensitivity of selective experiments.

Monitoring folding/unfolding transitions of proteins by capillary zone electrophoresis: measurement of deltaG and its variation along the pH scale.

Free-solution capillary zone electrophoresis (CZE) can be used to monitor folding/unfolding transitions of proteins and to construct the classical sigmoidal transition curve describing this isomerization process. By performing a series of CZE experiments along the pH scale (here between pH 2.5 and 6.0) it is possible to measure the parameter [urea]1/2, which represents the concentration of urea at the midpoint of each transition curve, and its dependence from the local pH value. The [urea]1/2 parameter provides an idea of the stability of the protein at a given pH; in the case of cytochrome c, for example, it shows that at and below pH 2 the protein will spontaneously unfold even in the absence of a denaturant. The equation describing the sigmoidal folding/unfolding transition can be used for deriving the term deltaG degrees, which refers to the intrinsic difference in the Gibb's free energy between the (total or partial) denatured state and the reference state, taken usually as the native configuration of a protein. The variation of deltaG degrees between the two extremes of our measurements (pH 2.5 and 6.0) along the stated pH interval has been measured (and theoretically calculated) to be of the order of 7-10 kcal/mol and is here interpreted by assuming that at pH 2.5 and below there is an additionally stretching of the polypeptide coil due to coulombic repulsion, as the unfolded chain looses its zwitterionic character and assumes a pure (or very nearly so) cationic surface. Given the minute amounts of sample required, the fully automated state of the analysis, the rapidity and ease of operation, it is hoped that the CZE technique will become more and more popular in the years to come for monitoring folding/unfolding transitions of proteins.