Aqueous solvent effects on the conformational space of tryptamine. Structural and electronic analysis

The TRA (3-[2-aminoethyl]indole) is an important neurotransmitter with a close structural and chemical similarity to the neurotransmitter serotonin (5-hydroxytryptamine), and to melatonin (5-methoxy-N-acetyltryptamine), which plays a key role in daily human behavior. Moreover, TRA, and other indolic compounds are very efficient antioxidants. In this work the conformational space of TRA was scanned in aqueous solution, simulating the solvent by the polarizable continuum model. Geometry optimizations were performed at B3LYP/6-31+G** level. Electronic distributions were analyzed at a better calculation level, thus improving the basis set (6-311++G**). A topological study based on Bader’s theory (atoms in molecules) and natural bond orbital (NBO) framework was performed. Structural changes found in solution were related with charge delocalization mechanisms, which explained the changes in the conformational relative population in aqueous phase. Solvent effects on molecular electrostatic potential (MEPs) were also quantified and rationalized through charge delocalization mechanisms, thus connecting changes in MEPs with changes in structure, bond polarization, orbital bonding populations, natural charges, and bond topological properties. Moreover, polarizabilities and dipolar moments were calculated. All conformers were taken into account. Our results are the first prediction of TRA polarizabilities. The results reported contribute to the understanding of the structure, stability and reactivity of TRA and other indole derivatives.     

The sensitivity of conformational free energies of the alanine dipeptide to atomic site charges

Different atomic point charge sets are obtained for the α_R and C_7.eq conformations of the alanine dipeptide by fitting the charges of each conformation to the respective ab initio electrostatic potential surfaces both individually and simultaneously, in both the united atom and the all-atom representations. Using these charge sets, the sensitivity of the relative conformational aqueous free energies to the atomic site charges is investigated. For this particular system, we find that the solute-water contributions to the conformational free energy differences have a rather weak dependence on site charges; the calculated intramolecular contributions, however, show a rather strong dependence on the atomic site charges. It is suggested that the calculated results for the alanine dipeptide using a single, simultaneously fit set of charges for both conformations are in better agreement with experiments than the calculations carried out with charges determined individually for each conformation. © 1997 John Wiley & Sons, Inc.     

Conformational analysis of endothelin-1: Effects of solvation free energy

In order to investigate conformational preferences of the 21-residue peptide hormone endothelin-1 (ET-1), an extensive conformational search was carried out in vacuo using a combination of high temperature molecular dynamics / annealing and a Monte Carlo / minimization search in torsion angle space. Fully minimized conformations from the search were grouped into families using a clustering technique based on rms fitting over the Cartesian coordinates of the atoms of the peptide backbone of the ring region. A wide range of local energy minima were identified even though two disulfide bridges (Cys¹-Cys¹⁵ and Cys³-Cys¹¹) constrain the structure of the peptide. Low energy conformers of ET-1 as a nonionized species in vacuo arestabilized by intramolecular interaction of the ring region (residues 1-15) with the tail (residues 16–21). Strained conformations for individual residues are observed. Conformational similarity to protein loops is established by matching to protein crystal structures In order to assess the influence of aqueous environment on conformational preference, the electrostatic contribution to the solvation energy was calculated for ET-1 as a fully ionized species (Asp⁸, Lys⁹, Glu¹⁰, Asp¹⁸, N- and C-terminus) using a continuum electrostatics model (DelPhi) for each of the conformed generated in vacuo, and the total solvation free energy was estimated by adding a hydrophobic contribution proportional to solvent accessible surface area. Solvation dramatically alters the relative energetics of ET-1 conformers from that calculated in vacuo. Conformers of ET-1 favored by the electrostatic salvation energy in water include conformers with helical secondary structure in the region of residues 9–15. Perhaps of most importance, it was demonstrated that the contribution tosolvation by an individual charge depends not only on its solvent accessibility but on the proximity of other charges, i.e., it is a cooperative effect. This was shown by the calculation of electrostatic solvation energy as afunction of conformation with individual charges systematically turned “on” and “off”. The cooperative effect of multiple charges on solvation demonstrated in this manner calls into question models that relate solvation energysimply to solvent accessibility by atom or residue alone. © 1995 John Wiley & Sons, Inc.     

Application of a universal solvation model to nucleic acid bases: comparison of semiempirical molecular orbital theory, ab initio Hartree-Fock theory, and density functional theory

The free energies of solvation of six nucleic acid bases (adenine, cytosine, hypoxanthine, guanine, thymine, and uracil) in water and chloroform are calculated using CM2 class IV charges and SM5.42R atomic surface tensions. Using any of three approximations to the electronic wave function (AM1, Hartree-Fock, or DFT), we obtain good agreement with experiment for five cases where the experimental results are known for the partition coefficients between the two solvents. Decomposition of the solvation effects into bulk electrostatic contributions and first-solvation-shell effects shows that the partitioning is dominated by the former, and this illustrates the importance of using accurate partial atomic charges for modeling these molecules in aqueous solution.     

Combined theoretical studies on solvation and hydrogen bond interactions in glycine tripeptide

The theoretical conformational analysis of glycine tripeptide (GT) has been carried out by molecular dynamics (MD) method in order to find minimum energy conformations. The MD studies on GT with water have been carried out for over 10 ns with a time step of 2 fs using fixed charge force field (AMBER ff03). By adding the solvation effect using water as a solvent, the GT conformers identified in this study exhibit α-helical conformation. Compared with the earlier reports, this MD study is able to identify the energetically favourable GT conformations. The obtained geometry of the five most stable GT conformations was optimised using the density functional theory method at B3LYP/6-311G** level of theory. Subsequently, the effects of solvation on the conformational characteristics of five most stable GT conformers with four water molecules (the number of water molecules in the first solvation shell of GT obtained from MD study) were investigated using the same method and the same level of theory. The effect of microsolvation on the fifth GT conformer has been studied with a cluster of 11 water molecules as the first hydration shell which generates folded structure. The interaction energies of all the complexes are calculated by correcting the basis set superposition error. The strong hydrogen bond mainly contributes to the interaction energies. The atoms in molecules theory and natural bond orbital analysis were used to study the origin of H-bonds. A good correlation between the structural parameters and the properties of charge density is found. NMR calculations show that the C = O carbons of the amine groups of the first and middle glycine fragments have maximum chemical shifts.     

Calculating proton uptake/release and binding free energy taking into account ionization and conformation changes induced by protein-inhibitor association: application to plasmepsin, cathepsin D and endothiapepsin-pepstatin complexes

The protein-inhibitor binding energies of enzymes are often pH dependent, and binding induces either proton uptake or proton release. The proton uptake/release and the binding energy for three complexes with available experimental data were numerically studied: pepstatin-cathepsin D, pepstatin-plasmepsin II and pepstatin-endothiapepsin. Very good agreement with the experimental data was achieved when conformational changes were taken into account. The role of the desolvation energy and the conformational changes was revealed by modeling the complex, the separated molecules in the complex conformation and the free molecules. It was shown that the conformational changes induced by the complex formation are as important for the proton transfer as the loss of solvation energy caused by the burial of interface residues. The residues responsible for the proton transfer were identified and their contribution to the proton uptake/release calculated. These residues were found to be scattered along the whole protein rather than being localized only at the active site. In the case of cathepsin D, these residues were found to be highly conserved among the cathepsin D sequences of other species. It was shown that conformation and ionization changes induced by the complex formation are critical for the correct calculation of the binding energy. Taking into account the electrostatics and the van der Waals (vdW) energies within the Boltzmann distribution of energies and allowing ionization and conformation changes to occur makes the calculated binding energy more realistic and closer to the experimental value. The interplay between electrostatic and vdW forces makes the pH dependence of the binding energy smoother, because the vdW force acts in reaction to the changes of the electrostatic energy. It was found that a small fraction of the ionizable groups remain uncharged in both the free and complexed molecules. The sequence and structural position of these groups aligns well within the three proteases, suggesting that these may have specific role.     

Variations in antigen-antibody association kinetics as a function of pH and salt concentration: a QSAR and molecular modeling study

The relationship between three environmental factors (ionic strength, pH, and temperature) and antigen-antibody binding kinetics was investigated using QSAR (quantitative structure-activity relationship) and molecular modeling approaches. The interaction used for this analysis is that between the camel antibody fragment cAbLys3 and lysozyme. Binding kinetics were measured using a Biacore 2000 instrument, at NaCl concentrations between 50 and 500 mM, at pH's between 5 and 10, and at temperatures between 15 and 30 degrees C, according to multivariate experimental designs. Variations in kinetic on- and off-rate parameters were up to 400- and 16-fold, respectively. Mathematical models that relate log k(on) to experimental conditions were developed. They indicated an influence of all three factors, with a clear dependency between pH and NaCl concentration for their effect on k(on). These models were able to predict on-rate parameters under new experimental conditions. Titration calculations using continuum electrostatics were performed on the crystallographic structures of the isolated and bound proteins to gain structural insight for the on-rate enhancement observed at pH <6.5 and low salt concentrations. These calculations rule out electrostatic steering linked to global and/or local charge variations in the molecules as the factor responsible for the on-rate enhancement at low pH. His 111 of cAbLys3, located at the binding interface, can adopt two side chain orientations with different intramolecular contacts. The results of the calculations suggest an alternative mechanism whereby the conformation of the interfacial His 111 depends on the charge, and these differences in conformation may influence the solvation energy and the subsequent binding kinetics. Our results stress the complex relationship between environmental conditions and molecular binding properties.     

Evaluation of catalytic free energies in genetically modified proteins

A combination of the empirical valence bond method and a free energy perturbation approach is used to simulate the activity of genetically modified enzymes. The simulations reproduce in a semiquantitative way the observed effects of mutations on the activity and binding free energies of trypsin and subtilisin. This suggests that we are approaching a stage of quantitative structure-function correlation of enzymes. The analysis of the calculations points towards the electrostatic energy of the reacting system as the key factor in enzyme catalysis. The changes in the charges of the reacting system and the corresponding changes in "solvation" free energy (generalized here as the interaction between the charges and the given microenvironment) are emphasized. It is argued that a reliable evaluation of these changes might be sufficient for correlating structure and catalysis. The use of free energy perturbation methods and thermodynamic cycles for evaluation of solvation energies and reactivity is discussed, pointing out our early contributions. The apparent elaborated nature of our treatment is clarified, explaining that such a treatment is essential for consistent calculations of chemical reactions in polar environments. The problems associated with seemingly more rigorous quantum mechanical methods are discussed, emphasizing the inconsistency associated with using gas phase charge distributions. The importance of dynamic aspects is examined by evaluating the autocorrelation of the protein "reaction field" on the reacting substrate. It is found that, at least in the present case, dynamic effects are not important. The nature of the catalytic free energy is considered, arguing that the protein provides preoriented dipoles (polarized to stabilize the transition state charge distribution) and small reorganization energy, thus reducing the activation free energy. The corresponding catalytic free energy is related to the folding free energy, which is being invested in aligning the active site dipoles.     

Zinc binding promotes greater hydrophobicity in Alzheimer's Aβ42 peptide than copper binding: Molecular dynamics and solvation thermodynamics studies

The aggregation of Aβ42 peptides is considered as one of the main causes for the development of Alzheimer's disease. In this context, Zn²⁺ and Cu²⁺ play a significant role in regulating the aggregation mechanism, due to changes in the structural and the solvation free energy of Aβ42. In practice, experimental studies are not able to determine the latter properties, since the Aβ42–Zn²⁺ and Aβ42–Cu²⁺ peptide complexes are intrinsically disordered, exhibiting rapid conformational changes in the aqueous environment. Here, we investigate atomic structural variations and the solvation thermodynamics of Aβ42_, Aβ42–Cu²⁺, and Aβ42–Zn²⁺ systems in explicit solvent (water) by using quantum chemical structures as templates for a metal binding site and combining extensive all-atom molecular dynamics (MD) simulations with a thorough solvation thermodynamic analysis. Our results show that the zinc and copper coordination results in a significant decrease of the solvation free energy in the C-terminal region (Met35-Val40), which in turn leads to a higher structural disorder. In contrast, the β-sheet formation at the same C-terminal region indicates a higher solvation free energy in the case of Aβ42_. The solvation free energy of Aβ42 increases upon Zn²⁺ binding, due to the higher tendency of forming the β-sheet structure at the Leu17-Ala42 residues, in contrast to the case of binding with Cu²⁺. Finally, we find the hydrophobicity of Aβ42–Zn²⁺ in water is greater than in the case of Aβ42–Cu²⁺.     

Functional analysis of genetic mutations in nucleotide binding domain 2 of the human retina specific ABC transporter

The rod outer segment (ROS) ABC transporter (ABCR) plays an important role in the outer segment of retinal rod cells, where it functions as a transporter of all-trans retinal, most probably as the complex lipid, retinylidene-phosphatidyl-ethanolamine. We report here a quantitative analysis of the structural and functional effects of genetic mutations, associated with several macular degenerations, in the second nucleotide-binding domain of ABCR (NBD2). We have analyzed the ATP binding, kinetics of ATP hydrolysis, and structural changes. The results of these multifaceted analyses were correlated with the disease severity and prognosis. Results presented here demonstrated that, in wild type NBD2, distinct conformational changes accompany nucleotide (ATP and ADP) binding. Upon ATP binding, NBD2 protein changed to a relaxed conformation where tryptophans became more solvent-exposed, while ADP binding reverses this process and leads back to a taut conformation that is also observed with the unbound protein. This sequence of conformational change appears to be important in the energetics of the ATP hydrolysis and may have important structural consequences in the ability of the NBD2 domain to act as a regulator of the nucleotide-binding domain 1. Some of the mutant proteins displayed strikingly different patterns of conformational changes upon nucleotide binding that pointed to unique structural consequences of these genetic mutations. The ABCR dysfunctions, associated with various retinopathies, are multifaceted in nature and include alterations in protein structure as well as the attenuation of ATPase activity and nucleotide binding.     

Solvation energetics and conformational change in EF-hand proteins

Calmodulin and other members of the EF-hand protein family are known to undergo major changes in conformation upon binding Ca(2+). However, some EF-hand proteins, such as calbindin D9k, bind Ca(2+) without a significant change in conformation. Here, we show the importance of a precise balance of solvation energetics to conformational change, using mutational analysis of partially buried polar groups in the N-terminal domain of calmodulin (N-cam). Several variants were characterized using fluorescence, circular dichroism, and NMR spectroscopy. Strikingly, the replacement of polar side chains glutamine and lysine at positions 41 and 75 with nonpolar side chains leads to dramatic enhancement of the stability of the Ca(2+)-free state, a corresponding decrease in Ca(2+)-binding affinity, and an apparent loss of ability to change conformation to the open form. The results suggest a paradigm for conformational change in which energetic strain is accumulated in one state in order to modulate the energetics of change to the alternative state.     

Dissecting the mechanism of Epac activation via hydrogen-deuterium exchange FT-IR and structural modeling

Exchange proteins directly activated by cAMP (Epac) make up a family of cAMP binding domain-containing proteins that play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Ras-proximate proteins. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen-deuterium (H-D) exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and structural modeling. Our studies show that unlike that of cAMP-dependent protein kinase (PKA), the classic intracellular cAMP receptor, binding of cAMP to Epac does not induce significant changes in overall secondary structure and structural dynamics, as measured by FT-IR and the rate of H-D exchange, respectively. These results suggest that Epac activation does not involve significant changes in the amount of exposed surface areas as in the case of PKA activation, and conformational changes induced by cAMP in Epac are most likely confined to small local regions. Homology modeling and comparative structural analyses of the CBDs of Epac and PKA lead us to propose a model of Epac activation. On the basis of our model, Epac activation by cAMP employs the same underlying structural principal utilized by PKA, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different. In addition, we predict that during Epac activation the first beta-strand of the switchboard switches its conformation to a alpha-helix, which folds back to the beta-barrel core of the CBD and interacts directly with cAMP to form the base of the cAMP-binding pocket.     

Theoretical investigation of the triphosphate forms of azidothymidine and thymidine

In this paper we investigate (using AM1 semi-empirical as well as HF methods at the STO-3G, 3-21G, 6-31G, 6-31G* and 6-31+G** level) the conformations, geometrical parameters, Mulliken charges, and solvation effects of the triphosphate form of AZT (AZTTP), as well as the thymidine nucleotide (dTTP) structure. Our calculated geometrical parameters and Mulliken charges, with and without solvation effects, are correlated with recent experimental results.     

Interaction of toxic metal ions Cd, Hg, and Pb with light-harvesting proteins of chloroplast thylakoid membranes. An FTIR spectroscopic study

The interaction of divalent metal ions Cd, Hg, and Pb with the light-harvesting proteins (LHC-II) of chloroplast thylakoid membranes was investigated in aqueous solution with metal ion concentrations of 0.01 to 20 mM, using Fourier Transform infrared (FTIR) difference spectroscopy. Correlations between the metal ion binding mode, protein conformational transitions, and structural variations are established.

Infrared difference spectroscopic evidence has shown strong Hg ion binding with different protein subunits at very low metal ion concentration with drastic structural modifications of interacted proteins. The Cd ion binding was observed at higher metal ion concentration with major protein conformational changes, while Pb ion interaction was less effective on protein conformation. The major metal ion binding sites were those of the protein carbonyl group or the nitrogen atom and/or both, while the sulphur donor sites were also the target of Hg-protein complexation.     

Interactions of Alzheimer amyloid-beta peptides with glycosaminoglycans effects on fibril nucleation and growth.

Proteoglycans and their constituent glycosaminoglycans are associated with all amyloid deposits and may be involved in the amyloidogenic pathway. In Alzheimer's disease, plaques are composed of the amyloid-beta peptide and are associated with at least four different proteoglycans. Using CD spectroscopy, fluorescence spectroscopy and electron microscopy, we examined glycosaminoglycan interaction with the amyloid-beta peptides 1-40 (Abeta40) and 1-42 (Abeta42) to determine the effects on peptide conformation and fibril formation. Monomeric amyloid-beta peptides in trifluoroethanol, when diluted in aqueous buffer, undergo a slow random to amyloidogenic beta sheet transition. In the presence of heparin, heparan sulfate, keratan sulfate or chondroitin sulfates, this transition was accelerated with Abeta42 rapidly adopting a beta-sheet conformation. This was accompanied by the appearance of well-defined amyloid fibrils indicating an enhanced nucleation of Abeta42. Incubation of preformed Abeta42 fibrils with glycosaminoglycans resulted in extensive lateral aggregation and precipitation of the fibrils. The glycosaminoglycans differed in their relative activities with the chondroitin sulfates producing the most pronounced effects. The less amyloidogenic Abeta40 isoform did not show an immediate structural transition that was dependent upon the shielding effect by the phosphate counter ion. Removal or substitution of phosphate resulted in similar glycosaminoglycan-induced conformational and aggregation changes. These findings clearly demonstrate that glycosaminoglycans act at the earliest stage of fibril formation, namely amyloid-beta nucleation, and are not simply involved in the lateral aggregation of preformed fibrils or nonspecific adhesion to plaques. The identification of a structure-activity relationship between amyloid-beta and the different glycosaminoglycans, as well as the condition dependence for glycosaminoglycan binding, are important for the successful development and evaluation of glycosaminoglycan-specific therapeutic interventions.     

N-terminal myristoylation regulates calcium-induced conformational changes in neuronal calcium sensor-1

Neuronal calcium sensor-1 (NCS-1), a Ca(2+)-binding protein, plays an important role in the modulation of neurotransmitter release and phosphatidylinositol signaling pathway. It is known that the physiological activity of NCS-1 is governed by its myristoylation. Here, we present the role of myristoylation of NSC-1 in governing Ca(2+) binding and Ca(2+)-induced conformational changes in NCS-1 as compared with the role in the nonmyristoylated protein. The (45)Ca binding and isothermal titration calorimetric data show that myristoylation increases the degree of cooperativity; thus, the myristoylated NCS-1 binds Ca(2+) more strongly (with three Ca(2+) binding sites) than the non-myristoylated one (with two Ca(2+) binding sites). Both forms of protein show different conformational features in far-UV CD when titrated with Ca(2+). Large conformational changes were seen in the near-UV CD with more changes in the case of nonmyristoylated protein than the myristoylated one. Although the changes in the far-UV CD upon Ca(2+) binding were not seen in E120Q mutant (disabling EF-hand 3), the near-UV CD changes in conformation also were not influenced by this mutation. The difference in the binding affinity of myristoylated and non-myristoylated proteins to Ca(2+) also was reflected by Trp fluorescence. Collisional quenching by iodide showed more inaccessibility of the fluorophore in the myristoylated protein. Mg(2+)-induced changes in near-UV CD are different from Ca(2+)-induced changes, indicating ion selectivity. 8-Anilino-1-naphthalene sulfonic acid binding data showed solvation of the myristoyl group in the presence of Ca(2+), which could be attributed to the myristoyl-dependent conformational changes in NCS-1. These results suggest that myristoylation influences the protein conformation and Ca(2+) binding, which might be crucial for its physiological functions.     

Protein and DNA reactions stimulated by electromagnetic fields

The stimulation of protein and DNA by electromagnetic fields (EMF) has been problematic because the fields do not appear to have sufficient energy to directly affect such large molecules. Studies with electric and magnetic fields in the extremely low-frequency range have shown that weak fields can cause charge movement. It has also been known for some time that redistribution of charges in large molecules can trigger conformational changes that are driven by large hydration energies. This review considers examples of direct effects of electric and magnetic fields on charge transfer, and structural changes driven by such changes. Conformational changes that arise from alterations in charge distribution play a key role in membrane transport proteins, including ion channels, and probably account for DNA stimulation to initiate protein synthesis. It appears likely that weak EMF can control and amplify biological processes through their effects on charge distribution.     

X-ray diffraction studies on muscle regulation

Using the intensity of the outer part of the second actin layer line as an indicator of thin filament conformation in vertebrate muscle we were able to identify the four different states of rest, and the three states induced by the presence of Ca²⁺ ions, rigor bridge attachment and actively cycling bridges, respectively. These findings are in qualitative agreement with a number of biochemical studies by Eisenberg and Greene and others, indicating that activation of the thin filament depends both on Ca²⁺ ions and crossbridge binding. Yet quantitatively, the biochemical data and our structural data are contradictory. Whereas the biochemical studies suggest a strong coupling between structural changes of the thin filament and the ATPase activity, the structural studies indicate that this is not necessarily the case.Troponin molecules also change their conformation upon activation depending on both Ca²⁺ ions and crossbridge binding as demonstrated by the early part of the time course of the thin filament meridional reflections in contracting frog muscle.Low ionic strength which has been shown by Brenner and collaborators to increase weakly binding crossbridges in relaxed rabbit psoas muscle does not influence the intensity of the second actin layer line in this muscle. Yet in contracting frog muscle the increase of the second actin layer line increases very rapidly in one step, suggesting that weak binding bridges which are attached to actin prior to force production may indeed influence the thin filament conformation. It therefore appears that weakly bound bridges in the low ionic strength state do not have the same effect on the thin filament conformation as weakly bound bridges in an actively contracting muscle.Arthropod muscles like the thin filament regulated lobster muscles differ from vertebrate muscle in not showing an increase of the second layer line during contraction, which may have to do with differences in crossbridge attachment. The myosin-regulated molluscan muscle ABRM shows a large increase on the second actin layer line upon phasic contraction and a much smaller increase in catch or rigor, indicating that actively cycling bridges influence the thin filament conformation differently than catch or rigor bridges.Several pieces of evidence which we have briefly outlined in this paper suggest that the thin filament conformational changes we have observed do not arise solely from tropomyosin movements and that conformational changes of actin domains should be considered.