Differences In Free Energy Changes Research Articles

Free Energy Changes at State-Transition Critical Points As a Patient-Specific Biomarker in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy with a poor overall survival rate. AML is a highly heterogeneous disease driven by combinations of genomic mutations, epigenetic alterations, and biochemical signaling. The heterogeneity underlying AML needs novel approaches for individualized clinical assessment and treatment selection. However, approaches that rely on differential expression or correlation analysis lack an underlying theoretical model of the disease to inform the interpretation of ongoing processes in each individual which is crucial for personalized diagnostics and treatment selection. We have previously shown that the state-transition (ST) theory, state-space could predict AML disease evolution using the mRNA of a conditional Cbfb-MYH11 (CM) knock-in mouse model. The ST model represents AML evolution from health to disease as a state transition of the transcriptome state described as a particle undergoing Brownian motion in a double-well quasi-potential, where critical points define states of healthy, c1, transition to AML, c2, and overt AML, c3. We integrated two approaches, the ST model and surprisal analysis (SA) from thermodynamics, to create a patient-specific characterization that can be used to identify the individualized state of the disease. The publicly available datasets of human blood including normal samples, AML samples of bone marrow (BM), and peripheral blood (PB) were utilized for the methods. A total of 858 samples from three publicly available RNA-seq datasets were used. The Therapeutically Applicable Research to Generate Effective Treatments (TARGET) dataset is a pediatric study, from which we analyzed 84 normal samples and 126 AML inv(16) samples, consisting of 105 BM and 21 PB samples with ages from 0.3 to 28 years. Two additional datasets from the BEATAML study and the Cancer Genome Atlas (TCGA), were downloaded from the Genomic Data Commons (GDC) portal using GDCRNATools. The BEATAML dataset included 21 normal and 476 AML samples consisting of 226 PB and 243 BM samples with ages from 2 to 87, and TCGA dataset contained 151 AML PB samples, with ages 21 to 88 years. We used a pseudotime approach to infer disease dynamics for ST using KIT and CD33. We found via t-test analysis that KIT expression between normal and AML samples was significantly different for both TARGET (p<0.01) and BEATAML (p<0.01). CD33 expression between normal and AML samples was significant in BEATAML (p=0.012), but not TARGET (p=0.30). We combined the ST with SA to analyze free energy changes (FEC) that occurred at ST critical points, which predicted AML progression as normal hematopoiesis, c1, transition to AML, c2, and overt AML, c3. Significant differences in FEC at the critical points c1, c2, and c3 were observed by t-test comparisons as c1 vs. c2 (p <0.001) and c1 vs. c3 (p <0.001) in TARGET dataset, and c2 vs. c3 (p < 0.01) in the BEATAML dataset. Our results showed that the transition disease state, c2, had higher FEC as compared to normal states, c1, suggesting that samples were less stable from a thermodynamic perspective. Certain samples at the more advanced state, c3, exhibited a trend toward decreasing FEC levels, suggesting a process of state stabilization at more advanced stages of the disease. SA and Gene Ontology analysis suggested that interleukin pathways such as IL1, IL2, IL8 and IL10, MAPK, NFkB, and migration-related pathways, which are known to be involved in AML progression, were found in multiple unbalanced processes in SA characterizing AML across all three datasets. The IL1 pathway was found in 3 unbalanced processes characterizing TARGET dataset and in four processes characterizing TCGA and BEATAML. IL2 related categories were found among induced transcripts in 4 unbalanced processes characterizing TARGET dataset, in 6 characterizing TCGA, and in 4 processes characterizing BEATAML. We observed that AML samples with comparable FEC levels or clinical characteristics could be defined by different sets of unbalanced processes, implying that this information might be used to determine tailored therapies in subgroups of individuals with similar clinical or thermodynamic characteristics. We showed that mapping FEC into an AML state-space given by ST critical points could provide a high resolution, patient-specific disease characterization, and these methods could be used in personalized diagnostics and individualized treatment procedures.

Quantum Chemical Insight into 1,2-Shift Rearrangement in Bromination of Allylaryls.

In this work, we have provided mechanistic insight into the addition of bromine to an allylic double bond of allylaryl derivatives using experimental and DFT-based electronic structure methods. The experimental yields indicate the influence of the functional group on the aryl ring on the ratio of 1,2-dibromo and 1,3-dibromo adducts formed in the reaction. The optimized geometry and the electron density maps of the allylaryls and their cationic intermediates from DFT simulations revealed that electron-rich aryl rings promoted formation of cationic spiro[2.5] intermediate II, whereas electron-poor aryl rings resulted in formation of bromonium intermediate I. It was observed that electron-rich allylaryls promoted the 1,2-shift of the aryl ring that resulted in bond formation between the carbon atom (C1) on the aryl ring and the central carbon atom (C3) in the allylic double bond and formed spiro[2.5] intermediate II, a trend which was confirmed by harmonic oscillator model of aromaticity index. Also, Wiberg bond order analysis is in good agreement with the experimental work. Thermochemical analysis indicates that smaller C1···C3 distance resulted in favorable values for the difference in free energy change (ΔΔG). The favorable ΔΔG values are a result of higher electron density on the aryl ring, making it more nucleophilic toward C3 carbon and promoting 1,2-shift that led to formation of the spiro[2.5] intermediate. Thus, the underlying mechanism indicates that the electron-rich allylaryls promote the formation of 1,3-dibromo compounds through formation and stabilization of the spiro[2.5] intermediate II.

Comprehensive Evaluation of the Binding of Lipocalin-Type Prostaglandin D Synthase to Poorly Water-Soluble Drugs

Low water solubility of candidate drug compounds is a major problem in pharmaceutical research and development. We developed a novel drug delivery system (DDS) for poorly water-soluble drugs using lipocalin-type prostaglandin D synthase (L-PGDS), which belongs to the lipocalin superfamily and binds a large variety of hydrophobic molecules. In this study, we comprehensively evaluated the capability of L-PGDS to bind and solubilize various poorly water-soluble drugs using structure-based docking. Docking simulations of 2892 commercially available approved drugs indicated that L-PGDS shows higher binding affinities for various drugs compared with 2-hydroxypropyl-β-cyclodextrin. Five drugs selected from the top 100 with the highest binding affinities for L-PGDS exhibited very low solubility in PBS (pH 7.4). However, in the presence of 1 mM L-PGDS, the apparent solubility of all drugs improved markedly, from 19.5- to 166-fold. Calorimetric experiments on two drugs, telmisartan and imatinib, revealed that L-PGDS forms a 1:2 complex with each drug, with dissociation constants of 0.4-40.0 μM. Kinetic simulations of drug dissolution with L-PGDS indicated that the difference in free energy change (ΔΔG) between the insoluble state and the L-PGDS-bound state are within the range from -10 to +5 kJ mol-1. The ΔΔG value is a critical factor in evaluating whether a poorly water-soluble drug can be solubilized by L-PGDS. Collectively, these results demonstrate that in silico docking is a promising approach for identifying drug molecules suitable for the L-PGDS-based DDS.

Molecular population dynamics of DNA structures in a bcl-2 promoter sequence is regulated by small molecules and the transcription factor hnRNP LL.

Minute difference in free energy change of unfolding among structures in an oligonucleotide sequence can lead to a complex population equilibrium, which is rather challenging for ensemble techniques to decipher. Herein, we introduce a new method, molecular population dynamics (MPD), to describe the intricate equilibrium among non-B deoxyribonucleic acid (DNA) structures. Using mechanical unfolding in laser tweezers, we identified six DNA species in a cytosine (C)-rich bcl-2 promoter sequence. Population patterns of these species with and without a small molecule (IMC-76 or IMC-48) or the transcription factor hnRNP LL are compared to reveal the MPD of different species. With a pattern recognition algorithm, we found that IMC-48 and hnRNP LL share 80% similarity in stabilizing i-motifs with 60 s incubation. In contrast, IMC-76 demonstrates an opposite behavior, preferring flexible DNA hairpins. With 120–180 s incubation, IMC-48 and hnRNP LL destabilize i-motifs, which has been previously proposed to activate bcl-2 transcriptions. These results provide strong support, from the population equilibrium perspective, that small molecules and hnRNP LL can modulate bcl-2 transcription through interaction with i-motifs. The excellent agreement with biochemical results firmly validates the MPD analyses, which, we expect, can be widely applicable to investigate complex equilibrium of biomacromolecules.

The Amber ff99 Force Field Predicts Relative Free Energy Changes for RNA Helix Formation

The ability of the Amber ff99 force field to predict relative free energies of RNA helix formation was investigated. The test systems were three hexaloop RNA hairpins with identical loops and varying stems. The potential of mean force of stretching the hairpins from the native state to an extended conformation was calculated with umbrella sampling. Because the hairpins have identical loop sequence, the differences in free energy changes are only from the stem composition. The Amber ff99 force field was able to correctly predict the order of stabilities of the hairpins, although the magnitude of the free energy change is larger than that determined by optical melting experiments. The two measurements cannot be compared directly because the unfolded state in the optical melting experiments is a random coil, while the end state in the umbrella sampling simulations was an elongated chain. The calculations can be compared to reference data by using a thermodynamic cycle. By applying the thermodynamic cycle to the transitions between the hairpins using simulations and nearest neighbor data, agreement was found to be within the sampling error of simulations, thus demonstrating that ff99 force field is able to accurately predict relative free energies of RNA helix formation.

Structural Stability as a Probe for Molecular Evolution of Homologous Albumins Studied by Spectroscopy and Bioinformatics

Equilibrium unfolding by guanidinium hydrochloride (GuHCl) and urea as well as evolutionary trends of two homologous albumins, pig serum albumin (PSA) and rabbit serum albumin (RSA), has been studied with circular dichroism, tryptophanyl fluorescence and bioinformatics. GuHCl cannot distinguish the contribution of electrostatic interactions to the proteins which were otherwise effectively monitored by urea. Higher differences in free energy changes due to urea than GuHCl show electrostatic interactions among charged amino acids are possibly responsible for higher structural stability of RSA in comparison to PSA. From the sequence of HSA and RSA, deletion of arginine at position 117 and the presence of one extra tryptophan at position 135 may possess some clue for lesser stability of PSA. Here, for comparison, chemical unfolding data of HSA and BSA had been taken into consideration. We found that thermodynamically RSA and PSA are closer to HSA and BSA, respectively, in accordance with their sequence homologies. Taxonomically, rabbit belongs to lagomorph which is closer to hominids than ungulates. Hence, on the basis of these thermodynamic data of protein denaturation of different species we can use this new approach to analyze the phylogenetic relationship among the major clades of eutherian mammals to obtain their evolutionary trends.

Free Energy Contributions to Direct Readout of a DNA Sequence

The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol(-1)), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol(-1) from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken.

NMR studies of chiral discrimination relevant to the enantioseparation of N‐acylarylalkylamines by an (R)‐phenylglycinol‐derived chiral selector

Recently, it was reported that the chiral recognition ability of (R)-N-3,5-dinitrobenzoyl phenylglycinol derivative was examined as a new HPLC chiral stationary phase (CSP 1) for the resolution of racemic N-acylnaphthylalkylamines. However, the mechanism of chiral discrimination on the CSP remained elusive until now. In this study, a spectroscopic investigation of the chiral discrimination mechanism of CSP 1 was undertaken using mixtures of (R)-N-3,5-dinitrobenzoyl phenylglycinol-derived chiral selector (2) and each of the enantiomers of N-acylnaphthylalkylamines (3) by NMR study. First, the differences in free energy changes (DeltaDeltaG) upon diastereomeric complexation in solution between the complex of each isomer with chiral selector 2 by NMR titration were calculated. The values were then compared with those estimated by chiral HPLC. The chemical shift changes of each proton on the chiral selector and analytes were also checked and it was found that the chemical shift changes decreased continuously as the acyl group on analytes increased in length. This observation was consistent with the HPLC data. From these experimental results, the interaction mechanism of chiral discrimination between the chiral selector and the analytes is more precisely explained.

Regulation of Glycosaminoglycan Function by Osmotic Potentials: MEASUREMENT OF WATER TRANSFER DURING ANTITHROMBIN ACTIVATION BY HEPARIN

The sulfated glycosaminoglycan heparin is an important anticoagulant, widely used to treat and to prevent arterial thrombosis. Heparin triggers conformational changes in, and the functional activation of, the serine proteinase inhibitor antithrombin. We investigated water-transfer reactions during the activation process to explore the possibility that functional interaction between antithrombin and sulfated glycosaminoglycans can be regulated by osmotic potentials. Volume of water transferred upon heparin binding was measured from differences in free energy change, Delta(Delta G), with osmotic stress, pi. Osmotic stress was induced with chemically inert probes that are geometrically excluded from the water-permeable spaces of antithrombin and from intermolecular spaces formed during the association reaction. The free energy change, Delta G, for the antithrombin/heparin interaction was calculated from the dissociation constant, determined by functional titrations of heparin with antithrombin at fixed concentrations of the coagulation protease factor Xa. The effect of osmotic stress was independent of the chemical nature of osmotic probes but correlated with their radius up to radius >17 A. In mixtures including a large and a small probe, the effect of the large probe was not modified by the small probe added at a large molar excess. With an osmotic probe of 4-A radius, the Delta(Delta G)/pi slope corresponds to a transfer of 119 +/- 25 water molecules to bulk solution on formation of the complex. Analytical characterization of water-permeable volumes in x-ray-derived bound and free antithrombin structures revealed complex surfaces with smaller hydration volumes in the bound relative to the free conformation. The residue distribution in, and atomic composition of, the pockets containing atoms from residues implicated in heparin binding were distinct in the bound versus free conformer. The results demonstrate that the heparin/antithrombin interaction is linked to net water transfer and, therefore, can be regulated in biological gels by osmotic potentials.

Thermodynamic considerations of arteriovenous gradients of hydrogen ion concentration and carbon dioxide tension.

It is shown that, in a multicompartmental homeostatic system, the extent of interaction between any two compartments can be assessed by determination of the difference in free energy change of one particular reaction, or a series of coupled reactions, operative in both of the compartments under consideration. Hydrogen ion concentration and carbon dioxide tension have been used to determine free energy change difference relationships between the venous and arterial compartments (-deltadeltaG(a-v)) of the circulatory system. Data from the literature (from two studies of congestive heart failure and one study of experimentally induced cardiac arrest) are used to calculate -deltadeltaG(a-v). It was found that in control subjects -deltadeltaG(a-v) is close to zero, whereas in congestive heart failure or cardiac arrest, the value rises to 150 cal mol(-1) or more, whereas in blood, the approach towards equilibrium between hydrogen and bicarbonate ions and dissolved carbon dioxide (aqueous CO2) is known to be only moderately rapid. It is concluded that, in the system under study, and with respect to the reaction H+ + HCO3- = CO2 + H2O, a high value for the free energy change difference between the two compartments (high -deltadeltaG(a-v)) must be due to an insufficient blood circulation rate. Accordingly, -deltadeltaG(a-v) is probably a quantitative measure of cardiac insufficiency.

Crystallization of hafnia and zirconia during the pyrolysis of acetate gels

Hafnia and zirconia gels were prepared by drying hafnyl or zirconyl acetate solutions. Hafnia and zirconia gels contain both hydroxyl group and bidentate acetates which are directly bonded to the metal ions. Thermal decomposition and crystallization behavior of the gels were investigated through XRD, FT-IR, and TEM. Hafnium-containing gels crystallized directly into stable monoclinic hafnia around 500–540 °C, while zirconium-containing gels first formed metastable tetragonal zirconia around 450 °C. The dissimilar crystallization behavior of the gels into metastable, tetragonal zirconia or into stable, monoclinic hafnia can be explained through the difference in free-energy changes of the tetragonal-to-monoclinic phase transformation.

Isotopic Fractionation of Fentanyl and Its Deuterated Analogues by Capillary Gas Chromatography.

Isotopic fractionation of fentanyl (FT) and its deuterated analogues by gas chromatography using capillary columns (CBP1 and CBP5) has been investigated. Seven kinds of analogues were labeled with 5 to 19 deuterium atoms at the anilino, propionyl and/or phenylethyl group of FT. The retention times of deuterated FT in CBP1 and CBP5 columns are inversely proportional to the number of labeled deuterium atoms in the molecule. The difference in free energy changes (ΔΔG) had a linear relationship with the number of labeled deuterium atoms, except for labeling at anilino and phenylethyl group. The contribution of a deuterium atom to the ΔΔG value was estimated to be 1.13 cal/mol in CBP1 and 1.40 cal/mol in CBP5, respectively. While, its contribution in the propiony group was 2.84 cal/mol in CBP1 and 2.48 cal/mol in CBP5, respectively. An important factor in separation by GC may differences in interactions between the stationary phase of the column with the three dimensional protrusive moiety in the molecule.

On the mechanism of preferential incorporation of dAMP at abasic sites in translesional DNA synthesis. Role of proofreading activity of DNA polymerase and thermodynamic characterization of model template-primers containing an abasic site.

DNA polymerase preferentially incorporate dAMP opposite abasic sites (A-rule). The mechanism of the A-rule can be studied by analyzing three dissected stages of the reaction including (i) initial nucleotide insertion, (ii) proofreading excision of the inserted nucleotide and (iii) extension of the nascent primer terminus. To assess the role of the stage (ii) in the A-rule, kinetic parameters of the proofreading excision of primer terminus nucleotides opposite abasic sites were determined using E.coli DNA polymerase I Klenow fragment. The relative efficiency of the excision (Vmax/Km) revealed that removal of A was the least favored of the four nucleotides, but the differences in the efficiencies between excision of A and the other nucleotides was less than 2-fold. In addition, in an attempt to reconcile kinetic data associated with the stage (i) or (ii), the differences in free energy changes (delta delta G degrees) for the formation of model template-primer termini containing XN pairs (X = abasic site, N = A, G, C or T) were determined by temperature dependent UV-melting measurements. The order of delta delta G degrees was XG > XA = XC > or = XT, with delta delta G degrees being 0.5 kcal/mol for the most stable XG and the least stable XT. Based on these data, the role of the stage (ii) and energetic aspects of the A-rule are discussed.

Energy coupling in DNA gyrase: a thermodynamic limit to the extent of DNA supercoiling.

ATP alpha S (Rp) has been shown to support the supercoiling of plasmid pBR322 catalyzed by Escherichia coli DNA gyrase at comparable rates to the natural substrate ATP and is able to promote the introduction of one more superhelical turn than ATP. The difference in free energy change between consecutive rounds of supercoiling in gyrase-mediated reactions is calculated to be 2.6 kJ mol-1. The difference in free energy of hydrolysis of ATP and ATP alpha S (Rp) has been determined from the difference in the equilibrium constants for the phosphorylation of arginine established by arginine kinase. This equilibrium constant has been found to be displaced by a factor of about 1.5, corresponding to a greater free energy of hydrolysis of ATP alpha S (Rp) compared to ATP of approximately 1 kJ mol-1. This difference in free energy can be tentatively ascribed to a relative destabilization of the MgATP alpha S (Rp) complex with respect to MgATP. Assuming that the stoichiometry of the coupled reactions requires two ATPs hydrolyzed per round of supercoiling, ATP alpha S (Rp) should be capable of providing an additional ca. 2 kJ mol-1 of free energy for DNA supercoiling, which is in good agreement with estimates for the additional free energy required to achieve a further round of supercoiling. These results provide direct evidence to support the proposal that the extent of DNA supercoiling by DNA gyrase is limited by the free energy of hydrolysis of the nucleotide.

Enhanced thermodynamic stabilities of yeast iso-1-cytochromes c with amino acid replacements at positions 52 and 102

We have determined the structures and thermodynamic stabilities of the wild type Asn-52 and unusually thermostable mutant Ile-52 yeast iso-1-cytochromes c (Das, G., Hickey, D. R. McLendon, D., McLendon, G., and Sherman, F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 496-499). Although both structures were similar, Water-166, buried within the wild type protein, is excluded from the Ile-52 mutant, which substantially reorganizes the local hydrogen bonding. Wild type Cys-102 was replaced with alanine or serine to eliminate dimerization in vitro. The Cys-102 (wild type), Ala-102, and Ser-102 proteins were equally stable, whereas the chemically modified Cys-102-SCH3 was less stable. The order of stability observed with replacements at positions 52 and 102 was as follows: Ile-52 Ala-102 greater than Ala-52 Ala-102 greater than Asn-52 Ala-102 ("normal") greater than Gly-52 Ala-102. No significant stabilization was attributed to potential energy interactions expressed as helix-forming propensities of replacements at position 52. A high correlation between differences in free energy changes and transfer free energies suggests hydrophobic interactions are the main factor for enhancing stability in the Ile-52 mutant. Additional possible contributions to the thermostability of the Ile-52 variant are energetic effects due to packing and hydrogen bonding changes surrounding position 52.

Thermodynamic stabilities of yeast iso-1-cytochromes c having amino acid substitutions for lysine 32.

Iso-1-cytochromes c having lysine 32 replaced by leucine, glutamine, tyrosine, and tryptophan were prepared from strains of bakers' yeast, Saccharomyces cerevisiae, and chemically blocked at cysteine 107 with methyl methanethiolsulfonate to prevent dimerization. These modified ferricytochromes c were guanidine denatured, and the unfolding thermodynamics were determined by circular dichroism and fluorescence measurements. Thermal unfolding was also monitored by absorbance measurements. The guanidine denaturation midpoints for the altered proteins are smaller than the wild type, while the orders of stability from unfolding free energy changes are: Lys-32 (wild type) approximately Leu-32 approximately Gln-32 (circular dichroism), greater than Gln-32 (fluorescence) greater than Tyr-32 approximately Trp-32. Midpoints and differences in free energy changes for thermal unfolding parallel the fluorescence free energy changes for guanidine-induced unfolding. Thus, the blocked Leu-32 and Lys-32 proteins are equally stable with respect to both chemical and thermal denaturation. The reported data indicate that single replacements may significantly modify protein stability, and that substitution for an evolutionarily retained residue in normal cytochrome c structures does not always destabilize the protein. In addition, in vitro thermal stabilities approximately correlate with in vivo specific activities.

Iron-histidine stretching vibration in the deoxy state of insect hemoglobins with different O2 affinities and Bohr effects.

Resonance Raman spectroscopy has been employed to detect the iron-proximal histidine stretching mode in deoxyhemoglobins from insect larvae of Chironomus thummi thummi (CTT). With the excitation of 413.1 nm, we observe a sharp and intense line in the 220-224 cm-1 region. The assignment of this line to the Fe-N epsilon (His) stretching mode was made on the basis of a 3-cm-1 shift upon 57Fe/54Fe isotope substitution. The Fe-N epsilon (His) vibration is used to monitor the possible changes in the Fe-N epsilon (His) bond strength (hence bone length) in the deoxy state of the monomeric (CTT I, III, and IV) and dimeric (CTT II) insect hemoglobins. As these hemoglobins differ in O2 affinity, off-rate and on-rate constants, and in the Bohr effect, they are excellent model systems for investigating the mechanism of protein control of the heme reactivity. Some of these hemoglobins (CTT III, IV, and II) are allosteric, exhibiting two interconvertible conformational states with high and low O2 affinity at high and low pH, respectively. The Fe-N epsilon (His) stretching frequency does not correlate with the O2 affinity, the on-rate and the off-rate constants for different hemoglobins, for different conformational states, and for modified hemoglobins with different heme peripheral groups. This vibrational mode is insensitive to deuteration of the heme vinyl groups. It is important to note that the Fe-N epsilon (His) bonds in the high pH (high-affinity) and the low pH (low-affinity) states are identical. This implies that the O2 molecule, prior to binding, "sees" identical binding sites. Thus, the difference in free energy changes upon O2 binding is manifested only in the oxy form.