Thermodynamics of unpaired terminal nucleotides on short RNA helixes correlates with stacking at helix termini in larger RNAs.

Free energies for stacking of unpaired nucleotides (dangling ends) at the termini of oligoribonucleotide Watson-Crick helixes (DeltaG(0)37,stack) depend on sequence for 3' ends but are always small for 5' ends. Here, these free energies are correlated with stacking at helix termini in a database of 34 RNA structures determined by X-ray crystallography and NMR spectroscopy. Stacking involving GA pairs is considered separately. A base is categorized as stacked by its distance from (相似文献   

Thermostability of double-stranded deoxyribonucleic acids: effects of covalent additions of a psoralen

We have carried out a thermodynamic study on the effects of covalent additions of the psoralen derivative HMT, 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, on the stability of double-stranded deoxyoligonucleotides. This was done with two systems. The first was a double-stranded DNA formed by two non-self-complementary oligonucleotides, 5'-GAAGCTACGAGC-3' and 5'-GCTCGTAGCTTC-3', where we site specifically placed an HMT molecule on the thymidine residue in oligonucleotide 5'-GAAGCTACGAGC-3' as either a furan-side monoadduct or a pyrone-side monoadduct. The second was a double-stranded DNA formed by a self-complementary oligonucleotide, 5'-GGGTACCC-3', where we placed an HMT molecule on the thymidine residue of each strand as a furan-side monoadduct or cross-linked the two strands with an HMT molecule linked to the two thymidines. We found that HMT cross-linking of the two strands stabilizes the double helix formed by 5'-GGGTACCC-3', as one might expect. Less predictable results were that the monoaddition of a psoralen stabilizes the double helix formed by the two non-self-complementary oligonucleotides by as much as 1.3 kcal/mol as a furan-side monoadduct and 0.7 kcal/mol as a pyrone-side monoadduct at 25 degrees C in 50 mM NaCl. In contrast, the monoaddition of a psoralen on each of the two thymidines in the double helix formed by 5'-GGGTACCC-3' destabilizes the helix by 1.8 kcal/mol at 25 degrees C in 1 M NaCl. This destabilization arises from an unfavorable enthalpy change (8.6 kcal/mol) and a favorable entropy change (23 cal/K X mol) due to the two HMT molecules at the centers of each strand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free energy contributions of G.U and other terminal mismatches to helix stability

Thermodynamic parameters of helix formation were measured spectroscopically for seven hexaribonucleotides containing a GC tetramer core and G.U or other terminal mismatches. The free energies of helix formation are compared with those for the tetramer core alone and with those for the hexamer with six Watson-Crick base pairs. In 1 M NaCl, at 37 degrees C, the free energy of a terminal G.U mismatch is about equal to that of the corresponding A.U pair. Although other terminal mismatches studied add between -1.0 and -1.6 kcal/mol to delta G0 37 for helix formation, all are less stable than the corresponding Watson-Crick pairs. Comparisons of the stability increments for terminal G.U mismatches and G.C pairs suggest when stacking is weak the additional hydrogen bond in the G.C pair adds roughly -1 kcal/mol to the favorable free energy of duplex formation.     

Strong interaction between disulfide derivatives and aromatic groups in peptides and proteins

The intermolecular interaction energy of complexes of dimethyldisulfide with benzene and cyclohexane, respectively, was computed as function of the relative distance and orientation within each pair of molecules. The energy of the most stable orientation of the dimethyldisulfide-cyclohexane complex is ?2.57 kcal/mol, while that of the most stable orientation of the dimethyldisulfide-benzene complex is ?3.33 kcal/mol. The energy difference of ～0.8 kcal/mol is due to favorable specific nonbonded interactions between the sulfur atoms and the atoms of the aromatic ring. Proper parameterization of empirical interatomic energies, used in computations in this laboratory, accounts for these interactions without the need for a $\text{special}$ sulfur-aromatic potential energy function.     

Replication protein A interactions with DNA. III. Molecular basis of recognition of damaged DNA

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (subunits of 70, 32, and 14 kDa) that is required for cellular DNA metabolism. RPA has been reported to interact specifically with damaged double-stranded DNA and to participate in multiple steps of nucleotide excision repair (NER) including the damage recognition step. We have examined the mechanism of RPA binding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containing damage. We show that the affinity of RPA for damaged dsDNA correlated with disruption of the double helix by the damaged bases and required RPAs ssDNA-binding activity. We conclude that RPA is recognizing single-stranded character caused by the damaged nucleotides. We also show that RPA binds specifically to damaged ssDNA. The specificity of binding varies with the type of damage with RPA having up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We show that this specific binding was absolutely dependent on the zinc-finger domain in the C-terminus of the 70-kDa subunit. The affinity of RPA for damaged ssDNA was 5 orders of magnitude higher than that of the damage recognition protein XPA (xeroderma pigmentosum group A protein). These findings suggest that RPA probably binds to both damaged and undamaged strands in the NER excision complex. RPA binding may be important for efficient excision of damaged DNA in NER.     

Measurement of thermodynamic parameters for hydrophobic mismatch 2: intermembrane transfer of a transmembrane helix

Hydrophobic matching between proteins and lipids is essential for the thermodynamic stability of integral membrane proteins. However, there is no direct thermodynamic information available about the intermembrane transfer of proteins between membranes with different hydrophobic thicknesses, which is crucial for understanding hydrophobic mismatch. This article reports the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the intermembrane transfer of the inert hydrophobic model transmembrane helix NBD-(AALALAA)(3)-NH(2) (NBD: 7-nitro-2-1,3-benzoxadiazol-4-yl), which is exchangeable between vesicles, from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to dimonounsaturated-phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 37-58 degrees C. The transfer free energies were calculated from equilibrium values of the extent of helix transfer from donor to acceptor lipid vesicles, as monitored by a decrease in fluorescence resonance energy transfer from the NBD group to a lipid-labeled Rhodamine in the donor upon transfer to the quencher-free acceptor. Under hydrophobic mismatch conditions up to approximately 7 A, the helix partitioning became unfavorable up to +7 kJ mol(-)(1), hampered by an increase in entropic (up to +20 kJ mol(-)(1)) and enthalpic (up to +66 kJ mol(-)(1)) terms in thinner and thicker membranes, respectively. Together with the results that H/D exchange at the membrane interface was accelerated in thinner membranes the obtained thermodynamic parameters were reasonably explained assuming that hydrophobic mismatch induces aqueous exposure or membrane burial of the helix termini, resulting in excess energies originating from the hydration of terminal hydrophobic residues or the unfavorable Born energy of terminal partial charges of the helix macrodipole.     

N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring. Evaluation of the mechanistic basis for their enhanced biological properties

Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.     

NMR structures of apo L. casei dihydrofolate reductase and its complexes with trimethoprim and NADPH: contributions to positive cooperative binding from ligand-induced refolding, conformational changes, and interligand hydrophobic interactions

In order to examine the origins of the large positive cooperativity (ΔG(0)(coop) = -2.9 kcal mol(-1)) of trimethoprim (TMP) binding to a bacterial dihydrofolate reductase (DHFR) in the presence of NADPH, we have determined and compared NMR solution structures of L. casei apo DHFR and its binary and ternary complexes with TMP and NADPH and made complementary thermodynamic measurements. The DHFR structures are generally very similar except for the A-B loop region and part of helix B (residues 15-31) which could not be directly detected for L. casei apo DHFR because of line broadening from exchange between folded and unfolded forms. Thermodynamic and NMR measurements suggested that a significant contribution to the cooperativity comes from refolding of apo DHFR on binding the first ligand (up to -0.95 kcals mol(-1) if 80% of A-B loop requires refolding). Comparisons of Cα-Cα distance differences and domain rotation angles between apo DHFR and its complexes indicated that generally similar conformational changes involving domain movements accompany formation of the binary complexes with either TMP or NADPH and that the binary structures are approaching that of the ternary complex as would be expected for positive cooperativity. These favorable ligand-induced structural changes upon binding the first ligand will also contribute significantly to the cooperative binding. A further substantial contribution to cooperative binding results from the proximity of the bound ligands in the ternary complex: this reduces the solvent accessible area of the ligand and provides a favorable entropic hydrophobic contribution (up to -1.4 kcal mol(-1)).     

Thermodynamic analysis of binding between mouse major urinary protein-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole

The mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT) binds to an occluded, nonpolar cavity in the mouse major urinary protein-I (MUP-I). The thermodynamics of this interaction have been characterized using isothermal titration calorimetry (ITC). MUP-I-SBT binding is accompanied by a large favorable enthalpy change (DeltaH = -11.2 kcal/mol at 25 degrees C), an unfavorable entropy change (-TDeltaS = 2.8 kcal/mol at 25 degrees C), and a negative heat capacity change [DeltaC(p)() = -165 cal/(mol K)]. Thermodynamic analysis of binding between MUP-I and several 2-alkyl-4,5-dihydrothiazole ligands indicated that the alkyl chain contributes more favorably to the enthalpy and less favorably to the entropy of binding than would be expected on the basis of the hydrophobic desolvation of short-chain alcohols. However, solvent transfer experiments indicated that desolvation of SBT is accompanied by a net unfavorable change in enthalpy (DeltaH = +1.0 kcal/mol) and favorable change in entropy (-TDeltaS = -1.8 kcal/mol). These results are discussed in terms of the possible physical origins of the binding thermodynamics, including (1) hydrophobic desolvation of both the protein and the ligand, (2) formation of a buried water-mediated hydrogen bond network between the protein and ligand, (3) formation of strong van der Waals interactions, and (4) changes in the structure, dynamics, and/or hydration of the protein upon binding.     

Improved free energies for G.C base-pairs

Thermodynamic parameters of helix formation are reported for seven oligoribonucleotides containing only G.C pairs. These data are used with the nearest-neighbor model to calculate enthalpies and free energies of base-pair formation for G.C pairs. For helix initiation, the free energy change at 37 degrees C, delta G(0)37, is +3.9 kcal/mol; for helix propagation, the delta G(0)37 values are -2.3, -3.2 and -3.3 kcal/mol for C-G, G-G and G-C neighbors, respectively.     

Cooperativity of papain-substrate interaction energies in the S2 to S2' subsites

Enzyme-substrate contacts in the hydrolysis of ester substrates by the cysteine protease papain were investigated by systematically altering backbone hydrogen-bonding and side-chain hydrophobic contacts in the substrate and determining each substrate's kinetic constants. The observed specificity energies [defined as delta delta G obs = -RT ln [(kcat/KM)first/(kcat/KM)second)]] of the substrate backbone hydrogen bonds were -2.7 kcal/mol for the P2 NH and -2.6 kcal/mol for the P1 NH when compared against substrates containing esters at those sites. The observed binding energies were -4.0 kcal/mol for the P2 Phe side chain, -1.0 kcal/mol for the P1' C=O, and -2.3 kcal/mol for the P2' NH. The latter three values probably all significantly underestimate the incremental binding energies. The P2 NH, P2 Phe side-chain, and P1 NH contacts display a strong interdependence, or cooperativity, of interaction energies that is characteristic of enzyme-substrate interactions. This interdependence arises largely from the entropic cost of forming the enzyme-substrate transition state. As favorable contacts are added successively to a substrate, the entropic penalty associated with each decreases and the free energy expressed approaches the incremental interaction energy. This is the first report of a graded cooperative effect. Elucidation of favorable enzyme-substrate contacts remote from the catalytic site will assist in the design of highly specific cysteine protease inhibitors.     

Molecular modelling studies unveil potential binding sites on human serum albumin for selected experimental and in silico COVID-19 drug candidate molecules

Human serum albumin (HSA) is the most prevalent protein in the blood plasma which binds an array of exogenous compounds. Drug binding to HSA is an important consideration when developing new therapeutic molecules, and it also aids in understanding the underlying mechanisms that govern their pharmacological effects. This study aims to investigate the molecular binding of coronavirus disease 2019 (COVID-19) therapeutic candidate molecules to HSA and to identify their putative binding sites. Binding energies and interacting residues were used to evaluate the molecular interaction. Four drug candidate molecules (β-D-N4-hydroxycytidine, Chloroquine, Disulfiram, and Carmofur) demonstrate weak binding to HSA, with binding energies ranging from −5 to −6.7 kcal/mol. Ivermectin, Hydroxychloroquine, Remdesivir, Arbidol, and other twenty drug molecules with binding energies ranging from −6.9 to −9.5 kcal/mol demonstrated moderate binding to HSA. The strong HSA binding drug candidates consist of fourteen molecules (Saquinavir, Ritonavir, Dihydroergotamine, Daclatasvir, Paritaprevir etc.) with binding energies ranging from −9.7 to −12.1 kcal/mol. All these molecules bind to different HSA subdomains (IA, IB, IIA, IIB, IIIA, and IIIB) through molecular forces such as hydrogen bonds and hydrophobic interactions. Various pharmacokinetic properties (gastrointestinal absorption, blood-brain barrier permeation, P-glycoprotein substrate, and cytochrome P450 inhibitor) of each molecule were determined using SwissADME program. Further, the stability of the HSA-ligand complexes was analyzed through 100 ns molecular dynamics simulations considering various geometric properties. The binding free energy between free HSA and compounds were calculated using Molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) and molecular mechanics generalized Born surface area (MM/GBSA) approach. The findings of this study might be useful in understanding the mechanism of COVID-19 drug candidates binding to serum albumin protein, as well as their pharmacodynamics and pharmacokinetics.Keyword: Human serum albumin, HAS, Serum protein, COVID-19, Molecular docking, Molecular dynamics simulation, Pharmacokinetics, Pharmacodynamics     

HhaI DNA methyltransferase uses the protruding Gln237 for active flipping of its target cytosine

Access to a nucleotide by its rotation out of the DNA helix (base flipping) is used by numerous DNA modification and repair enzymes. Despite extensive studies of the paradigm HhaI methyltransferase, initial events leading to base flipping remained elusive. Here we demonstrate that the replacement of the target C:G pair with the 2-aminopurine:T pair in the DNA or shortening of the side chain of Gln237 in the protein severely perturb base flipping, but retain specific DNA binding. Kinetic analyses and molecular modeling suggest that a steric interaction between the protruding side chain of Gln237 and the target cytosine in B-DNA reduces the energy barrier for flipping by 3 kcal/mol. Subsequent stabilization of an open state by further 4 kcal/mol is achieved through specific hydrogen bonding of the side chain to the orphan guanine. Gln237 thus plays a key role in actively opening the target C:G pair by a "push-and-bind" mechanism.     

Role of Two Strictly Conserved Residues in Nucleotide Flipping and N-Glycosylic Bond Cleavage by Human Thymine DNA Glycosylase

Thymine DNA glycosylase (TDG) promotes genomic integrity by excising thymine from mutagenic G·T mismatches arising by deamination of 5-methylcytosine, and follow-on base excision repair enzymes restore a G·C pair. TDG cleaves the N-glycosylic bond of dT and some other nucleotides, including 5-substituted 2′-deoxyuridine analogs, once they have been flipped from the helix into its active site. We examined the role of two strictly conserved residues; Asn¹⁴⁰, implicated in the chemical step, and Arg²⁷⁵, implicated in nucleotide flipping. The N140A variant binds substrate DNA with the same tight affinity as wild-type TDG, but it has no detectable base excision activity for a G·T substrate, and its excision rate is vastly diminished (by ∼10^4.4-fold) for G·U, G·FU, and G·BrU substrates. Thus, Asn¹⁴⁰ does not contribute substantially to substrate binding but is essential for the chemical step, where it stabilizes the transition state by ∼6 kcal/mol (compared with 11.6 kcal/mol stabilization provided by TDG overall). Our recent crystal structure revealed that Arg²⁷⁵ penetrates the DNA minor groove, filling the void created by nucleotide flipping. We found that the R275A and R275L substitutions weaken substrate binding and substantially decrease the base excision rate for G·T and G·BrU substrates. Our results indicate that Arg²⁷⁵ promotes and/or stabilizes nucleotide flipping, a role that is most important for target nucleotides that are relatively large (dT and bromodeoxyuridine) and/or have a stable N-glycosylic bond (dT). Arg²⁷⁵ does not contribute substantially to the binding of TDG to abasic DNA product, and it cannot account for the slow product release exhibited by TDG.     

Electrostatic stabilization in four-helix bundle proteins.

Charge substitutions generated by site-directed mutagenesis at the termini of adjacent anti-parallel alpha-helices in a four-helix bundle protein were used to determine a precise value for the contribution of indirect charge-charge interactions to overall protein stability, and to simulate the electrostatic effects of alpha-helix macrodipoles. Thermodynamic double mutant cycles were constructed to measure the interaction energy between such charges on adjacent anti-parallel helices in the four-helix bundle cytochrome b562 from Escherichia coli. Previously, theoretical calculations of helix macrodipole interactions using modeled four-helix bundle proteins have predicted values ranging over an order of magnitude from 0.2 to 2.5 kcal/mol. Our system represents the first experimental evidence for electrostatic interactions such as those between partial charges due to helix macrodipole charges. At the positions mutated, we have measured a favorable interaction energy of 0.6 kcal/mol between opposite charges simulating an anti-parallel helix pair. Pairs of negative or positive charges simulating a parallel orientation of helices produce an unfavorable interaction of similar magnitude. The interaction energies show a strong dependence upon ionic strength, consistent with an electrostatic effect. Indirect electrostatic contacts do appear to confer a limited stabilization upon the association of anti-parallel packing of helices, favoring this orientation by as much as 1 kcal/mol at 20 mM K phosphate.     

A molecular thermodynamic view of DNA-drug interactions: a case study of 25 minor-groove binders

Developing a molecular view of the thermodynamics of DNA recognition is essential to the design of ligands for regulating gene expression. In a first comprehensive attempt at sketching an atlas of DNA-drug energetics, we present here a detailed thermodynamic view of minor-groove recognition by small molecules via a computational study on 25 DNA-drug complexes. The studies are configured in the MMGBSA (Molecular Mechanics-Generalized Born-Solvent Accessibility) framework at the current state of the art and facilitate a structure-energy component correlation. Analyses were conducted on both energy minimized structures of DNA-drug complexes and molecular dynamics trajectories developed for the purpose of this study. While highlighting the favorable role of packing, shape complementarity, and van der Waals and hydrophobic interactions of the drugs in the minor groove in conformity with experiment, the studies reveal an interesting annihilation of favorable electrostatics by desolvation. Structural modifications attempted on the ligands point to the requisite physico-chemical factors for obtaining improved binding energies. Hydrogen bonds predicted to be important for specificity based on structural considerations do not always turn out to be significant to binding in post facto analyses of molecular dynamics trajectories, which treat thermal averaging, solvent, and counterion effects rigorously. The strength of the hydrogen bonds retained between the DNA and drug during the molecular dynamics simulations is approximately 1kcal/mol. Overall, the study reveals the compensatory nature of the diverse binding free energy components, possible threshold limits for some of these properties, and the availability of a computationally viable free energy methodology which could be of value in drug-design endeavors.     

Position dependence of amino acid intrinsic helical propensities II: non-charged polar residues: Ser, Thr, Asn, and Gln.

The assumption that the intrinsic alpha-helical propensities of the amino acids are position independent was critical in several helix/coil transition theories. In the first paper of these series, we reported that this is not the case for Gly and nonpolar aliphatic amino acids (Val, Leu, Met, and Ile). Here we have analyzed the helical intrinsic propensities of noncharged polar residues (Ser, Thr, Asn, and Gln) at different positions of a model polyalanine-based peptide. We found that Thr is more favorable (by approximately 0.3 kcal/mol) at positions N1 and N2 than in the helix center, although for Ser, Asn, and Gln the differences are smaller (+/-0.2 kcal/mol), and in many cases within the experimental error. There is a reasonable agreement (+/-0.2 kcal/mol) between the calculated free energies, using the ECEPP/2 force field equipped with a hydration potential, and the experimental data, except at position N1.