Cyclic oxyphosphoranes as model intermediates during splicing and cleavage of RNA: ab initio molecular orbital calculations on the conformational analysis.

Ab initio molecular orbital calculations have been carried out on hydrated adducts of methyl ethylene phosphate as a model intermediate during cleavage of RNA. Upon rotating the apical methoxyl group two kinds of stable conformers and two kinds of rotational transition states are located, the most stable conformation being gs-G where the dihedral angle between the apical methyl group and the basal ring oxygen is calculated to be 76 degrees. In this gs-G conformation one of the lone pairs on the apical oxygen is oriented antiperiplanar to the basal ring ester bond. The torsional energy required to rotate the methyl group about the phosphorus-apical oxygen bond leading to ts-C conformation, where the methyl group is eclipsed with the ring oxygen, is calculated to be 5.2 kcal/mol. Judging from the published substrate's coordinates in the RNase environment, the expected pentacoordinate-intermediate/transition state during the cleavage of RNA appears to be, in fact, the most stable gs-G conformation.     

The conformation of end-groups is one determinant of carotenoid topology suitable for high fidelity molecular recognition: A study of β- and ε-end-groups

Conformation affects a carotenoid’s ability to bind selectively to proteins. We calculated adiabatic energy profiles for rotating the ring end-groups around the C6C7 bond and for flexing of the ring with respect to the polyene chain. The choice of computational methods is important. A low, 4.2 kcal/mol barrier to rotation exists for a β-ring. An 8.3 kcal/mol barrier exists for rotation of an ε-ring. Rotation of the ε-ring is sensitive to substitution at C3. In the absence of external forces neither β- nor ε-rings are rotationally constrained. The nearly parallel alignment of the β-ring to the C6C7 bond axis contrasts to the more perpendicular orientation of the ε-ring. Flexion of a β-ring to the minimized ε-ring conformation requires ∼23 kcal/mol; extension of the ε-ring to the minimized β-ring conformation requires ∼8 kcal/mol. Selectivity associated with β- versus ε-rings is dominated by the inability of the β-ring to flex to minimize protein/ring steric interactions and maximize van der Waal’s attractions with the binding site.     

H nuclear magnetic resonance study of restricted internal rotation of N-6,N-6-dimethyladenine in aqueous solution.

Kinetics of internal rotation about the C(6)-N(6) bond of N-6,N-6-dimethyladenine (M2-6A) was investigated by -1H nuclear magnetic resonance line-shape analysis of the methyl resonances (220 MHz). Rates of rotation were determined for M2-6A deuterated at N(1) and for neutral M2-6A. Activation parameters for monodeuterated M2-6A at 22 degrees are Ea = 13.8kcal/mol, log A = 12.6, incrementG++=14.9 kcal/mol, incrementH++ = 13.1 kcal/mol, incrementS++ = minus 5.8 eu; for neutral M2-6A: Ea = 15.5 kcal/mol, log A = 14.9, incrementG++ = 12.6 kcal/mol, incrementH++ = 14.9 kcal/mol, incrementS++ =7.8 eu. Vertical stacking of bases interferes with internal rotation of the dimethylamino group.     

Application of ab initio molecular-orbital calculations to the structural moieties of carbohydrates. Part VI

Ab initio RHF/4–31G molecular-orbital calculations have been conducted on methoxymethyl formate and methoxymethyl acetate as models for examining the anomeric effect and stereochemistry of 1-O-acetylglycopyranoses. The results indicate that, as with the methyl glycopyranosides, the α-⁴C₁(D) configurations are more stable than the β-⁴C₁(D), except that the energy difference is more dependent on the disposition about the glycosidic bond. The lowest-energy conformations occur with glycosidic torsion-angles of ?  180°, where the anomeric energy is about 4 kcal/mol. There is a secondary energy-minimum at ?  90°, for which the anomeric energy is less, about 2 kcal/mol. This orientation corresponds to the conformation most commonly observed in the crystal structures of peracetylated glycopyranoses. Small differences in the CO single-bond lengths, which are observed experimentally in both the α and β anomers, are reproduced by the theoretical calculations.     

B3LYP/6-311++G** study of alpha- and beta-D-glucopyranose and 1,5-anhydro-D-glucitol: 4C1 and 1C4 chairs, (3,O)B and B(3,O) boats, and skew-boat conformations

Geometry optimization, at the B3LYP/6-311++G** level of theory, was carried out on 4C1 and 1C4 chairs, (3,O)B and B(3,O) boats, and skew-boat conformations of alpha- and beta-D-glucopyranose. Similar calculations on 1,5-anhydro-D-glucitol allowed examination of the effect of removal of the 1-hydroxy group on the energy preference of the hydroxymethyl rotamers. Stable minimum energy boat conformers of glucose were found, as were stable skew boats, all having energies ranging from approximately 4-15 kcal/mol above the global energy 4C1 chair conformation. The 1C4 chair electronic energies were approximately 5-10 kcal/mol higher than the 4C1 chair, with the 1C4 alpha-anomers being lower in energy than the beta-anomers. Zero-point energy, enthalpy, entropy, and relative Gibbs free energies are reported at the harmonic level of theory. The alpha-anomer 4C1 chair conformations were found to be approximately 1 kcal/mol lower in electronic energy than the beta-anomers. The hydroxymethyl gt conformation was of lowest electronic energy for both the alpha- and beta-anomers. The glucose alpha/beta anomer ratio calculated from the relative free energies is 63/37%. From a numerical Hessian calculation, the tg conformations were found to be approximately 0.4-0.7 kcal/mol higher in relative free energy than the gg or gt conformers. Transition-state barriers to rotation about the C-5-C-6 bond were calculated for each glucose anomer with resulting barriers to rotation of approximately 3.7-5.8 kcal/mol. No energy barrier was found for the path between the alpha-gt and alpha-gg B(3,O) boat forms and the equivalent 4C1 chair conformations. The alpha-tg conformation has an energy minimum in the 1S3 twist form. Other boat and skew-boat forms are described. The beta-anomer boats retained their starting conformations, with the exception of the beta-tg-(3,O)B boat that moved to a skew form upon optimization.     

Correlation Times and Adiabatic Barriers for Methyl Rotation in SNase

The relation of rotational correlation times to adiabatic rotational barriers for alanine methyl groups in staphylococcal nuclease (SNase) is investigated. The hypothesis that methyl rotational barriers may be useful probes of local packing in proteins is supported by an analysis of ten X-ray crystal structures of SNase mutants. The barrier heights are consistent across a set of ten structures of a native SNase and mutants containing single-point mutations or single or double insertions, most in a ternary SNase complex. The barriers for different methyls have a range of 7.5 kcal/mol, which at 300 K would correspond to a five-order-of-magnitude range in correlation time. It is demonstrated that adiabatic rotational barriers can fluctuate significantly during an MD simulation of hydrated SNase, but that a Boltzmann weighted average is predictive of rotational correlation times determined from correlation functions. Even if a given methyl is on average quite sterically hindered, infrequently sampled low-barrier conformations may dominate the Boltzmann distribution. This result is consistent with the observed uniformity of NMR correlation times for (13)C-labeled methyls. The methyl barriers in simulation fluctuate on multiple time scales, which can make the precise relationship between methyl rotational correlation time and methyl rotation barriers complicated. The implications of these issues for the interpretation of correlation times determined from NMR and simulation are discussed.     

An assessment of the reaction energetics for cytochrome P450-mediated reactions

Regioselectivity is used to determine the absolute energetic differences for four different reactions catalyzed by P450. Abstraction of a hydrogen from a benzylic carbon containing a chlorine has a 1.0 kcal/mol lower barrier than abstraction from a simple benzylic carbon, which in turn is 0.4 to 0.9 kcal/mol lower than abstraction from the methyl group of an aromatic ether and 0.1 to 0.6 kcal/mol easier than aromatic hydroxylation. Isotope effects are used to determine if the enzyme-substrate complexes leading to each product, from a given substrate, are in rapid equilibrium. For all enzymes isotopically sensitive branching is observed from the benzylic carbon upon deuterium incorporation at that position to each of the other positions, indicating that each product arises from the same active oxygen species. The energetic differences determined experimentally are accurately reproduced by theoretical hydrogen atom abstractions at both the AM1 semiempirical and DFT levels of theory.     

The conformational properties of the glycosidic linkage

Stereochemical properties of the glycosidic linkage have been studied by the quantum-chemical PCILO method, using 2-methoxytetrahydropyran as a model. Calculations of the two-dimensional, conformational (Φ, Ψ) maps showed that the rotation around the C-1---O-1 bond is more hindered than that around the O-1---C-6 bond, and that there are differences in the shape of the energy curve for the axial and equatorial forms of 2-methoxytetrahydropyran. The observed population of the five stable conformers at equilibrium (GG:GT:TG¹:TG₂:TT = 70.8:6.0:19.9:2.0:1.3) is consistent with the prediction of the anomeric and exo-anomeric effects. The calculated abundance (76.8%) of the axial form of 2-methoxytetrahydropyran is comparable with experimental results (77–80%) obtained by n.m.r. measurements in non-polar solvents. The energies found for individual conformers made it possible to calculate the magnitude of the anomeric effect (3 kJ/mol) and to determine, for the first time, the values of the exo-anomeric effect for axial (6 kJ/mol) and equatorial 2-methoxytetrahydropyran (7 kJ/mol). The calculated variations of the geometry arising from rotation around the C-1---O-1 bond are consistent with results obtained by statistical analysis of experimental data for - and β-glycosides. The results obtained, indicating that the energy, geometry, and electronic structure of glycosides are largely affected by the conformation of the acetal segment, are discussed from the point of view of conformational analysis of oligo- and poly-saccharides.     

Cis-trans isomerization and puckering of proline residue

We report here the results on N-acetyl-L-proline-N'-methylamide (Ac-Pro-NHMe) calculated at the HF/6-31+G(d) level with the conductor-like polarizable continuum model (CPCM) of self-consistent reaction field methods to investigate the changes of backbone and prolyl ring along the cis-trans isomerization of the prolyl peptide bond. From the potential energy surface, the barrier to ring flip from the down-puckered conformation to the up-puckered one is estimated to be 2.5 and 3.2 kcal/mol for trans and cis conformers of Ac-Pro-NHMe, respectively. In particular, the ring flip seems to be inaccessible in the intermediate regions between trans and cis conformations, because of higher barriers (approximately 13-19 kcal/mol) to rotation of the prolyl peptide bond. The torsion angles for backbone and prolyl ring vary largely around the transition states at omega' approximately 120 degrees and -70 degrees for the prolyl peptide bond. Three kinds of puckering amplitudes show the same trend of puckering along the cis-trans isomerization although their absolute values are different. In particular, trans and cis conformations have the almost same degree of puckering. The cis populations and barriers to rotation of the prolyl peptide bond for Ac-Pro-NHMe are increased with the increase of solvent polarity, which is mainly ascribed to the decreases of relative free energies for cis conformations and the increase of relative free energies for transition states.     

Tryptophan-47 rotational isomerization in variant-3 scorpion neurotoxin. A combination thermodynamic perturbation and umbrella sampling study.

A combination thermodynamic perturbation and umbrella sampling study predicts two free energy wells for the rotational isomerization of the variant-3 scorpion neurotoxin tryptophan-47 indole side chain. One well has the indole side chain in the crystallographic orientation; the other has the indole rotated approximately 220 degrees to form a new conformation with a relative free energy of 3 +/- 2 kcal/mol. The activation barrier is 8.5 kcal/mol from the crystallographic well, from which transition state theory predicts a rate of escape of 2 x 10(5) s-1. Correlations in the displacements of side chains neighboring tryptophan-47 and the isomerization reaction coordinate last up to 20 ps. Favorable conditions of experimental verification are discussed.     

Dynamic analysis of efficient heme rotation in myoglobin by NMR spectroscopy.

Sperm whale myoglobin was reconstituted with 1,4,5,8-tetramethylhemin. The hyperfine-shifted proton NMR signals from the prosthetic group exhibit remarkable pattern changes around 15 degrees C, while the globin resonances are normal to obey the Curie law. The NMR anomaly specifically observed for the heme signals suggests a slow to rapid rotational transition of the hemin about the iron-histidine bond. The temperature-dependent pattern changes were quantitatively analyzed by a dynamic NMR method. Two sets of analyses with the heme-methyl and pyrrole-proton lines consistently afforded delta H not equal to = 16.3 kcal/mol, delta S not equal to = 14.0 e.u., delta G not equal to = 12.1 kcal/mol at 298 K, and a frequency of 90 degrees heme rotation 5600 s-1 at 20 degrees C. The relatively large activation entropy suggests that structural rearrangements at the direct heme vicinity are involved and that efficient heme rotation is accomplished by a number of fluctuative local heme-globin contacts within a conserved crevice structure.     

A quantum mechanics/molecular mechanics study of the catalytic mechanism and product specificity of viral histone lysine methyltransferase

There are three reaction steps in the S-adenosylmethionine (AdoMet) methylation of lysine-NH2 catalyzed by a methyltransferase. They are (i) combination of enzyme.Lys-NH3+ with AdoMet, (ii) substrate ionization to provide enzyme.AdoMet.Lys-NH2, and (iii) methyl transfer providing enzyme.AdoHcy.Lys-N(Me)H2+ and the dissociation of AdoHcy. In this study of the viral histone methyltransferase (vSET), we find that substrate ionization of vSET.Lys27-NH3+, vSET.Lys27-N(Me)H2+, and vSET.Lys27-N(Me)2H+ takes place upon combination with AdoMet. The presence of a water channel allows dissociation of a proton to the solvent. There is no water channel in the absence of AdoMet. That the formation of a water channel is combined with AdoMet binding was first discovered in our investigation of Rubisco large subunit methyltransferase. Via a quantum mechanics/molecular mechanics (QM/MM) approach, the calculated free energy barrier (DeltaG++) of the first methyl transfer reaction catalyzed by vSET [Lys27-NH2 + AdoMet --> Lys27-N(Me)H2+ + AdoHcy] equals 22.5 +/- 4.3 kcal/mol, which is in excellent agreement with the free energy barrier (21.7 kcal/mol) calculated from the experimental rate constant (0.047 min-1). The calculated DeltaG++ of the second methyl transfer reaction [AdoMet + Lys27-N(Me)H --> AdoHcy + Lys27-N(Me)2H+] at the QM/MM level is 22.6 +/- 3.6 kcal/mol, which is in agreement with the value of 22.4 kcal/mol determined from the experimental rate constant (0.015 min-1). The third methylation [Lys27-N(Me)2 + AdoMet --> Lys27-N(Me)3+ + AdoHcy] is associated with a DeltaG++ of 23.1 +/- 4.0 kcal/mol, which is in agreement with the value of 23.0 kcal/mol determined from the experimental rate constant (0.005 min-1). Our computations establish that the first, second, and third methyl transfer steps catalyzed by vSET are linear SN2 reactions with the bond making being approximately 50% associative.     

A theoretical study of glucosamine synthase

Continuing our theoretical studies of glucosamine synthase catalysis, we have carried out MNDO and ab initio calculations of the first stage of the reaction, which involves the attack of a cysteine thiol group from the enzyme active site on the side chain carboxyamide group of glutamine, producing ammonia and thioester. The reactants were modelled by methyl mercaptate and acetamide, respectively. For two considered mechanisms of the reaction the energy surfaces were evaluated. Mechanism I, proposed by Chmara et al. (1985) involves the nucleophilic attack of a deprotonated thiol group on the carbonyl carbon atom. Mechanism II, postulated in our previous work (Tempczyk et al. 1989), assumes the concerted binding of the mercaptate sulphur to the carbonyl carbon and the sulfhydryl hydrogen to the amide nitrogen with simultaneous breaking of the S-H bond. The energy surface of mechanism I shows no minimum on the approach of the mercaptide anion towards the carbonyl carbon, which is also consistent with ab initio calculations in a 4-31 G basis set. Therefore, mechanism I seems to be unlikely. The same analysis of mechanism II shows that it leads to the desired products: methyl thioacetate and ammonia. The presence of a sulfhydryl hydrogen causes apparent pyramidicity of the amido nitrogen and lengthening of the C-N bond in the transition state, making conditions for the release of the ammonia molecule. The MNDO calculated energy barrier of the reaction is 50.1 kcal/mol and the approximate 4-31 G ab initio barrier (at the MNDO geometries of the substrate complex and the transition state) is 63 kcal/mol. The biggest energy contribution to the barrier comes from the breaking of the S-H bond, which also causes a large charge separation in the transition state. The latter affect may result in the stabilisation of the transition state in a real enzymatic environment when compared to the gas phase, e.g. by the interaction of the reacting center with a pair of oppositely charged amino acid side chains such as lysinium and glutamate (aspartate), which are present in the enzyme studied. To estimate the magnitude of this effect, molecular mechanics calculations were carried out on the reaction center at the transition state in our proposed model of the enzymatic active site. The site was supplemented by ammonium and acetate ion, which were to mimic the lysinium and glutamate/aspartate side chains. A transition state stabilization energy of 20 kcal/mol was obtained and this lowers the energy barrier to about 30 kcal/mol. This value is within the thermal energy range of an average protein and indicates that our mechanism is a possible route of glucosamine synthase catalysis. Offprint requests to: E. Borowski     

Flexible-geometry conformational energy maps for the amino acid residue preceding a proline.

Previously calculated conformational energy maps suggest that the alpha-helical conformation for the residue preceding a proline is disfavored relative to the extended conformation by more than 7 kcal/mol. In known protein structures this conformation is observed, however, to occur for about 9% of all prolines. In addition, introduction or removal of prolines at theoretically unfavorable positions in proteins and peptides can have modest effects on stability and structure. To investigate the discrepancy between calculation and experiment, we have determined how the conformation of the proline affects the calculated energy. We have also explored the effect of bond length and bond angle relaxation on the conformational energy map. The conformational energy of the preceding residue is found to be unaffected by the conformation of the proline, but the effect of allowing covalent bond relaxation is dramatic. If bond lengths and angles, and dihedral angles within the pyrrolidine ring, are allowed to relax, a calculated energy difference between the alpha and beta conformations of 1.1 kcal/mol is obtained, in reasonable agreement with experiment. The detailed shape of the calculated energy surface is also in excellent agreement with the observed conformational distributions in known protein structures.     

Catalysis of a rotational transition in a peptide by crystal forces

Detailed examination of the dynamics trajectories of the isolated cyclic peptide cyclo-(Ala-Pro-D-Phe)2 and of the molecule in its crystalline environment led to the unexpected observation that the methyl groups of the alanine residues rotated more frequently during a simulation in the crystal environment than in a simulation of the isolated peptide. In effect, the crystal environment is "catalyzing" the rotational isomerization of the methyl groups. In order to understand how the crystal forces increase the rate of this rotation, and to explore any possible analogy to the inducing of strained conformations of ligands by enzymes, the barriers to rotation in the two environments were studied by using the torsion angle forcing method. The crystal forces induce a different, higher energy, conformation of the peptide than is found for the isolated molecule, and the different rates of rotation have been explained in terms of the resulting specific intramolecular interactions that, it turns out, give rise to the lower rotational barrier. Molecular dynamics simulations of the peptide were also run at higher temperatures in order to calculate the barriers to rotation through the use of Arrhenius plots. The barriers obtained in this way agree well with the barriers obtained through an adiabatic reaction path derived by rotating the methyl through the barrier while minimizing all remaining degrees of freedom. The rates of rotation calculated from these adiabatic barriers also agree well with the rates observed during the 300 K simulations.     

Computational modeling of substrate specificity and catalysis of the β-secretase (BACE1) enzyme

In this combined MD simulation and DFT study, interactions of the wild-type (WT) amyloid precursor protein (APP) and its Swedish variant (SW), Lys670 → Asn and Met671 → Leu, with the beta-secretase (BACE1) enzyme and their cleavage mechanisms have been investigated. BACE1 catalyzes the rate-limiting step in the generation of 40-42 amino acid long Alzheimer amyloid beta (Aβ) peptides. All key structural parameters such as position of the flap, volume of the active site, electrostatic binding energy, structures, and positions of the inserts A, D, and F and 10s loop obtained from the MD simulations show that, in comparison to the WT-substrate, BACE1 exhibits greater affinity for the SW-substrate and orients it in a more reactive conformation. The enzyme-substrate models derived from the MD simulations were further utilized to investigate the general acid/base mechanism used by BACE1 to hydrolytically cleave these substrates. This mechanism proceeds through the following two steps: (1) formation of the gem-diol intermediate and (2) cleavage of the peptide bond. For the WT-substrate, the overall barrier of 22.4 kcal/mol for formation of the gem-diol intermediate is 3.3 kcal/mol higher than for the SW-substrate (19.1 kcal/mol). This process is found to be the rate-limiting in the entire mechanism. The computed barrier is in agreement with the measured barrier of ca. 18.00 kcal/mol for the WT-substrate and supports the experimental observation that the cleavage of the SW-substrate is 60 times more efficient than the WT-substrate.     

NMR study of stacking interactions and conformational adjustments in the dinucleotide-carcinogen adduct 2'-deoxycytidylyl-(3----5)-2'-deoxy-8-(N-fluoren-2-ylac etamido)guanosi ne

The conformation and dynamics of the dinucleotide d-CpG modified at the C(8) position of the guanine ring by the carcinogen 2-(acetylamino)fluorene has been investigated by high-field 1H NMR spectroscopy. A two-state analysis of chemical shift data has enabled estimation of the extent of intramolecular stacking in aqueous solution as a function of temperature. The stacking, which is mostly fluorene-cytosine, is virtually complete in the low-temperature range. The 500-MHz 1H NMR spectrum consists of two subspectra near ambient temperatures due to a 14.3 +/- 0.3 kcal/mol barrier to internal rotation about the amide bond in the stacked form. A large barrier to internal rotation about the guanyl-nitrogen bond at C(8) has also been ascertained, but separate NMR subspectra were not detected due to the predominance of one of the torsional diastereomers (alpha' = 90 degrees) in the fully stacked state. Problems of self-association and chemical exchange were identified and overcome to enable analysis of the sugar-phosphate backbone conformation utilizing coupling constants. For the exocyclic C(4')-C(5') bond of the deoxyguanosine moiety, there is a high gauche+ (gamma = 60 degrees) conformer population, which is uncommon for a purine nucleotide with a syn orientation about the glycosyl bond. The gauche- conformation (gamma = 300 degrees), which is normally present in syn purine nucleotides in solution, was not detected. The exocyclic C(5')-O(5') torsion of the deoxy-guanosine moiety remains near the classical energy minimum (beta = 180 degrees) in the major stacked conformations. The sugar ring of the deoxycytidine moiety is predominantly in the C2'-endo conformation, while the deoxyguanosine ring is a mixture of conformations, one of which appears to be unusually puckered. The results support intercalation models of modified DNA and suggest a looped-out structure, with the modified guanine being the first base in the loop. Such structures could explain the relatively rapid rate of repair and the frame-shift mutations of this type of adduct.     

Stereoelectronic control in peptide bond formation. Ab initio calculations and speculations on the mechanism of action of serine proteases.

Ab initio molecular orbital calculations have been performed on the reaction profile for the addition/elimination reaction between ammonia and formic acid, proceeding via a tetrahedral intermediate: NH3 + HCO2H----H2NCH(OH)2----NH2CHO + H2O. Calculated transition state energies for the first addition step of the reaction revealed that a lone pair on the oxygen of the OH group, which is antiperiplanar to the attacking nitrogen, stabilized the transition state by 3.9 kcal/mol, thus supporting the hypothesis of stereoelectronic control for this reaction. In addition, a secondary, counterbalancing stereoelectronic effect stabilizes the second step, water elimination, transition state by 3.1 kcal/mol if the lone pair on the leaving water oxygen is not antiperiplanar to the C-N bond. The best conformation for the transition states was thus one with a lone pair antiperiplanar to the adjacent scissile bond and also one without a lone-pair orbital on the scissile bond oxygen or nitrogen antiperiplanar to the adjacent polar bond. The significance of these stereoelectronic effects for the mechanism of action of serine proteases is discussed.     

CNDO/2 molecular orbital calculations on the antifolate DAMP and some related species: structural geometries, ring distortions, change distributions and conformational characteristics

Geometry-optimized CNDO/2 molecular orbital calculations were carried out on 2, 4-diamino-5-(1-adamantyl 1)-6-methyl pyrimidine (DAMP), a potent inhibitor of mammalian dihydrofolate reductase which is now in clinical trials, and on its inactive 5-(1-naphthyl) analogue (DNMP-1). Crystallographic data show that DAMP (as the ethylsulfonate salt) has a severely distorted, N1 protonated, pyrimidine ring and has steric crowding of the 6-methyl and adamantyl hydrogens whereas DNMP-2 (as a methanol complex) has a planar, nonprotonated pyrimidine ring that is nearly perpendicular to the naphthalene ring. The CNDO/2 results largely reproduce the crystal structure geometry and show that the ring distortions in DAMP are initiated by steric conflicts between the adamantyl group and the 4- and 6-substituents on the ring. In DNMP-1, the non-interfering naphthyl ring induces little strain within the pyrimidine ring and the effect of protonation is negligible. Rotation about the bond joining the two ring groups is restricted in DAMP by a broad barrier of ca. 8.0 kcal mol-1, and no conformation was successful in relieving steric conflicts and hence reducing the ring distortions. In DNMP-1, rotation is less hindered overall with a broad region of accessible conformational space and a maximum barrier of ca. 7.2 kcal mol-1 for the coplanar conformation. The electronic charge distributions of DAMP and DNMP-1 are almost identical and protonation is preferred at N1 rather than at N3 by ca. 3.7 kcal mol-1 for both DAMP and DNMP-1. The calculations establish that the present methodology can be useful as a predictive tool with regard to the structure and conformational characteristics of these and related species.     

Nuclear magnetic resonance studies of hemoproteins. IV. Hindered rotation of heme side methyl group as a probe for studying van der Waals contacts in the heme side environments of myoglobin derivatives.

220 MHz roton NMR spectral evidence for restricted rotation of one methyl group in the heme side chain of ferric horse cyanomyoglobin is reported here. Temperature dependence of this methyl proton signal was computer-simulated, yielding 14,8 kcal/mol for the methyl hindered rotation. Ionic additives such as NaCl and (NH4) 2 minus SO4 caused a slackening of this restriction of methyl rotation, evidenced from collapse of methyl signal doubling by the addition of these ionic substances. This is discussed in terms of breaking of the salt bridge formed between one of the propionate COO minus group of heme and a part of the apoprotein which might lead to constraint of one of the heme side methyl groups. The peculiarity of hyperfine-shifted methyl proton signals for other myoglobin complexes such as azide and imidazole derivatives is also discussed briefly in terms of constraint of heme side methyl group buried in a hydrophobic cleft.