phi-Values beyond the ribosomally encoded amino acids: kinetic and thermodynamic consequences of incorporating trifluoromethyl amino acids in a globular protein

The consequences of the substitution of 4,4,4-trifluorovaline for valine on the folding kinetics and thermodynamics of a globular protein are presented. Variants of the N-terminal domain of L9, a small alpha-beta protein, were prepared in which V3 or V21 was replaced by trifluorovaline. CD and NMR demonstrate that the structure is not perturbed. Both are more stable, the V3 variant by 0.8 kcal mol-1 and the V21 variant by 1.4 kcal mol-1. The increase of stability is significantly larger than that observed in coiled-coils on a per trifluoromethyl group basis. Folding is two-state, and the variants both fold faster than the wild type. The Phi-values are 0.16 and 0.11, respectively.     

Design of a hyperstable protein by rational consideration of unfolded state interactions

Stabilization of proteins is a long-sought objective. Targeting the unfolded state interactions of a protein is not a method used for this purpose, although many proteins are known to contain such interactions. The N-terminal domain of ribosomal protein L9 (NTL9) has a lysine residue at position 12, which makes strong non-native interactions in the unfolded state. Substitution of a d-alanine for G34 in NTL9 is known to stabilize the protein by reducing the entropy of the unfolded state. Here we combine these two mutations to design a hyperstable protein. The structure of the variant is the same as that of wild-type as judged by 2D NMR. The variant is hyperstable as judged by denaturation experiments, where complete thermal unfolding of the protein does not occur in native buffer.     

Cu^2＋诱导甘氨酸质子迁移机理的理论研究

采用CCSD/6-31＋＋G＊＊//B3LYP/6-31＋＋G＊＊方法研究了Cu2＋诱导甘氨酸质子迁移的机理.优化得到了7个中性配合物和1个两性配合物,其中两性配合物最稳定,结合能为215.93 kcal mol-1.中性构型间通过分子内单键的旋转相互转化,C-N、C-C和C-O键旋转的能垒范围分别为1.62～2.49、0.27～7.80和2.27～16.97 kcal mol-1;中性构型N6经质子迁移变为两性构型,能垒为33.82 kcal mol-1.Cu2＋作用于甘氨酸,使甘氨酸N5原子负电荷减少超过0.5,降低了N5对H6原子的库仑吸引,钝化了共价键B（O3–H6）,动力学上不利于H6质子迁移;但是H6质子迁移后,形成的两性构型Z1却是热力学最稳定体系.     

On the stability of glycine-water clusters with excess electron: implications for photoelectron spectroscopy

Calculations are presented for the glycine-(H(2)O)(n) (-) (n=0-2) anionic clusters with excess electron, with the glycine core in the canonical or zwitterion form. A variety of conformers are predicted, and their relative energy is examined to estimate thermodynamic stability. The dynamic (proton transfer) pathways between the anionic clusters with the canonical and the zwitterion glycine core are examined. Small barrier heights for isomerization from the zwitterion glycine-(H(2)O)(2) (-) anion to those with canonical glycine core suggest that the former conformers may be kinetically unstable and unfavorable for detection of neutral glycine zwitterion-(H(2)O)(n) (n=1,2) clusters by photodetachment, in accordance with the photoelectron spectroscopic experiments by Bowen and co-workers [Xu et al., J. Chem. Phys. 119, 10696 (2003)]. The calculated stability of the glycine-(H(2)O)(n) (-) anion clusters with canonical glycine core relative to those with zwitterion core indicates that the observation of the anionic conformers with the canonical glycine core would be much more feasible, as revealed by Johnson and co-workers [Diken et al. J. Chem. Phys. 120, 9902 (2004)].     

The influence of hydrogen bonding interactions on the C-H bond activation step in class I ribonucleotide reductases

In order to model the C-H bond activation step in ribonucleotide reductases the hydrogen atom abstraction reaction from cis-tetrahydrofuran-2,3-diol (7) by methylthiyl (8) radical has been studied with theoretical methods. In order to identify an appropriate theoretical method for this system, the hydrogen transfer reaction between radical 8 and methanol (9) to give methanol radical (10) and methyl thiol (11) has been studied at several different levels of theory. While the reaction energy for this process is predicted equally well by the Becke3LYP and BH and HLYP hybrid functional methods, the reaction barrier is predicted to be significantly lower by the former. Compared to results obtained at CCSD(T)/cc-pVTZ level the BH and HLYP functional is better suited for the calculation of activation barriers for hydrogen abstraction reactions. This latter method was subsequently used to study the reaction of radical 8 with cis-tetrahydrofuran-2,3-diol 7 in the absence and in the presence of additional functional groups (acetate and acetamide) as models for the substrate reaction of class I ribonucleotide reductases (RNRs). The reaction barrier is lowest in those systems, in which acetate forms a double hydrogen bonded complex with the hydroxy groups of diol 7 (+8.2 kcal mol-1) and increases somewhat for side-on complexes between substrate 7 and acetate featuring only one hydrogen bond (+10.5 kcal mol-1). The barrier reduction of 6.5 kcal mol-1 obtained through complexation of diol 7 with acetate appears to be due to the formation of short strong hydrogen bonds in the transition. These effects can also be found in reactions of thiyl radical 8 with complexes of diol 7 with acetamide, but to a much smaller extent. The lowest reaction barrier is in this case calculated for the side-on complex (+11.2 kcal mol-1), while the bridging orientation between diol 7 and acetamide leads to a reaction barrier (+13.4) that is only slightly lower than that for the uncatalyzed process (+14.7 kcal mol-1). With respect to the structure of the active site of the RNR R1 subunit, only the side-on complexes appear to be relevant for the enzyme-catalyzed process. Under this condition the influence of the E441 side chan and thus the impact of the E441Q mutation in the initial C-H bond activation step will be rather small.     

Paralog-selective ligands for bcl-2 proteins

There is considerable current interest in molecules that bind intra- or extracellular protein surfaces and inhibit protein-protein interactions. Previously we have reported that miniature proteins based on pancreatic-fold polypeptides can recognize even shallow alpha-helix binding clefts with high affinity and selectivity against unrelated proteins. One such miniature protein, PPBH3-1, binds the anti-apoptotic protein paralogs Bcl-2 and Bcl-XL with nanomolar affinity and a DeltaDeltaG = 1.2 kcal.mol-1 preference for Bcl-XL. Here we describe the directed evolution of PPBH3-1 into two new miniature proteins, PPBH3-5 and PPBH3-6, whose paralog specificity is reversed relative to PPBH3-1. PPBH3-5 and PPBH3-6 bind Bcl-2 with nanomolar affinity and a DeltaDeltaG = 0.9-1.3 kcal.mol-1 preference for Bcl-2 over Bcl-XL. Experiments with Bcl-XL variants suggest that PPBH3-5 and PPBH3-6 achieve high paralog specificity by exploiting subtle structural or electrostatic differences in the Bcl-2 and Bcl-XL molecular landscapes. PPBH3-5 and PPBH3-6 may have unique applications as early examples of nonnatural ligands that interact selectively with Bcl-2 proteins.     

Quantum chemical study of the structure and thermochemistry of the five-membered nitrogen-containing heterocycles and their anions and radicals

The nitrogen-containing heterocycles are of interest as high-energy-density materials for use as propellants and explosives, while the pyrolysis of these compounds is also important in understanding the evolution of unwanted NO and NO2 (NOx) from organic fuels such as coal and biomass. We have used ab initio and density functional methods to study the molecular structures and thermochemical properties of the five-membered nitrogen-containing heterocycles and their anions and radicals corresponding to respective heterolytic and homolytic loss of a hydrogen atom from either a nitrogen or carbon site. Many of these thermochemical properties have not previously been measured, especially for the heterocycles containing three and four nitrogen atoms. Using the theoretical methods CBS-APNO, G3, and G3B3, we calculate enthalpies of formation of 26.5, 42.4, 31.9, 63.7, 46.8, 81.0, and 79.0 kcal mol-1 for pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, 1H-tetrazole, and 2H-tetrazole. A correlation is developed between the number of nitrogen atoms in a heterocycle and its enthalpy, and we extrapolate this relationship to predict the enthalpy of formation of pentazole. N-H BDEs in the heterocycles typically increase with the number of nitrogen atoms in the molecule, while C-H BDEs are similar in all of the studied heterocycles, at around 120 kcal mol-1. In all cases the N-H BDEs are weaker than the C-H BDEs, suggesting abstraction of the N-H hydrogen atom is more likely. Deprotonation enthalpies and free energies reveal that the N-H protons become more acidic with increasing number of nitrogen atoms in the heterocycle. C-H protons are less acidic than N-H protons by ca. 49 kcal mol-1, or ca. 35 kcal mol-1 when adjacent to the NH group. Trends in N-H and C-H acidities can be qualitatively explained by electrostatic effects and electron affinities. From its use as a reference species in our calculations, we identify that the experimental enthalpy of pyrimidine (1,3-diazine) may be in error by ca. 1-3 kcal mol-1, and we recommend an enthalpy of formation of 44.8 +/- 1.0 kcal mol-1.     

Configurational stability of chlorophosphines

The configurational stability of chlorophosphines is investigated. Several mechanisms involving chlorophosphine monomer, dimers, and adducts with HCl are evaluated by density functional theory calculations. The presence of HCl in the medium is found to catalyze the P-center chiral inversion at room temperature. The reaction involves a two-step mechanism with low transition states (10 kcal.mol-1) and a stabilized achiral intermediate (-2.6 kcal.mol-1). Further calculations and experiments on the halogen exchange with HBr corroborate this mechanism, with bromophosphines being formed instantaneously. Finally, to avoid the racemization, the borane is found to be a very promising protecting group for the configurational stability of the P-chirogenic chlorophosphines.     

Conformational studies by dynamic NMR. 79. Dimesityl sulfine revisited: detection of the helical antipodes and determination of their enantiomerization pathways.

By means of low-temperature NMR spectra, it is demonstrated that dimesityl sulfine (Mes2C=SO) adopts in solution the same chiral propeller conformation (C1 symmetry) determined by X-ray diffraction in the crystalline state. With the help of MM calculations, it has been also shown that a correlated rotation (cog wheel effect) of the two mesityl rings reverses the molecular helicity according to an enantiomerization process entailing a one-ring flip pathway with delta G++ = 5.9 kcal mol-1 and a two-ring flip pathway with delta G++ = 13.8 kcal mol-1. On the contrary the Z- and E-isomers of mesityl phenyl sulfine (MesPhC=SO) adopt essentially achiral conformations (Cs symmetry), having the Ph-CSO rotation barriers equal to 5.2 and 5.8 kcal mol-1, respectively, and the mesityl-CSO rotation barriers equal to 21.3 and 15.1 kcal mol-1, respectively.     

Remote substituent effects on allylic and benzylic bond dissociation energies. Effects on stabilization of parent molecules and radicals

The effect of remote substituents on bond dissociation energies (BDE) is examined by investigating allylic C-F and C-H BDE, as influenced by Y substituents in trans-YCH=CHCH2-F and trans-YCH=CHCH2-H. Theoretical calculations at the full G3 level model chemistry are reported. The interplay of stabilization energies of the parent molecules (MSE) and of the radicals formed by homolytic bond cleavage (RSE) and their effect on BDE are established. MSE values of allyl fluorides yield an excellent linear free energy relationship with the electron-donating or -withdrawing ability of Y and decrease by 4.2 kcal mol-1 from Y = (CH3)2N to O2N. RSE values do not follow a consistent pattern and are of the order of 1-2 kcal mol-1. A decrease of 4.1 kcal mol-1 is found in BDE[C-F] from Y = CH3O to NC. BDE[YCH=CHCH2-H] generally increases with decreasing electron-donating ability of Y for electron-donating groups and does not follow a consistent pattern with electron-withdrawing groups, the largest change being an increase of 3.6 kcal mol-1 from Y = (CH3)2N to CF3. The G3 results are an indicator of benzylic BDE in p-YC6H4CH2-F and p-YC6H4CH2-H, via the principle of vinylogy, demonstrated by correlating MSE of the allylic compounds with physical properties of their benzylic analogues.     

Low-lying electronic states and dissociation energies of the monochlorides of Cr, Mn, Fe, Co, and Ni

Published equilibrium data involving the gaseous monochlorides of Cr, Mn, Fe, Co, and Ni have been re-examined by thermochemical analysis, using more recent information on the low-lying electronic states, yielding D degrees 0 values in kcal mol-1 of CrCl (90.0), MnCl (79.8), FeCl (79.3), CoCl (81.3), and NiCl (88.1). Although this revised approach is believed to yield more reliable values of the FeCl, CoCl, and NiCl dissociation energies, results show that use of M+ electronic levels in place of the adopted MCl values leads to alternate D degrees 0(MCl) values agreeing within 1.6 kcal mol-1, providing a useful check on electronic-level contributions to the thermochemical calculations.     

Zwitterion-dianion complexes and anion-anion clusters with negative dissociation energies

Clusters of oxalate and malonate dianions with glycine in its zwitterionic form were found by ab initio and density functional theory calculations. Proton transfer is impeded by an electrostatic barrier, but the resulting anion-anion pairs form complexes despite having negative dissociation energies. A variety of X-/Y- species with exothermic dissociation energies ranging from 10 to 141 kcal mol-1, but large enough barriers to be experimentally produced, are reported. These dianions may represent an unrecognized control element in nature and provide a unique opportunity to probe electrostatic effects and a wealth of novel clusters.     

Azido Homoalanine is a Useful Infrared Probe for Monitoring Local Electrostatistics and Sidechain Solvation in Proteins

The use of IR probes to monitor protein structure, deduce local electric field, and investigate the mechanism of enzyme catalysis and protein folding has attracted increasing attention. Here, the azidohomoalanine (Aha) is considered as a useful IR probe. The intricate details of the distinct effects of backbone peptide bonds and H-bonded water molecules on the azido stretch mode of the IR probe Aha were revealed by carrying out QM/MM MD simulations of two variants of the protein NTL9, NTL9-Met1Aha and NTL9-Ile4Aha and comparing the resulting simulated IR spectra with experiments.     

Rate-determining cooperative effects of bimolecular reactions in solution

An expression for the rate constant of condensed-phase bimolecular reactions is derived. The key feature of the proposed model is the formulation of the energy-dependent rate constant in terms of the diffusion rate and the ratio of the volume in phase space that leads to product over the total volume. The dependence of the bimolecular rate constant by the reduced barrier x = E thermod/kT is given in explicit form in terms of the incomplete and the complete gamma functions of Euler. The performance of the proposed model is tested against the experimental rate constants for the Menschutkin reaction by fitting the parameters of the expression for the rate constant to experimental data at various temperatures. The potential energy barrier obtained from the regression (16.75 kcal mol-1) is close to the independently computed value at the CPCM B3LYP/CRENBL.6-311(+)G(d) level of theory (16.84 kcal mol-1). The corresponding fitting to the transition state theory expression affords the lower value of 14.65 kcal mol-1.     

Thermochemistry of methyl and ethyl nitro, RNO2, and nitrite, RONO, organic compounds

Computational quantum theory is employed to determine the thermochemical properties of n-alkyl nitro and nitrite compounds: methyl and ethyl nitrites, CH3ONO and C2H5ONO, plus nitromethane and nitroethane, CH3NO2 and C2H5NO2, at 298.15 K using multilevel G3, CBS-QB3, and CBS-APNO composite methods employing both atomization and isodesmic reaction analysis. Structures and enthalpies of the corresponding aci-tautomers are also determined. The enthalpies of formation for the most stable conformers of methyl and ethyl nitrites at 298 K are determined to be -15.64 +/- 0.10 kcal mol-1 (-65.44 +/- 0.42 kJ mol-1) and -23.58 +/- 0.12 kcal mol-1 (-98.32 +/- 0.58 kJ mol-1), respectively. DeltafHo(298 K) of nitroalkanes are correspondingly evaluated at -17.67 +/- 0.27 kcal mol-1 (-74.1 +/- 1.12 kJ mol-1) and -25.06 +/- 0.07 kcal mol-1 (-121.2 +/- 0.29 kJ mol-1) for CH3NO2 and C2H5NO2. Enthalpies of formation for the aci-tautomers are calculated as -3.45 +/- 0.44 kcal mol-1 (-14.43 +/- 0.11 kJ mol-1) for aci-nitromethane and -14.25 +/- 0.44 kcal mol-1 (-59.95 +/- 1.84 kJ mol-1) for the aci-nitroethane isomers, respectively. Data are evaluated against experimental and computational values in the literature with recommendations. A set of thermal correction parameters to atomic (H, C, N, O) enthalpies at 0 K is developed, to enable a direct calculation of species enthalpy of formation at 298.15 K, using atomization reaction and computation outputs.     

Surprisingly long-lived ascorbyl radicals in acetonitrile: concerted proton-electron transfer reactions and thermochemistry

Proton-coupled electron transfer (PCET) reactions and thermochemistry of 5,6-isopropylidene ascorbate (iAscH-) have been examined in acetonitrile solvent. iAscH- is oxidized by 2,4,6-tBu3C6H2O. and by excess TEMPO. to give the corresponding 5,6-isopropylidene ascorbyl radical anion (iAsc.-), which persists for hours at 298 K in dry MeCN solution. The stability of iAsc.- is surprising in light of the transience of the ascorbyl radical in aqueous solutions and is due to the lack of the protons needed for radical disproportionation. A concerted proton-electron transfer (CPET) mechanism is indicated for the reactions of iAscH-. Redox potential, pKa and equilibrium measurements define the thermochemical landscape for 5,6-isopropylidene ascorbic acid and its derivatives in MeCN. These measurements give an O-H bond dissociation free energy (BDFE) for iAscH- of 65.4 +/- 1.5 kcal mol-1 in MeCN. Similar studies on underivatized ascorbate indicate a BDFE of 67.8 +/- 1.2 kcal mol-1. These values are much lower than the aqueous BDFE for ascorbate of 74.0 +/- 1.5 kcal mol-1 derived from reported data.     

Differential ordering of the protein backbone and side chains during protein folding revealed by site-specific recombinant infrared probes

The time scale for ordering of the polypeptide backbone relative to the side chains is a critical issue in protein folding. The interplay between ordering of the backbone and ordering of the side chains is particularly important for the formation of β-sheet structures, as the polypeptide chain searches for the native stabilizing cross-strand interactions. We have studied these issues in the N-terminal domain of protein L9 (NTL9), a model protein with mixed α/β structure. We have developed a general approach for introducing site-specific IR probes for the side chains (azide) and backbone ((13)C═(18)O) using recombinant protein expression. Temperature-jump time-resolved IR spectroscopy combined with site-specific labeling enables independent measurement of the respective backbone and side-chain dynamics with single residue resolution. We have found that side-chain ordering in a key region of the β-sheet structure occurs on a slower time scale than ordering of the backbone during the folding of NTL9, likely as a result of the transient formation of non-native side-chain interactions.     

Gas-phase ionic syntheses of amino acids: beta versus alpha

Both theoretical and experimental studies are reported for the gas-phase reactions of protonated hydroxylamine with acetic and propanoic acids which yield protonated glycine and alanine, GlyH+ and AlaH+, respectively. The key step for these reactions is an insertion of the amino group into a C-H bond. For the formation of AlaH+, the reaction barrier for insertion into a Cbeta-H bond is ca. 5 kcal.mol-1 lower than that for the insertion into a Calpha-H bond; the product beta-AlaH+ is ca. 6 kcal mol-1 lower in energy than alpha-AlaH+. Thus, both kinetics and thermodynamics favor formation of the beta-form. The energetic preference for the beta-form is due to more efficient hydrogen bonding between the amino group and the carbonyl oxygen in the limiting transition structure and in the beta-AlaH+ product. These theoretical results are in excellent accord with selected ion flow tube measurements of the gas-phase synthesis which show striking specificity for the beta-isomer according to multi-collision-induced dissociation of the AlaH+ product ion. The results suggest that Gly and beta-Ala found in carbonaceous chondrite meteorites are products of interstellar chemistry.     

Aggregation and C-N rotation of the lithium salt of N,N-dimethyldiphenylacetamide

[formula: see text] Two methyl 1H NMR signals for the Li salt of N,N-dimethyldiphenylacetamide are observed at low temperature and assigned to the monomer and dimer. From line shape analysis, the dimerization constant (K1,2) is 40 +/- 10 M-1 at 200 K (delta G degree = 1.5 kcal mol-1, delta H degree = 0.8 kcal mol-1, delta S degree = 12 eu) and the activation parameters are delta H++ = 5.5 kcal mol-1 and delta S++ = -18 eu. The C-N bond rotation is too fast to observe on the NMR time scale, indicating a rotation barrier of less than 10 kcal mol-1.     

Transesterification and amide cis-trans isomerization in Zn and Cd complexes of the chelating amino acid ligand Boc-Asp(Dpa)-OBzl

The amino acid derivative Boc-Asp-OBzl (Boc=N-butyloxycarbonyl; Asp=aspartic acid; Bzl=benzyl) was functionalized by coupling its carboxylate side chain to dipicolylamine. This yielded the tridentate nitrogen donor ligand Boc-Asp(Dpa)-OBzl (-OBzl). The compound -OBzl contains three different carbonyl groups: a tertiary amide linkage between Asp and Dpa, a C-terminal benzyl ester function, and an N-terminal urethane protecting group. NMR spectra were used to compare the reactivity of these moieties. The Boc protecting group gives rise to two isomers, (E, 9%) and (Z, 91%). Coordination of Cd(NO3)2 and Zn(NO3)2 yielded the complexes and. These compounds have significantly reduced barriers to rotation about the tertiary amide C-N bond compared with the free ligand (-OBzl:18.5 kcal mol-1 in CDBr3;: 12.9 kcal mol-1 in (CD3)2CO;: 13.8 kcal mol-1 in (CD3)2CO). Both complexes readily undergo transesterification in methanol or CD3OD. Experimental pseudo-first order rate constants were determined in CD3OD and (CD3)2CO:CD3OD (3:1;). It was found that the zinc complex (k=(2.28+/-0.02)x10(-4) s-1) is significantly more reactive than the cadmium complex (k=(1.41+/-0.03)x10(-6) s-1). In order to study their tertiary amide cis-trans isomerization, the cadmium complex [(-OCH3)Cd(NO3)2] was synthesized, and the zinc complex [(-OCD3)Zn(NO3)2] was generated in situ in (CD3)2CO:CD3OD (3:1). The barriers to rotation were determined (:14.1 kcal mol-1 in CD3OD;: 13.4 kcal mol-1 in (CD3)2CO:CD3OD (3:1)). Our results show that the stronger Lewis-acid zinc(II) is significantly more active than cadmium(II) in the acceleration of the transesterification. This is in marked contrast to the tertiary amide bond rotation which is comparably fast with both metal ions.