Electron-proton Distances Research Articles

Alternative wavefunction ansatz for including explicit electron-proton correlation in the nuclear-electronic orbital approach

The nuclear-electronic orbital (NEO) approach treats specified nuclei quantum mechanically on the same level as the electrons with molecular orbital techniques. The explicitly correlated Hartree-Fock (NEO-XCHF) approach was developed to incorporate electron-nucleus dynamical correlation directly into the variational optimization of the nuclear-electronic wavefunction. In the original version of this approach, the Hartree-Fock wavefunction is multiplied by (1+Ĝ), where Ĝ is a geminal operator expressed as a sum of Gaussian type geminal functions that depend on the electron-proton distance. Herein, a new wavefunction ansatz is proposed to avoid the computation of five- and six-particle integrals and to simplify the computation of the lower dimensional integrals involving the geminal functions. In the new ansatz, denoted NEO-XCHF2, the Hartree-Fock wavefunction is multiplied by √(1+Ĝ) rather than (1+Ĝ). Although the NEO-XCHF2 ansatz eliminates the integrals that are quadratic in the geminal functions, it introduces terms in the kinetic energy integrals with no known analytical solution. A truncated expansion scheme is devised to approximate these problematic terms. An alternative hybrid approach, in which the kinetic energy terms are calculated with the original NEO-XCHF ansatz and the potential energy terms are calculated with the NEO-XCHF2 ansatz, is also implemented. Applications to a series of model systems with up to four electrons provide validation for the NEO-XCHF2 approach and the treatments of the kinetic energy terms.

Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of α-Synuclein

alpha-Synuclein (alpha-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related alpha-synulceinopathies. However, whether phosphorylation promotes or inhibits alpha-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of alpha-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type alpha-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated alpha-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of alpha-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of alpha-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern alpha-syn aggregation and toxicity in PD and related alpha-synulceinopathies.

EDTA-derivatized deoxythymidine as a tool for rapid determination of protein binding polarity to DNA by intermolecular paramagnetic relaxation enhancement.

EDTA-derivatized deoxythymidine (dT-EDTA), incorporated into DNA and complexed to Fe2+ in the presence of dithiothreitol, is a widely used reagent for sequence-specific cleavage of duplex DNA. Using HPLC/electrospray mass spectrometry, we show that cleavage is specific to Fe2+, and no cleavage occurs when DNA-EDTA is complexed to other metal ions such as Ca2+, Mn2+, and Fe3+ even after many days. Because dT-EDTA can be incorporated at any desired position of a synthetic oligonucleotide, DNA-EDTA is ideally suited for the measurement of intermolecular paramagnetic relaxation enhancement effects between a paramagnetic ion chelated to DNA-EDTA and a bound protein. Measurements on the SRY/DNA-EDTA complex using two double-stranded oligonucleotides bearing dT-EDTA at opposite ends of the sequence indicate that intermolecular 1HN-T2 enhancement by chelated Mn2+ can be used to readily ascertain the polarity of protein binding to DNA and to derive quantitative long-range distance information for structure refinement. In the case of the SRY-DNA complex, excellent agreement between observed and calculated 1HN-T2 paramagnetic relaxation enhancement data can be achieved with insignificant shifts in the atomic coordinates ( approximately 0.25 A for all heavy atoms) while simultaneously satisfying all other experimental restraints. A unique feature of DNA-EDTA is that the relaxation enhancement effect can be tuned by judicious choice of the paramagnetic metal ion, thereby permitting a wide range of long-range intermolecular electron-proton distances, ranging from approximately 9 to 35 A, to be probed.

Structure of spin-labeled methylmethanethiolsulfonate in solution and bound to TEM-1 beta-lactamase determined by electron nuclear double resonance spectroscopy.

Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully applied to investigate secondary structure and conformational transitions of proteins. In these studies, it is expected that the spin-label moiety adopts different conformations dependent on its local environment. To determine the conformation of SLMTS in solution reacted with L-cysteine (SLMTCys) and bound in the active site of the Glu240Cys mutant of TEM-1 beta-lactamase, we have synthesized SLMTS both of natural abundance isotope composition and in site-specifically deuterated forms for electron nuclear double resonance (ENDOR) studies. ENDOR-determined electron-proton distances from the unpaired electron of the nitroxyl group of the spin-label to the methylene and methyl protons of SLMTS showed three conformations of the oxypyrrolinyl ring with respect to rotation around the S-S bond dependent on the solvent dielectric constant. For SLMTCys, two conformations of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H(alpha) and H(beta1,2) of cysteine. To determine SLMTS conformation reacted with the Glu240Cys mutant of TEM-1 beta-lactamase, enzyme was overexpressed in both ordinary and perdeuterated minimal medium. Resonance features of H(alpha) and H(beta1,2) of the Cys240 residue of the mutant and of the side-chain methylene protons within the spin-label moiety yielded electron-proton distances that sterically accommodated the two conformations of free SLMTCys in solution.

Rydberg states of the hydrogen atom near a metal surface

We consider n = 20 Rydberg states of the hydrogen atom interacting with a nearby metal surface over an atom - surface distance range of 105 nm - 530 nm. We show that the traditional quadratic approximation to the potential is insufficient to follow the true potential and one needs to include higher order terms to obtain accurate energies for these Rydberg states. We include terms up to eighth order in the electron-proton distance in the potential and demonstrate by means of basis set calculations that the resulting spectra differ significantly from those obtained in the quadratic approximation. We observe an energy spread of the n = 20 manifold which is roughly twice that given by the quadratic approximation. Observed level repulsions are attributed to z-parity symmetry breaking. We compare our results with those from degenerate perturbation theory.

Catalytic conformation of carboxypeptidase A. Structure of a true enzyme reaction intermediate determined by electron nuclear double resonance.

The structure of a catalytically competent reaction intermediate of carboxypeptidase A (CPA) formed with the specific spin-label ester substrate O-[3-(2,2,-5,5-tetramethyl-1-oxypyrrolinyl)propen-2-oyl]-L-b eta- phenyllactate through application of cryoenzymological methods has been determined by electron nuclear double resonance (ENDOR) and molecular modeling. It is shown that the reaction intermediate is best identified as a mixed-anhydride acylenzyme derivative in which the side chain of Glu-270 is acylated by the spin-label substrate, in agreement with previous cryoenzymological and spectroscopic studies from this laboratory. From the observed proton ENDOR shifts corresponding to principal hyperfine coupling components and assigned by selective deuteration, the dipolar hyperfine coupling components were calculated to estimate electron-proton distances. With these ENDOR-determined distances as constraints, the conformation of the substrate free in solution and in the active site of CPA has been determined on the basis of torsion angle search calculations. With a catalytically active, acetylated form of CPA, we have also assigned the position of the side chain of Tyr-198 with respect to the nitroxyl group. The positional assignments of both substrate and active-site residues in a true reaction intermediate provide important constraints in defining the structural basis of action of CPA.

Structure and conformation of nitroxyl spin-label compounds in frozen solutions by electron nuclear double resonance spectroscopy

The structure and conformation of carboxylic acid, formyl, and propenoic acid derivatives of the nitroxyl spin-label 2,2,5,5-tetramethyl-1-oxypyrroline have been determined by electron nuclear double resonance (ENDOR) spectroscopy. From ENDOR spectra of the spin-label compounds in frozen solutions, we have assigned the resonance absorption features for each class of protons. The ENDOR spectra were analyzed on the basis of their dependence onH0. The maximum and minimum ENDOR shifts for each proton were shown to correspond to axially symmetric principal hyperfine coupling (hfc) components, from which the dipolar contributions were estimated to calculate electron-proton separations. Conformational analysis on the basis of torsion angle search calculations constrained by the ENDOR determined electron-proton distances revealed that in all three spin-label compounds the side chains are in a planar conformation with respect to the oxypyrrolinyl ring. In the carboxylic acid and formyl derivatives the C=O group is in as-trans conformation with respect to the vinyl group of the spin-label, while in the spin-labeled propenoic acid the conformation is found to be all planartrans-s-cis.

Studies of Electrons Trapped in X-Irradiated Rhamnose Crystals

The electrons trapped in single crystals of rhamnose X-irradiated at low temperature were studied by ENDOR spectroscopy. Hyperfine couplings of protons in the environs of the electron have been determined from ENDOR measurements, including those of some of the more remote carbon-bound hydrogen atoms. The likely site of electron trapping in the crystal structure of rhamnose was inferred from calculations of the electric potential generated by the dipoles of hydroxy groups about preexisting void spaces. Electron-proton distances for nonexchangeable hydrogen atoms from points within the void were calculated from the crystal structure and compared with distances obtained from hyperfine couplings. Good agreement was obtained between experimental and calculated values.

Application of the semicontinuum potential model to deduce localized electron trapping sites in single crystals of d-sorbitol

The semicontinuum model of electron solvation [J. Am. Chem. Soc. 95, 1398 (1976)] has been used to calculate the stability of the trapped electron in single crystals of D-sorbitol at 4.2 K. Experimental ENDOR results suggest an environment of two OH dipoles, with the protons at equal distances from the electron. The geometry of the most probable site in a lattice undistorted by irradiation results in an unbound electron state. This arises because the dominant term in the total energy is the dipole–dipole interaction which is attractive for that geometry. If the oxygen atoms remain fixed at crystal lattice positions but the protons are allowed to rearrange so as to point towards the electron, a minimum in the total energy is obtained, but the electron–proton distance is much shorter than the experimental value. However, good agreement with experiment is obtained for configurations of two or four OH dipoles pointing towards the trapped electron when lattice rearrangement is allowed. These calculations suggest that dipole rearrangement occurs during electron trapping in single crystals even at 4.2 K.

Temperature dependence of electron spin–lattice relaxation of presolvated and solvated electrons in methanol–water matrices

In 1:1 volume ratio methanol–water matrices both solvated and presolvated electrons, with equilibrium and nonequilibrium solution shells, respectively, can be stabilized by radiolysis at 77 K. The temperature dependence of the electron spin–lattice relaxation of these two types of electrons has been studied by the saturation recovery method. The relaxation rate remains fast at low temperatures and shows a large component linear in temperature which dominates up to 30 K. This is interpreted in terms of the recently developed model of tunneling relaxation involving modulation of the electron nuclear dipolar interaction by tunneling of nuclei in the radical’s environment. [J. Phys. Chem. 81, 456 (1977)]. The tunneling relaxation coefficient for presolvated electrons is about 76% of that for solvated electrons. This is explained in terms of the average electron–proton distance changes that occur during electron solvation.

Electron—proton distance changes during solvation of electrons in 3-methylhexane and other organic glasses by matrix ENDOR and second moment EPR lineshape analyses

Matrix proton ENDOR signals have been measured for stabilized electrons produced by irradiation at 4 K and at 77 K in 3-methylhexane glass. The matrix proton ENDOR linewidths are smaller when irradiation is carried out at 77 K. This difference is analyzed to show a 0.15 Å change in the average electron—proton distance as the electron produced at 4 K is solvated by warming to 77 K. Similar distance changes have been deduced from a second moment EPR lineshape analysis for electrons in several organic glasses for which the proton hyperfine interaction is mainly dipolar.

Hydrogen spin flip satellite lines associated with the PO 2−3 radical in a single crystal of Na 2HPO 3· 5H 2O : Calculated and experimental intensities

Intensities of satellite lines have been calculated for PO 2− 3 in a single crystal of Na 2HPO 3·5H 2O, based on the known crystallographic structure and unpaired spin distribution in the radical. It was assumed that water protons contribute to the satellite intensity. Variation of the satellite intensity was calculated in the bc plane and data were compared with measured intensities at two orientations. At X-band (9 GHz) the experimental intensity is greater than the calculated intensity by a factor of 3 to 4, probably because of overlap with and borrowed intensity from the main line and the inadequacy of the high field approximation. At Q-band (35 GHz) there is good agreement between the calculated and experimental intensities within 10–20 %. It seems that measurements at higher microwave frequency are more reliable than those at lower microwave frequency for the derivation of the electron-proton distances.

Electron spin echo envelope modulation of trapped radicals in disordered glassy systems: Application to the molecular structure around excess electrons in γ-irradiated 2-methyltetrahydrofuran glass

Three specifically deuterated 2-methyltetrahydrofurans (MTHF) have been synthesized to delineate the proton (deuteron) arrangement and hence the molecular arrangement around a trapped excess electron in γ-irradiated glassy MTHF at 77 K. The electron–deuteron anisotropic hyperfine interaction was detected by its modulation of the two-pulse electron spin echo envelope. Comparison of experimental and simulated modulation patterns shows that the excess electron is located about 3.7 Å below the center of the plane of the ring carbons in MTHF on the side opposite the methyl group and that three or four MTHF molecules constitute the first solvation shell. A second moment analysis of the EPR linewidths of the excess electron in the variously deuterated MTHF’s independently supports the same structural conclusions. The shortest electron–proton distance is then ∼3.1 Å, which compares moderately well with previous, less accurate ENDOR results. This structure seems somewhat more expanded than that indicated by theoretical calculations based on the semicontinuum model, but does not seem incompatible with that theoretical model. A small positive isotropic coupling to the ring protons (∼1 MHz) is also indicated by the spin echo results.