Synthesis of PAF,an Antifungal Protein from P. chrysogenum,by Native Chemical Ligation: Native Disulfide Pattern and Fold Obtained upon Oxidative Refolding

The folding of disulfide proteins is of considerable interest because knowledge of this may influence our present understanding of protein folding. However, sometimes even the disulfide pattern cannot be unequivocally determined by the available experimental techniques. For example, the structures of a few small antifungal proteins (PAF, AFP) have been disclosed recently using NMR spectroscopy but with some ambiguity in the actual disulfide pattern. For this reason, we carried out the chemical synthesis of PAF. Probing different approaches, the oxidative folding of the synthetic linear PAF yielded a folded protein that has identical structure and antifungal activity as the native PAF. In contrast, unfolded linear PAF was inactive, a result that may have implications concerning its redox state in the mode of action.     

Characterization of the 310-helix in model peptides by HRMAS NMR spectroscopy

A tetra- and a hepta-homopeptide from the C(alpha)-tetrasubstituted Aib (alpha-aminoisobutyric acid) residue were covalently linked to the POEPOP resin by the fragment-condensation approach. The conformational preferences of the two model peptides were determined for the first time on a solid support by means of high-resolution magic angle spinning NMR spectroscopy. The results obtained indicate that the Aib homopeptides adopt a regular 3(10)-helical structure even when they are covalently bound to a polymeric matrix, and thus confirm the remarkable conformational stability of the peptides rich in this amino acid. An ATR-FTIR spectroscopic investigation, performed in parallel, also confirmed that these polymer-bound peptides do indeed adopt a helical conformation. The results of this study open the possibility to exploit the peptide-resin conjugates based on C(alpha)-tetrasubstituted alpha-amino acids as helpful, structurally organized templates in molecular recognition studies or as catalysts in asymmetric synthesis.     

核磁共振技术(NMR)在组合化学中的应用

对科学产生最大影响的分析方法是核磁共振技术(NMR),它被广泛用于许多领域.本文结合作者的研究结果评述了NMR在组合化学中的应用,着重于NMR在固相合成的应用、液态NMR和NMR在高通量筛选中的应用.     

Matrix‐Free DNP‐Enhanced NMR Spectroscopy of Liposomes Using a Lipid‐Anchored Biradical

Magic‐angle spinning dynamic nuclear polarization (MAS‐DNP) has been proven to be a powerful technique to enhance the sensitivity of solid‐state NMR (SSNMR) in a wide range of systems. Here, we show that DNP can be used to polarize lipids using a lipid‐anchored polarizing agent. More specifically, we introduce a C16‐functionalized biradical, which allows localization of the polarizing agents in the lipid bilayer and DNP experiments to be performed in the absence of excess cryo‐protectant molecules (glycerol, dimethyl sulfoxide, etc.). This constitutes another original example of the matrix‐free DNP approach that we recently introduced.     

Context-dependence of the contribution of disulfide bonds to beta-hairpin stability

Incorporation of disulfide bonds to stabilize protein and peptide structures is not always a successful strategy. To advance current knowledge on the contribution of disulfide bonds to beta-hairpin stability, a previously reported beta-hairpin-forming peptide was taken as a template to design a series of Cys-containing peptides. The conformational behavior of these peptides in their oxidized, disulfide-cyclized peptides, and reduced, linear peptides, was investigated on the basis of NMR parameters: NOEs, and 1H and 13C chemical shifts. We found that the effect of disulfide bonds on beta-hairpin stability depends on its location within the beta-hairpin structure, being very small or even destabilizing when connecting two hydrogen-bonded facing residues. When the disulfide bond is linking non-hydrogen-bonded facing residues, we estimated that its contribution to the free-energy change of beta-hairpin folding is approximately -1.0 kcal mol(-1). This value is larger than those reported for most beta-hairpin-stabilizing cross-strand side-chain-side-chain interactions, except for some aromatic-aromatic interactions, in particular the Trp-Trp one, and the cation-pi interaction between Trp and the non-natural methylated Arg/Lys. As disulfide bonds are frequently used to stabilize peptide conformations, our conclusions can be useful for peptide, peptidomimetic, and protein design, and may even extend to other chemical cross-links.     

Orientational effect of aryl groups on 77Se NMR chemical shifts: experimental and theoretical investigations

The orientational effect of p-YC6H4 (Ar) on delta(Se) is elucidated for ArSeR, based on experimental and theoretical investigations. The effect is examined in the cases in which Se--CR in ArSeR is either in the Ar plane (pl) or is perpendicular to the plane (pd). 9-(Arylselanyl)anthracenes (1) and 1-(arylselanyl)anthraquionones (2) are employed to establish the effect in pl and pd, respectively. Large upfield shifts are observed for Y=NMe2, OMe, and Me, and large downfield shifts for Y=COOEt, CN, and NO2 in 1, relative to Y=H, as is expected. Large upfield shifts are brought by Y=NMe2, OMe, Me, F, Cl, and Br, and downfield shifts by Y=CN and NO2 in 2, relative to Y=H, with a negligible shift by Y=COOEt. Absolute magnetic shielding tensors of Se (sigma(Se)) are calculated for ArSeR (R=H, Me, and Ph), assuming pl and pd, based on the DFT-GIAO method. Observed characters are well explained by the total sigma(Se). Paramagnetic terms (sigmap(Se)) are governed by (sigmap(Se)xx+sigmap(Se)yy), in which the direction of np(Se) (constructed by 4pz(Se)) is set to the z axis. The main interaction in pl is the np(Se)-pi(C6H4)-pz(Y) type. The Y dependence in pl occurs through admixtures of 4pz(Se) in pi(SeC6H4Y) and pi*(SeC6H4Y), modified by the conjugation, with 4px(Se) and 4py(Se) in sigma(CSeX) and sigma*(CSeX) (X=H or C) under a magnetic field. The main interaction in pd is the sigma(CSeX)-pi(C6H4)-px(Y) type, in which Se-X is nearly on the x axis. The Y dependence in pd mainly arises from admixtures of 4pz(Se) in np(Se) with 4px(Se) and 4py(Se) in modified sigma*(CSeX), since np(Se) is filled with electrons. It is demonstrated that the effect of Y on sigmap(Se) in the pl conformation is the same regardless of whether Y is an electron-donor or electron-acceptor, whereas for pd conformations the effect is greater when Y is an electron donor, as observed in 1 and 2, respectively. Contributions of each molecular orbital and each transition on sigmap(Se) are evaluated, which enables us to recognize and visualize the effect clearly.     

1-Cyclohepta-2,4,6-trienyl-selanes--a 77Se NMR study: indirect nuclear 77Se--13C spin-spin coupling constants and application of density functional theory (DFT) calculations

1-Cyclohepta-2,4,6-trienyl-selanes Se(C(7)H(7))(2) (2c), R--Se--C(7)H(7) with R = Bu, (t)Bu, Ph, 4-F--C(6)H(4) (12a,b,c,d) were prepared by the reaction of the corresponding silanes, Si(SeMe(3))(2) and R--Se--SiMe(3), respectively, with tropylium bromide C(7)H(7)Br. In spite of the low stability of the selanes even in dilute solutions and at low temperature, they could be characterised by their (1)H, (13)C and (77)Se NMR parameters. Coupling constants (1)J((77)Se,(13)C) were measured and calculated by DFT methods at the B3LYP/6-311+G(d,p) level of theory. The comparison of experimental and calculated coupling constants (1)J((77)Se,(13)C) included numerous selenium carbon compounds with largely different Se--C bonds, revealing a satisfactory agreement. Both the spin-dipole (SD) and the paramagnetic spin-orbital (PSO) terms contributed significantly to the spin-spin coupling interaction, in addition to the Fermi contact (FC) term.     

How (77)Se NMR chemical shifts originate from pre-alpha, alpha, beta, and gamma effects: interpretation based on molecular orbital theory

Plain rules founded in a theoretical background are presented that can be used to determine the structure of selenium compounds on the basis of delta(Se) data and to predict delta(Se) data from a given structure with satisfactory accuracy. As a first step to establish such rules, the origin of delta(Se) is elucidated on the basis of MO theory. The Se(2-) ion was chosen as the standard for the analysis. The concept of the pre-alpha effect is proposed, which is defined as the downfield shift due to protonation of a lone-pair orbital of Se. The pre-alpha effect of two protons in H(2)Se is explained by the generation of double sigma(Se--H) and sigma*(Se--H) through protonation of the spherical Se(2-) ion. The orbitals, together with n(p)(Se), result in effective transitions for the pre-alpha effect. The alpha effect is the downfield shift caused by the replacement of Se--H by Se--Me. The extension of HOMO-2 [4p(y)(Se)], HOMO-1 [4p(x)(Se)], and HOMO [4p(z)(Se)] over the whole Me(2)Se molecule is mainly responsible for the alpha effect. The beta effect originates not from the occupied-to-unoccupied (psi(i)-->psi(a)) transitions but from the occupied-to-occupied (psi(i)-->psi(j)) transitions. Although psi(i)-->psi(j) transitions contribute to upfield shifts in Me(2)Se, the magnitudes become smaller as the methyl protons are substituted by Me groups one after another. The gamma effect of upfield shifts is also analyzed, although complex. The effect of p(Se)-pi(C==C) conjugation is analyzed in relation to the orientational effect. Contributions from each MO (psi(i)) and each psi(i)-->psi(a) transition are evaluated separately, by using a utility program derived from the Gaussian 03 program suite (NMRANAL-NH03G). The treatment enables us to visualize and understand the origin of (77)Se NMR chemical shifts.     

Asymmetry and Non‐Adiabaticity in Fragmentation of Disulfide Bonds upon Electron Capture

Although it has been generally assumed that electron attachment to disulfide derivatives leads to a systematic and significant activation of the S? S bond, we show, by using [CH₃SSX] (X=CH₃, NH₂, OH, F) derivatives as model compounds, that this is the case only when the X substituents have low electronegativity. Through the use of MP2, QCI and CASPT2 molecular orbital (MO) methods, we elucidate, for the first time, the mechanisms that lead to unimolecular fragmentation of disulfide derivatives after electron attachment. Our theoretical scrutiny indicates that these mechanisms are more intricate than assumed in previous studies. The most stable products, from a thermodynamic viewpoint, correspond to the release of neutral molecules; CH₄, NH₃, H₂O, and HF. However, the barriers to reach these products depend strongly on the electronegativity of the X substituents. Only for very electronegative substituents, such as OH or F, the loss of H₂O or HF is the most favorable process, and likely the only one observed. This is possible because of two concomitant factors, 1) the extra electron for [CH₃SSX]^? (X=OH, F) occupies a σ*(S? X) MO, which favors the cleavage of the S? X bond, and 2) the activation barriers associated with the hydrogen transfer process to produce H₂O and HF are rather low. Only when the substituents are less electronegative (X=H, CH₃, NH₂) the extra electron is located in a σ*(S? S) orbital and the cleavage of the disulfide bridge becomes the most favorable process. The intimate mechanism associated with the S? S bond dissociation process also depends strongly on the nature of the substituent. For X=H or CH₃ the process is strictly adiabatic, while for X=NH₂ it proceeds through a conical intersection ( CI ) associated with the charge reorganization necessary to obtain, from a molecular anion with the extra electron delocalized in a σ*(S? S) antibonding orbital, two fragments with the proper charge localization.     

Direct Measurement of Dihedral Angles with High-Resolution NMR Spectroscopy

Help in determining biomolecular structure by NMR spectroscopy is found in a new method recently proposed by Reif, Hennig, and Griesinger, which enables the direct measurement of angles between bond vectors (see picture; X, Y=¹³C, ¹⁵N). This work may be another milestone in the progress of NMR spectroscopy toward simpler and more generally applicable structure determination of biomolecules.