Nowe metody wyznaczania parametru asymetrii tensora gradientu pola elektrycznego w spektroskopii NQR i ich zastosowanie do badania układów nieuporządkowanych

Wydział Fizyki

Use of 45° pulse pair as a filter for pure-phase two-dimensional NMR spectroscopy

Conventionally two-dimensional NMR spectra are recorded in the absolute-intensity mode (1-4). It has recently been demonstrated that absorption-mode 2D spectra have much higher resolution and are the preferred mode of presentation, especially for 2D spectra of biomolecules (5-7). Indeed, any experimental scheme which yields phasemixed lineshapes is subject to modification to yield pure-phase spectra, even at the expense of intensity and anomalous multiplet structure (8-10). For this purpose two types of filters are already known: the z filter (9, 20) and the purging pulse (8, 10). In this note, we propose a 45” pulse pair as a filter for obtaining pure-phase 2D spectra, mainly for experiments in which the above filters do not yield pure-phase spectra.

Coherence transfer via longitudinal spin order generalized pulse pair filtering for pure phase two-dimensional NMR spectroscopy

A generalized pulse pair has been suggested in which the longitudinal spin order is retained and the transverse components cancelled by random variation of the interval between pulses, in successive applications of the two-dimensional NMR algorithm. This method leads to pure phases and has been exploited to provide a simpler scheme for two-spin filtering and for pure phase spectroscopy in multiple-quantum-filtered two-dimensional NMR experiments.

Two-dimensional NMR spectroscopy of connected transitions by linear combination of flip angle dependent cosy: An alternative scheme for E.COSY

An alternative pulse scheme which simplifies and improves the recently proposed P.E.COSY experiment is suggested for the retention of connected or unconnected transitions in a coupled spin system. An important feature of the proposed pulse scheme is the improved phase characteristics of the diagonal peaks. A comparison of various experiments designed for this purpose, namely COSY-45, E.COSY, P.E.COSY and the present scheme (A.E.COSY), is also presented. The suppression of unconnected transitions and the measurement of scalar coupling constants and their relative signs are illustrated from A.E.COSY spectra of 2,3-dibromopropionic acid and 2-(2-thienyl)pyridine.

Investigation of DMSO-Induced Conformational Transitions in Human Serum Albumin Using Two-Dimensional Raman Optical Activity Spectroscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Two-dimensional IR correlation spectroscopy: Sequential events in the unfolding process of the λ Cro-V55C repressor protein

A question often posed in protein folding/unfolding studies is whether the process is fully cooperative or whether it contains sequential elements. To address this question, one needs tools capable of resolving different events. It seems that, at least in certain cases, two-dimensional (2D) IR correlation spectroscopy can provide answers to this question. To illustrate this point, we have turned to the Cro-V55C dimer of the λ Cro repressor, a protein known to undergo thermal unfolding in two discrete steps through a stable equilibrium intermediate. The secondary structure of this intermediate is compatible with that of a partially unfolded protein and involves a reorganization of the N terminus, whereas the antiparallel β-ribbon formed by the C-terminal part of each subunit remains largely intact. To establish whether the unfolding process involves sequential events, we have performed a 2D correlation analysis of IR spectra recorded over the temperature range of 20–95°C. The 2D IR correlation analysis indeed provides evidence for a sequential formation of the stable intermediate, which is created in three (closely related) steps. A first step entails the unfolding of the short N-terminal β-strand, followed by the unfolding of the α-helices in a second step, and the third step comprises the reorganization of the remaining β-sheet and of some unordered segments in the protein. The complete unfolding of the stable intermediate at higher temperatures also undergoes sequential events that ultimately end with the breaking of the H bonds between the two β-strands at the dimer interface.

The two-dimensional IR nonlinear spectroscopy of a cyclic penta-peptide in relation to its three-dimensional structure

A form of two-dimensional (2D) vibrational spectroscopy, which uses two ultrafast IR laser pulses, is used to examine the structure of a cyclic penta-peptide in solution. Spectrally resolved cross peaks occur in the off-diagonal region of the 2D IR spectrum of the amide I region, analogous to those in 2D NMR spectroscopy. These cross peaks measure the coupling between the different amide groups in the structure. Their intensities and polarizations relate directly to the three-dimensional structure of the peptide. With the help of a model coupling Hamiltonian, supplemented by density functional calculations, the spectra of this penta-peptide can be regenerated from the known solution phase structure. This 2D-IR measurement, with an intrinsic time resolution of less than 1 ps, could be used in all time regimes of interest in biology.

Two-dimensional infrared spectroscopy of peptides by phase-controlled femtosecond vibrational photon echoes

Two-dimensional infrared spectra of peptides are introduced that are the direct analogues of two- and three-pulse multiple quantum NMR. Phase matching and heterodyning are used to isolate the phase and amplitudes of the electric fields of vibrational photon echoes as a function of multiple pulse delays. Structural information is made available on the time scale of a few picoseconds. Line narrowed spectra of acyl-proline-NH2 and cross peaks implying the coupling between its amide-I modes are obtained, as are the phases of the various contributions to the signals. Solvent-sensitive structural differences are seen for the dipeptide. The methods show great promise to measure structure changes in biology on a wide range of time scales.

Drying process of microcrystalline cellulose studied by attenuated total reflection IR spectroscopy with two-dimensional correlation spectroscopy and principal component analysis

Molecular interactions between microcrystalline cellulose (MCC) and water were investigated by attenuated total reflection infrared (ATR/IR) spectroscopy. Moisture-content-dependent IR spectra during a drying process of wet MCC were measured. In order to distinguish overlapping O–H stretching bands arising from both cellulose and water, principal component analysis (PCA) and, generalized two-dimensional correlation spectroscopy (2DCOS) and second derivative analysis were applied to the obtained spectra. Four typical drying stages were clearly separated by PCA, and spectral variations in each stage were analyzed by 2DCOS. In the drying time range of 0–41 min, a decrease in the broad band around 3390 cm−1 was observed, indicating that bulk water was evaporated. In the drying time range of 49–195 min, decreases in the bands at 3412, 3344 and 3286 cm−1 assigned to the O6H6cdots, three dots, centeredO3′ interchain hydrogen bonds (H-bonds), the O3H3cdots, three dots, centeredO5 intrachain H-bonds and the H-bonds in Iβ phase in MCC, respectively, were observed. The result of the second derivative analysis suggests that water molecules mainly interact with the O6H6cdots, three dots, centeredO3′ interchain H-bonds. Thus, the H-bonding network in MCC is stabilized by H-bonds between OH groups constructing O6H6cdots, three dots, centeredO3′ interchain H-bonds and water, and the removal of the water molecules induces changes in the H-bonding network in MCC.

Identification and relative quantification of tyrosine nitration in a model peptide using two-dimensional infrared spectroscopy

Nitration of tyrosine in proteins and peptides is a post-translational modification that occurs under conditions of oxidative stress. It is implicated in a variety of medical conditions, including neurodegenerative and cardiovascular diseases. However, monitoring tyrosine nitration and understanding its role in modifying biological function remains a major challenge. In this work, we investigate the use of electron-vibration-vibration (EVV) two-dimensional infrared (2DIR) spectroscopy for the study of tyrosine nitration in model peptides. We demonstrate the ability of EVV 2DIR spectroscopy to differentiate between the neutral and deprotonated states of 3-nitrotyrosine, and we characterize their spectral signatures using information obtained from quantum chemistry calculations and simulated EVV 2DIR spectra. To test the sensitivity of the technique, we use mixed-peptide samples containing various levels of tyrosine nitration, and we use mass spectrometry to independently verify the level of nitration. We conclude that EVV 2DIR spectroscopy is able to provide detailed spectroscopic information on peptide side-chain modifications and to detect nitration levels down to 1%. We further propose that lower nitration levels could be detected by introducing a resonant Raman probe step to increase the detection sensitivity of EVV 2DIR spectroscopy. (Graph Presented).