Deuterated phospholipids as nonperturbing components for Raman studies of biomembranes.

The deuterated phospholipid, 1,2-dipalmitoyl-d62-phosphatidylcholine is shown by Raman spectroscopic measurements to be useful for obtaining information concerning phospholipid conformation in complex phospholipid and lipidprotein mixtures. The Raman bands of the deuterated phospholipid are assigned, and the sensitivity of these vibrational modes to conformational changes in the bilayer is demonstrated. Deuteration of the alkyl chains reveals the CH vibrations of the head group. A change in these bands is observed at the melting temperature and is assigned to alteration of the glycerol backbone conformation upon melting.     

Low-frequency Raman spectra of lysozyme.

New techniques in laser Raman spectroscopy are used to obtain spectra of aqueous solutions of lysozylme for frequency shifts as small as 5 cm^?1. In addition, Raman measurements are made on two crystalline forms of hen egg white lysozyme. The spectra obtained from the solution and from the crystal are found to be similar for frequencies above 100 cm^?1. However, a low-frequency band at 25 cm^?1 observed in crystalline lysozyme is not found in the solution, indicating that this band cannot be attributed to an internal molecular vibration.     

Efficient methods for obtaining phase-sensitive gradient-enhanced HMQC spectra

Summary Three improved versions of the gradient-enhanced HMQC experiment are presented which yield phase-sensitive spectra with increased sensitivity compared to the recently described field-gradient HMQC schemes. The first method uses a complex linear back-prediction in order to generate the FIDs at the t₁=0. With this approach, refocusing pulses on the heteronucleus are not necessary. The sequence is especially useful for larger proteins with short relaxation times for the coherences that evolve during t₁. In the other two methods lower and shorter gradient pulses or asymmetric gradients are used to optimize sensitivity.     

Low-frequency Raman spectra of lysozyme crystals and oriented DNA films: dynamics of crystal water.

We observed low-frequency Raman spectra of tetragonal lysozyme crystals and DNA films, with varying water content of the samples. The spectra are fitted well by sums of relaxation modes and damped harmonic oscillators in the region from approximately 1 cm(-1) to 250 cm(-1). The relaxation modes are due to crystal water, and the distribution of relaxation times is determined. In wet samples, the relaxation time of a small part of the water molecules is a little longer than that of bulk water. The relaxation time of a considerable part of the crystal water, which belongs mainly to the secondary hydration shell, is an order of magnitude longer than that of bulk water. Furthermore, the relaxation time of some water molecules in the primary hydration shell of semidry samples is shorter than we expected. Thus we have shown that low-frequency Raman measurements combined with properly oriented samples can give specific information on the dynamics of hydration water in the ps range. On the other hand, we concluded, based on polarized Raman spectra of lysozyme crystals, that the damped oscillators correspond to essentially intramolecular vibrational modes.     

Resonance Raman spectra of whole mitochondria.

The resonance Raman spectra of reduced cytochromes b and c and cytochrome oxidase in whole mitochondria have been recorded without any instrument modifications. The contributions of the individual cytochromes have been identified by comparison with the characteristic features observed in partially purified preparations including: (i) the strong dependence of the intensity patterns on excitation wavelength relative to the peak positions of the alpha, beta, and gamma absorption bands of the cytochromes; and (ii) the presence of marker bands for heme type. Since the Raman spectra can be used as an intrinsic indicator of interaction between hemes, the ability to record spectra in intact mitochondria opens the possibility to study heme-heme interactions in the functioning membrane in situ.     

Raman spectra of polyglycines

The laser-excited Raman spectrum of helical polyglycine II has been obtained. Oligomers of polyglycine are in the planar zigzag conformation and their Raman spectra are indicative of the spectrum of polyglycine I. The Raman spectra of polyglycines have bands complementary to the infrared which are sensitive to the conformation of the chain. The spectra of the oligomers have bands sensitive to the length of the polyglycine. The Raman spectra of di- and triglycine in aqueous solution suggest the conformation is neither planar nor helical.     

Raman spectra of poly-L-lysines

The Raman spectra of poly-L -lysine hydrochloride and poly-?-carbobenzoxy-L -lysine in the solid state have been obtained and are consistent with the presence of an α-helical structure. The Raman spectrum of poly-L -lysine in aqueous solution suggests the presence of random coil structures.     

Raman spectra of chemically modified lysozyme. Oxindole derivatives.

The Raman spectra of oxidation products of lysozyme have been investigated. The protein was oxidized by N-bromosuccinimide and dimethyl sulfoxide/HCl. Depending on the experimental conditions one to six tryptophan residues are oxidized to oxindole. The most prominent difference between the spectra of lysozyme and its oxindole derivatives is the strong band at 1017 cm^?1 which displaces the tryptophan peak at 1010 cm^?1. Other tryptophan bands are also weakened corresponding to the number of the tryptophan side chains destroyed. Shifts are observed in the amide I and in the amide III regions sensitive to conformational changes. These shifts indicate conformational differences in the higher oxidized species and in the native enzyme, although the amide III maxima overlap with a strong oxindole band. Similar effects are observed in the range of the C-C stretching vibrations of the peptide backbone. If more than one tryptophan side chain is oxidized changes have also been found in the S-S stretching range. The evaluation of this effect is difficult because of the strong oxindole vibration appearing in this region. In species oxidized by great excess of N-bromosuccinimide the tyrosine vibrations can no longer be detected, indicating the modification of this amino acid too.     

A method of mesa trimming with glass knives for obtaining large series of ultrathin sections

Ultrastructural analysis of tissue based on 3D reconstruction from serial ultrathin sections is one of the most adequate methods in studies of spatial organization of bio-objects. The sample preparation technique for 3D reconstruction includes the two most technically difficult procedures: an obtaining of stable ribbon of serial sections and mounting of this ribbon onto a slot grid coated with a support film. To mount the ribbon, special approaches and technical tools have been proposed and well evaluated. Much attention has also been paid to obtaining a large and stable ribbon, but this attention deals mainly with the choice of epoxy embedding media. The critical condition of obtaining the straight and stable ribbon is the precise parallelism of trailing and leading edges of mesa falling onto the knife cutting edge. The mesa trimming with dry diamond knife for cryoultratomy allows this condition to be maintained. In the present communication, the way of obtaining parallel sides of the mesa has been proposed with the aid of two forms of glass knives.     

Selective enhancement of Raman or fluorescence spectra of biomolecules using specifically annealed thick gold films

Annealing of "thick" metal films deposited onto a smooth dielectric substrate leads to high-order self-organization of metal clusters on the film surface. This work presents the first experimental evidence that the "thick" gold film (TGF) may be specifically annealed to serve as a substrate for surface-enhanced fluorescence or surface-enhanced Raman scattering (SERS) spectroscopy of the same molecule. High-resolved SERS spectra of mitoxantrone (mitox) were recorded on the TGF annealed at 340 degrees C whereas no Raman enhancement but an increase of mitox fluorescence signal were detected on the TGF annealed at 240 degrees C. The mitox fluorescence was further enhanced by deposition of monolayers of pentanethiol or poly-L-lysine on the surface of annealed TGF. The maximal fluorescence enhancement factor per mitox molecule of approximately 50 that was obtained on the annealed TGF covered with poly-L-lysine makes the system promising for applications in immunofluorescence assays and in microspectrofluorescence analysis.     

Laser Raman spectra of glucagon

Laser Raman spectroscopy study indicates that in concentrated fresh acidic solution (30 mg/ml), glucagon remains predominantly α-helix and not random-coil. The splitting of the amide III band into three components in the crystal at 1262, 1275, and 1295 cm^?1 is due to the α-conformation as expected. The presence of a small fraction of β-conformation is demonstrated by the appearance of the weak band at 1230 cm^?1 in the fresh solution. This study also established the frequencies of amide III′ bands for the α- and β-conformations of glucagon: 957 and 988 cm^?1 for α and β forms, respectively. The conformations of acidic and basic glucagon solutions are apparently different.     

Raman spectra of L-alanine oligomers

The Raman spectra have been obtained of di-, tri-, tetra-, penta-, and hexa-L -alanine in the solid state. Raman spectra of the dimer and trimer in aqueous solution are also reported. The oligomers of alanine exist as zwitterions in the solid state and aqueous solution. Spectral differences between the dipeptide, and other oligomers arise primarily from the conformationally sensitive amide modes. The dipeptide exists as a nonplanar structure in the solid state and the other oligomere as β conformations. Comparison of Raman spectra of tri-L -alanine in the solid state and in aqueous solution suggests a conformational change to a random coil upon dissolution.