Biospectroscopy最新文献

Charged groups reside mainly on protein surfaces, but for proteins that incorporate redox centers, a charge typically exists at the prosthetic group within the interior. How a protein accommodates a buried charge and the effect of redox changes on protein stability are thermodynamically related problems. To examine these problems in cytochrome c, the metal-free protein was used as a model. When pH is lowered, the neutral, monocation, and dication forms of the porphyrin are progressively formed as indicated by their characteristic absorption spectra. Infrared studies of the protein over this pH range show that the protein remains in a predominately α-helical structure, although the carboxyl groups of the dicarboxylic amino acids become protonated at lower pH. The monocation porphyrin form (which has not been previously reported in a protein and is a charge analogue of ferric heme) has a fluorescence maximum at 609 nm. The pKs for the respective one and two protonation of the porphyrin pyrrole Ns are 3.2 and 1.6 for the folded protein, and 4.4 and 3.1 for the unfolded protein. These values indicate that the protection of the polypeptide chain for protonation is ∼ 3 kcal. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 141–150, 1999

A new fluorescence method has been developed to measure simultaneously and independently the release of fluorophores from two vesicle populations. Calcein and sulforhodamine B were used as a probe couple: the leakage of these probes from vesicles can be recorded independently since they can be excited simultaneously at 510 nm, and their individual fluorescence can be isolated by measuring the fluorescence signal at 525 and 590 nm, using a T-shape fluorometer. Controls show that both probes are suitable for the leakage assay based on fluorescence self-quenching, that they do not interact physically or chemically at the concentrations used in the method, and that they leak in a similar fashion from a given vesicle type. This dual-probe technique is applied to examine the specificity of the release relative to the cholesterol content of the vesicles for melittin, a toxin. This new approach shows in a straightforward manner that melittin-induced release for a given population can be modulated by the presence of vesicles with another lipid composition and this competitive release is associated with a preferential distribution of the peptide on the targeted vesicles. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 133–140, 1999

In this article, the assignment of the ν(CH) stretching region of lipid molecules is revisited. This region is extensively used to follow lipid phase transitions, and especially the frequency shifts and bandwidth alterations in the ν_symCH₂ band have been utilized in this respect. Here, we propose and prove that behind these phenomena there are pairs of component bands in the cases of both the ν_symCH₂ and the ν_asCH₂ bands. The lower-frequency components of the pairs are assigned to the vibrations of CH₂ groups on trans segments of the fatty acyl chains, while the higher-frequency components of the pairs are assigned to CH₂ groups on gauche segments. To prove these assignments, we have shown that the νCH₂ frequencies are characteristic of the conformation of the lipid fatty acyl chain itself, and not the state of the whole lipid matrix. Curve fitting in fact revealed the conformer-specific components. With the use of singular value decomposition analysis we have demonstrated that the relative intensity changes in the components, and not the shifts in the whole bands, cause the observed shifts in the νCH₂ bands upon lipid phase transition. The results of this approach are presented for deuterium-saturated dioleoyl–phosphatidylcholine mixtures, for the gel → liquid-crystalline phase transition of dipalmitoyl–phosphatidylcholine multilayers, and for a biological membrane, barley thylakoid. This refined assignment offers physically plausible reasoning for the observed phenomena and is able to explain frequency shifts and bandwidth changes observed previously upon lipid phase transitions, including their nonconcerted temperature dependences. In biological membranes, this interpretation allows the separation of protein- and membrane-dynamics-induced lipid conformational changes. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 169–178, 1999

¹³C-enriched bacteriochlorophyll c (R[E, E] BChl c_F) was suspended in chloroform to form an aggregate showing the Q_y absorption at 705 nm. (1) The aggregate exhibited several largely split ¹³C-NMR signals suggesting the presence of nonequivalent BChl c molecules in the form of the piggyback dimer. (2) Changes in the ¹³C chemical shifts were traced when methanol was titrated to dissolve the aggregate, and the aggregation shifts (in reference to the monomeric state) were determined as a function of the amount of methanol titrated, and they were analyzed empirically. (3) The ring-current effects were calculated based on the loop-current approximation, and the results were compared with the observed aggregation shifts for ¹³C and ¹H nuclei (the ¹H aggregation shifts were determined by extrapolation of the data taken from Mizoguchi, T.; Limantara, L.; Matsuura, K.; Shimada, K.; Koyama, Y. J Mol Structure 1996, 379, 249–265). The results showed that the assembly of two straight columns consisting of the piggyback dimer stacked in the antiparallel orientation is the best choice as a model for the B705 aggregate. (4) Three-dimensional F1 ¹³C-edited F3 ¹³C-filtered heteronuclear single-quantum nuclear-Overhauser-effect spectroscopy was applied to the aggregate consisting of a 1 : 1 mixture of ¹³C-labeled and unlabeled BChl c in order to selectively detect the intermolecular ¹H–¹H NOE correlations. The NOE correlations were explained in terms of a straight column, supporting the above model. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 63–77, 1999

The freeze-trapped bacteriopheophytin a radical anion Φ has been investigated by ¹H-ENDOR/Special TRIPLE resonance spectroscopy in photosynthetic reaction centers of Rhodobacter sphaeroides, in which the Tyr at position M210 had been replaced by either Phe, Leu, His or Trp. In the wild type reaction center and the mutants YF(M210) and YW(M210) two distinct states of Φ, denoted I and I, can be stabilized below 200 K. The state I is metastable and relaxes to I as the temperature is raised from 135 K to 180 K. The difference in the electronic structure of Φ between the two states is interpreted in terms of a conformational change of Φ_A after freeze-trapping, involving a reorientation of the 3-acetyl group with respect to the macrocycle of the bacteriopheophytin. This interpretation is supported by the results of RHF-INDO/SP calculations. In the YH(M210) reaction center only one Φ state is obtained that is distinct from I and I, and the observed electronic structure indicates an almost in-plane orientation of the 3-acetyl group. This is consistent with the proposal that a hydrogen bond is formed between His M210 and the 3¹-keto oxygen of Φ_A that impedes the reorientation of the acetyl group. Only one Φ state is observed in the YL(M210) reaction center, which is similar to the metastable state I in the wild type complex. This result is interpreted in terms of a steric hindrance of the reorientation of the 3-acetyl group that is exerted by the side chain of Leu at position M210. Possible implications of these findings for the mechanism of electron transfer in bacterial reaction centers are discussed. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 35–46, 1999

With the aim to perform spectroscopic studies and spectral images inside living cells, a microspectrofluorometer has been designed for two-dimensional spectral imaging in the visible and in the near-UV region. The main advantage of the device relies on its ability to scan the laser beam along one direction of the sample. This scanning is optically coupled with one direction of a bidimensional detector, allowing an instantaneous recording of a one-dimensional spectral image. The overall scanning of the sample is achieved by means of submicrometric displacements of the stage in the perpendicular direction. The main characteristics and performances of the microspectrofluorometer in terms of sensitivity (detection of a few molecules), spatial resolution (0.5 × 0.5 × 1 μm), and spectral resolution (1 nm) are presented. Finally, applications of this new apparatus concerning in situ localization and spectral characterization of two dyes are shown with Drosophila salivary glands (ethidium bromide) and T47D tumor cells (Hoechst 33342). © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 101–115, 1999

Surface enhanced Raman spectroscopy (SERS) was used to characterize a homologous series of α,ω-amino acids on colloidal gold and silver. Raman and SER spectra of the α,ω-amino acids, NH₂(CH₂)_nCOOH (n = 3–7), are presented and analyzed, revealing the probable conformations of the molecules on the metal surfaces. The α,ω-amino acids interact with silver and gold through both the amine and carboxylate end groups, and modify the conformation of the molecular backbone in order to maximize these interactions. An odd–even effect is observed for backbone conformations of molecules adsorbed to the silver substrate. The anomolous SER spectrum of 5-aminopentanoic acid on gold suggests the possibility of condensation polymerization at the gold surface. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 9–17, 1999

A novel spectrophotometric method, based upon Raman spectroscopy, has been developed for accurate quantitative determination of nucleoside triphosphate phosphohydrolase (NTPase) activity. The method relies upon simultaneous measurement in real time of the intensities of Raman marker bands diagnostic of the triphosphate (1115 cm⁻¹) and diphosphate (1085 cm⁻¹) moieties of the NTPase substrate and product, respectively. The reliability of the method is demonstrated for the NTPase-active RNA-packaging enzyme (protein P4) of bacteriophage ϕ6, for which comparative NTPase activities have been estimated independently by radiolabeling assays. The Raman-determined rate for adenosine triphosphate substrate (8.6 ± 1.3 μmol · mg⁻¹ · min⁻¹ at 40°C) is in good agreement with previous estimates. The versatility of the Raman method is demonstrated by its applicability to a variety of nucleotide substrates of P4, including the natural ribonucleoside triphosphates (ATP, GTP) and dideoxynucleoside triphosphates (ddATP, ddGTP). Advantages of the present protocol include conservative sample requirements (∼ 10⁻⁶ g enzyme/protocol) and relative ease of data collection and analysis. The latter conveniences are particularly advantageous for the measurement of activation energies of phosphohydrolase activity. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 3–8, 1999