Vibrational coupling, isotopic editing, and beta-sheet structure in a membrane-bound polypeptide

The N-acetylated hexapeptide WLLLLL (AcWL5) partitions into lipid membranes and is believed to assemble into an antiparallel beta-sheet. As a test of this structural assignment, the peptide bonds of residues 2-6 were labeled with (13)C and allowed to adsorb onto a supported lipid membrane. Peptides bound to the membrane were examined for evidence of coupling between the labeled vibrational modes in adjacent beta-strands with internal reflection infrared spectroscopy. Experimental results indicate that the amide I absorption band in D(2)O (i.e., amide I') attributable to (13)C is selectively enhanced when the label is at any one of several positions along the peptide backbone. Simulations employing an excitonic model with through-bond and through-space interactions were performed on AcWL5 models in parallel and antiparallel beta-sheet configurations. The simulations yield spectra in good agreement with the experimental results, accounting for the enhancement of both (13)C band intensities and band frequencies. They also yield insight into the physical origin and structure selectivity of the distinctive amide I' band shapes that arise in isotopically edited spectra. It is concluded that the beta-sheet formed by membrane-bound AcWL5 is indeed antiparallel, and the enhancement of (13)C bands in the infrared spectra of these peptides is caused by both interstrand and intrastrand coupling to (12)C modes.     

Photoproperties of isolated cis and trans stilbene molecules

A detailed account is given of the experimental approach to measuring transient spectra of dilute gases using picosecond pulses. The picosecond continuum generated by Nd:glass laser pulses is used to probe gaseous samples and spectra are recorded in a double beam arrangement. The pump and probe pulses interact with the sample over a few centimeters by means of a dielectric waveguide. Picosecond time resolved spectra, relative fluorescence quantum yield measurements, and fluorescence spectra are reported for trans-stilbene under collision free conditions. The lifetime of the optically prepared states at 265 nm and 287 nm are 15 ps and 55 ps respectively, measured by the decay of the transient absorption. The deuteration effect is less than 20%. The variation of the fluorescence yield with vibrational energy excess in the excited state of trans is fitted to these lifetime measurements to yield the variation of nonradiative decay due to twisting of trans-stilbene. Cis-stilbene is suggested to twist in less than 1 ps. Consideration of the spectral results yields new information about the isomerization of stilbene, in particular that there exists a barrier to twisting in the isolated molecule and that vibrational energy redistribution at the trans configuration is probably not complete on the time scale of our experiments. A pictorial model for discussing constant energy relaxation phenomena is introduced.     

Three-dimensional tomography of the beryllium fermi surface: surface charge redistribution

The discontinuity in the lattice periodic potential at surfaces often leads to the creation of new electronic surface states. We developed a photoemission based Fermi surface tomography whose surface sensitivity allowed us to quantify the charge redistribution on the Be(0001) surface. The volume enclosed by the bulklike Fermi surface is significantly reduced at the surface, consistent with the charge transfer to the two surface states as estimated from the area within their two-dimensional Fermi contours. This result represents the first quantification of the charge redistribution on a natural surface termination.     

Electron Transfer Dissociation Mass Spectrometry of Hemoglobin on Clinical Samples

A mass spectrometry-based assay combining the specificity of selected reaction monitoring and the protein ion activation capabilities of electron transfer dissociation was developed and employed for the rapid identification of hemoglobin variants from whole blood without previous proteolytic cleavage. The analysis was performed in a robust ion trap mass spectrometer operating at nominal mass accuracy and resolution. Subtle differences in globin sequences, resulting with mass shifts of about one Da, can be unambiguously identified. These results suggest that mass spectrometry analysis of entire proteins using electron transfer dissociation can be employed on clinical samples in a workflow compatible with diagnostic applications.     

Wipe sampling procedure coupled to LC–MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC–MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm⁻²) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy.     

The polarized charge-transfer spectrum of crystalline anthracene-TNB complex

The intermolecular charge-transfer spectrum of the crystalline anthracene-TNB complex has been measured, using polarized light. The crystal spectrum differs from that of the solution to a much greater degree than is the case for ordinary aromatic systems. The transition is polarized along the intermolecular axis and is blue-shifted with respect to the solution. There is pronounced vibrational structure, including a prominent 250 cm^-1 interval. A Davydov splitting of approximately 200 cm^-1 is observed, and this is shown to be in reasonable agreement with the known crystal structure and with the symmetry assignment of the transition, which is A′ ← A′ for the complex in solution and A ← A in the crystal.     

Design, synthesis, and photochemical validation of peptide linchpins containing the S,S-tetrazine phototrigger

The design, solid-phase synthesis, and photochemical validation of diverse peptide linchpins, containing the S,S-tetrazine phototrigger, have been achieved. Steady state irradiation or femtosecond laser pulses confirm their rapid photofragmentation. Attachment of peptides to the C- and N-termini will provide access to diverse constrained peptide constructs that hold the promise of providing information about early peptide/protein conformational dynamics upon photochemical release.