'q-Titration' of long-chain and short-chain lipids differentiates between structured and mobile residues of membrane proteins studied in bicelles by solution NMR spectroscopy

'q-Titration' refers to the systematic comparison of signal intensities in solution NMR spectra of uniformly (15)N labeled membrane proteins solubilized in micelles and isotropic bicelles as a function of the molar ratios (q) of the long-chain lipids (typically DMPC) to short-chain lipids (typically DHPC). In general, as q increases, the protein resonances broaden and correspondingly have reduced intensities due to the overall slowing of protein reorientation. Since the protein backbone signals do not broaden uniformly, the differences in line widths (and intensities) enable the narrower (more intense) signals associated with mobile residues to be differentiated from the broader (less intense) signals associated with "structured" residues. For membrane proteins with between one and seven trans-membrane helices in isotropic bicelles, we have been able to find a value of q between 0.1 and 1.0 where only signals from mobile residues are observed in the spectra. The signals from the structured residues are broadened so much that they cannot be observed under standard solution NMR conditions. This q value corresponds to the ratio of DMPC:DHPC where the signals from the structured residues are "titrated out" of the spectrum. This q value is unique for each protein. In magnetically aligned bilayers (q>2.5) no signals are observed in solution NMR spectra of membrane proteins because the polypeptides are "immobilized" by their interactions with the phospholipid bilayers on the relevant NMR timescale (～10(5)Hz). No signals are observed from proteins in liposomes (only long-chain lipids) either. We show that it is feasible to obtain complementary solution NMR and solid-state NMR spectra of the same membrane protein, where signals from the mobile residues are present in the solution NMR spectra, and signals from the structured residues are present in the solid-state NMR spectra. With assigned backbone amide resonances, these data are sufficient to describe major features of the secondary structure and basic topology of the protein. Even in the absence of assignments, this information can be used to help establish optimal experimental conditions.     

Nanoscale dendritic organoplatinum antitumor drugs

Organoplatinum compounds exhibit broad antitumor activity. Cisplatin [cis- dichlorodiammine platinum (II)] is the most prominent of this family and is currently the most prescribed cancer drug. It is widely used for the treatment of human testicular, ovarian, bladder, head and neck carcinomas. However, it is an extremely toxic compound and side effects such as nephrotoxicity and myelotoxicity are major drawbacks for its use in clinical applications. A way to combat the side effects associated with the administration of platinum drugs would be to generate a prodrug conjugate that, when present in the extracellular fluid, would slowly release active platinum species at a level below the threshold for side effects. Traditionally, linear hydrophilic polymers have been examined for potential application as carrier platforms for these drugs. The advent of dendrimers, which are highly branched macromolecules with precisely controlled nanoscale size, shape and end-group functionality, has provided an excellent opportunity to design and formulate novel multivalent time-release drug systems. For example, treatment of a generation 4.5 (carboxylate surface) poly(amidoamine) [PAMAM] dendrimer with diaquo(1,2-diaminocyclohexane) platinum(II) generates a nanoparticle with forty platinum moieties attached at the surface. This polymer-drug conjugate displays a very good release profile for the platinum species. Under physiological conditions about 80% of the platinum units are released over the first 24 hours. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)     

Saturation Effects and the Colored Pump Noise in a Dye Laser

We investigate the statistical properties of two single-mode dye-laser models-the colored-loss-noise model and the colored-gain-noise model. Analytic expressions of the probability,the mean and the variance of the steady-state laser intensity are obtained through the unified expansion theory [Phys. Rev. A43 (1991) 700]. By comparison we find that when the cavity decay rate for the electric field is large and the pump parameter a₀ is small, the differences of the results of the two laser models are small. Otherwise, the differences are large. And the correlation time of the noise has very obvious influence on the results of the two models.     

Defining protease specificity with proteomics: a protease with a dibasic amino acid recognition motif is regulated by a two-component signal transduction system in Salmonella.

Microbial proteases play diverse and important roles in bacterial virulence but their detection and characterisation is often hampered by their limited abundance or lack of expression in the absence of suitable environmental signals. We describe here a sensitive proteomic approach to detect proteases that are under the control of a virulence regulator and to characterise their recognition motifs. Using MG++-depleted growth media or a mutant strain of Salmonella in which the PhoP-PhoQ virulence regulatory system is constitutively active, truncated forms of DnaK, elongation factor G, elongation factor Tu and ribosomal protein S1 proteins were detected. Two other global regulatory mutants and cells exposed to acid or to oxidative stress failed to produce the truncated proteins, indicating specific control of the protease activity by the PhoP-PhoQ system. Our results suggest that at least two proteases are induced. To define the proteolytic cleavage sites of one of the proteases, peptides from each of the truncated proteins were identified by tryptic mass fingerprinting/nanoelectrospray mass spectrometry and mapped onto the sequence of the intact protein. Alignment of the regions around the cut site indicates that the protease recognises a dibasic amino acid motif characteristic of the omptin protease family. The induction of such proteases in bacteria depleted of Mg++ ions may contribute to the PhoPQ-mediated resistance of Salmonella to cationic antimicrobial peptides. Additionally, our results suggest it would be prudent to keep the concentration of this ion above micromolar levels during bacterial sample preparation for proteomic analyses.     

Evolution of crystallinity in photodegraded polyethylene films studied by ft-raman spectroscopy

FT-Raman spectroscopic studies of photodegraded polyethylene films have enabled the evolution of the crystallinity process to be measured. Commercial polyethylene films of M_w=90 000 were exposed in a weathering UV-chamber under known conditions of exposure time and radiant energy. The spectral profiles were modelled using Fourier methods. The relative amounts of the orthorrombic crystalline phase, α_c, the amorphous phase, α_a and the interphase, α_b, were calculated using Raman bands at 1416 cm⁻¹ characteristic of the crystalline phase and the bands at 1080, 1305 cm⁻¹, characteristic of the amorphous phase. The interphase content can be calculated from the relationship αb= 1-(α_c+α_a). It was found that the weathering process affects only the relative intensities of the bands attributed to crystalline and amorphous fractions; the crystalline content increases at the expenses of the amorphous fraction. These results are discussed in terms of the changes in the intermolecular forces caused by radiation exposure.     

Controlled flux behaviour of membrane processes

Controlling ultrafiltration (UF) and microfiltration (MF) membrane fluxes at or around a region where fouling is minimal can provide an interesting and economic operating regime. Selectivity may be enhanced and cleaning may be easier. For a given flux it is sometimes possible to filter a product suspension at the same trans-membrane pressure (TMP) as for pure water (PWP), but this can require a lot of energy input to maintain cross-flow or high shear in other ways if high fluxes are required. The critical flux is the flux above which one starts to observe fouling. By operating at lower cross-flow velocities and just above the critical flux, and thus, with lower TMPs, periodic cleaning can be effected by temporarily stopping permeation. A change in feed rate demands a change in flux which is obtained by temporarily increasing energy inputs. Controlled flux improves macromolecular fractionation. As flux increases the rejection of high molecular weight materials decreases whilst that of lower molecular weight materials decreases. This paper discusses the causes of fouling and the use controlled flux operation to mitigate its effects.     

Lichen colonization of an active volcanic environment: a Raman spectroscopic study of extremophile biomolecular protective strategies

The Raman spectra of some sixteen lichen specimens from eight genera growing on basaltic lava and wood substrates on new lava fields on the Kilauea volcano, Kona, Hawaii, have been analysed in therms of the pigments and biomolecules produced in this extreme environment. Chlorophyll and a carotenoid, probably lutein or astaxanthin, are ubiquitous, but otherwise, a range of protective pigments have been identified, including atranorin, usnic acid, gyrophoric acid, parietin and pulvinic acid dilactone. In addition, some species were observed to produce calcium oxalate monohydrate and dihydrate. Clearly, several strategies for survival were being adopted by the lichens to combat radiation insolation, desiccation and high temperatures; in one case, of the same lichen species colonizing wood and lava in the same location, a different survival strategy could be detected related to their substrate, but further long‐term ecological experiments would be indicated to derive more detailed information on this aspect. Copyright © 2009 John Wiley & Sons, Ltd.