SDSL-ESR-based protein structure characterization

As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structur

The nature of chlorine-inhibition of photocatalytic degradation of dichloroacetic acid in a TiO2-based microreactor

Photocatalytic degradation of dichloroacetic acid (DCA) was studied in a continuous-flow set-up using a titanium microreactor with an immobilized double-layered TiO2 nanoparticle/nanotube film. Chloride ions, formed during the degradation process, negatively affect the photocatalytic efficiency and at a certain concentration (approximately 0.5 mM) completely stop the reaction in the microreactor. Two proposed mechanisms of inhibition with chloride ions, competitive adsorption and photogenerated-hole scavenging, have been proposed and investigated by adsorption isotherms and electron paramagnetic resonance (EPR) measurements. The results show that chloride ions block the DCA adsorption sites on the titania surface and reduce the amount of adsorbed DCA molecules. The scavenging effect of chloride ions during photocatalysis through the formation of chlorine radicals was not detected.Slovenian Research Agency/P2-0084Slovenian Research Agency/J2-4309Slovene Human Resources Development and Scholarship Fund/Ad Futur

Isolation of Bacterial Ribosomes with Monolith Chromatography

We report the development of a rapid chromatographic method for the isolation of bacterial ribosomes from crude cell lysates in less than ten minutes. Our separation is based on the use of strong anion exchange monolithic columns. Using a simple stepwise elution program we were able to purify ribosomes whose composition is comparable to those isolated by sucrose gradient ultracentrifugation, as confirmed by quantitative proteomic analysis (iTRAQ). The speed and simplicity of this approach could accelerate the study of many different aspects of ribosomal biology

Immobilized enzyme reactors for \u201comics\u201d approaches

During the last decade, miniaturization, availability of new analytical tools as well as introduction of high-resolution instrumentation on the market have made feasible an important step forward in the \u201comics\u201d field. However, notwithstanding the great advances, experimental workflows still suffer from several problems, which requires improved analytical strategies to be solved. In particular, most protocols in shotgun proteomics and glycomics commonly involve an enzyme-catalyzed key step, which bears several drawbacks such as long incubation times, loss of (expensive) material due to the non-reusability of the enzyme, poor reproducibility and low automation. To circumvent these problems, significant efforts have been recently made to develop more efficient procedures, limit interferences and enhance automation and throughput. Among these, developing immobilized enzyme reactors (IMERs) is an attractive approach. IMERs have shown several advantages over the traditional in-solution methods, among which high enzyme-to-substrate ratio and high efficiency, which translate into shorter analysis time, long term stability, higher reproducibility and higher automation. Thus IMERs, if correctly integrated into a LC-MS analytical platform, may lead to improved workflows in \u201comics\u201d protocols that involve enzyme-catalyzed steps. In particular, conventional workflows in proteomics include in-gel or in-solution protein digestion by proteases, most commonly by trypsin, while in glycomics Peptide-N-glycosidase F (PNGase F) is commonly used to release glycans from glycoproteins or glycopeptides. IMERs based on such enzymes may, therefore, find interesting applications in proteomics and glycomics. To achieve automation and a significant decrease in analysis time IMERs were prepared using short bed, high performance monolithic columns (CIMac\u2122 Analytical columns), which are suitable supports for IMER preparation when integration into a separative system is required and large molecules need to be analyzed. Different chemistries and immobilization protocols were used to achieve stable IMERs with high enzyme activity. Operational conditions to preserve IMER activity and achieve hyphenation with MS system were optimized. In particular, trypsin-IMERs were obtained by covalent immobilization on CIMac\u2122 analytical columns. Trypsin loading and surface chemistry were shown to be key factors for rapid and efficient protein digestion. Comparison with in solution digestion and a commercially available trypsin-column showed a high efficiency for the new developed IMER, with advantages over conventional methods. On the other hand, the PNGase F-IMER was obtained by oriented covalent immobilization of the target enzyme onto an epoxy CIMac\u2122 analytical column. The PNGase F-IMER could be inserted into a LC-ESI-Q-ToF system, enabling the on-line deglycosylation of target proteins and analysis of the released glycans without any derivatisation step

Analysis of side chain rotational restrictions of membrane-embedded proteins by spin-label ESR spectroscopy

Site-directed spin-labeling electron spin resonance (SDSL-ESR) is a promising tool for membrane protein structure determination. Here we propose a novel way to translate the local structural constraints gained by SDSL-ESR data into a low-resolution structure of a protein by simulating the restrictions of the local conformational spaces of the spin label attached at different protein sites along the primary structure of the membrane-embedded protein. We test the sensitivity of this approach for membrane-embedded M13 major coat protein decorated with a limited number of strategically placed spin labels employing high-throughput site-directed mutagenesis. We find a reasonably good agreement of the simulated and the experimental data taking a protein conformation close to the one determined by fluorescence resonance energy transfer analysis [P.V. Nazarov, R.B.M. Koehorst, W.L. Vos, V.V. Apanasovich, M.A. Hemminga, FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein, Biophys. J. 92 (2007) 1296–1305]

How perifosine affects liposome-encapsulated drug delivery across a cell barrier

BACKGROUND: The development of efficient drug delivery systems to transport therapeutics across barrier-forming cells remains a challenge. Recently it was shown that liposomes containing perifosine, a synthetic analog of lysophosphatidylcholine, efficiently deliver liposome encapsulated content across barrier-forming cells. METHODS: To elucidate the mechanism of the delivery, fluorescent and spin labeled analog of perifosine were synthesized and their transport from liposomes to the barrier-forming MDKC cells was measured. RESULTS & CONCLUSION: Perifosine analogs are rapidly transported from liposomes into cell membranes. The total amount of perifosine accumulated in plasma membranes seems to be the most important factor in efficient transepithelial transport of liposome-encapsulated substances. Lysolipid-containing liposomal formulations seem to be promising candidates as drug delivery systems in general

Motional Restrictions of Membrane Proteins: A Site-Directed Spin Labeling Study

Site-directed mutagenesis was used to produce 27 single cysteine mutants of bacteriophage M13 major coat protein spanning the whole primary sequence of the protein. Single-cysteine mutants were labeled with nitroxide spin labels and incorporated into phospholipid bilayers with increasing acyl chain length. The SDSL is combined with ESR and CD spectroscopy. CD spectroscopy provided information about the overall protein conformation in different mismatching lipids. The spin label ESR spectra were analyzed in terms of a new spectral simulation approach based on hybrid evolutionary optimization and solution condensation. This method gives the residue-level free rotational space (i.e., the effective space within which the spin label can wobble) and the diffusion constant of the spin label attached to the protein. The results suggest that the coat protein has a large structural flexibility, which facilitates a stable protein-to-membrane association in lipid bilayers with various degrees of hydrophobic mismatch