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The T-cell receptor-CD3 complex (TCR-CD3) serves a critical role in protecting organisms from infectious agents. The TCR is a heterodimer composed of α- and β-chains, which are responsible for antigen recognition. Within the transmembrane domain of the α-subunit, a region has been identified to be crucial for the assembly and function of the TCR. This region, termed core peptide (CP), consists of nine amino acids (GLRILLLKV), two of which are charged (lysine and arginine) and are crucial for the interaction with CD3. Earlier studies have shown that a synthetic peptide corresponding to the CP sequence can suppress the immune response in animal models of T-cell-mediated inflammation, by disrupting proper assembly of the TCR. As a step towards the understanding of the source of the CP activity, we focused on CP in egg phosphatidylcholine/cholesterol (9:1, mol/mol) model membranes and determined its secondary structure, oligomerization state, and orientation with respect to the membrane. To achieve this goal, 15-residue segments of TCRα, containing the CP, were synthesized and spin-labeled at different locations with a nitroxide derivative. Electron spin-echo envelope modulation spectroscopy was used to probe the position and orientation of the peptides within the membrane, and double electron-electron resonance measurements were used to probe its conformation and oligomerization state. We found that the peptide is predominantly helical in a membrane environment and tends to form oligomers (mostly dimers) that are parallel to the membrane plane.

Nitroxide spin-labelled lipid analogues are often used to study model membrane properties using EPR spectroscopy. Whereas in liquid phase membranes the spin label assumes, on average, its putative location, in gel phases and frozen membrane, depending on its position along the acyl chain, it may exhibit a different average location. Here we used 2H three-pulse Electron Spin Echo Envelope Modulation (ESEEM) of phospholipid spin probes, combined with various deuteration schemes to detect the effect of the model membrane curvature and cholesterol on vertical migrations of the spin label. We compared large and small unilamellar 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles with and without cholesterol (10%). The vertical displacement of the spin label was manifested as an apparently flat trans-membrane profile of water concentration and of label proximity to the head group choline. The spin-label propensity to migrate was found to increase with vesicle curvature and decrease in the presence of c...

The organization and orientation of membrane−inserted helices is important for better understanding the mode of action of membrane−active peptides and of protein−membrane interactions. Here we report on the application of ESEEM (electron spin−echo envelope modulation) and DEER (double electron−electron resonance) techniques to probe the orientation and oligomeric state of an α−helical trans−membrane model peptide, WALP23, under conditions of negative mismatch between the hydrophobic cores of the model membrane and the peptide. Using ESEEM, we measured weak dipolar interactions between spin−labeled WALP23 and (2)H nuclei of either the solvent (D(2)O) or of lipids specifically deuterated at the choline group. The ESEEM data obtained from the deuterated lipids were fitted using a model that provided the spin label average distance from a layer of (2)H nuclei in the hydrophilic region of the membrane and the density of the (2)H nuclei in the layer. DEER was used to probe oligomerization through the dipolar interaction between two spin−labels on different peptides. We observed that the center of WALP23 does not coincide with the bilayer midplane and its N−terminus is more buried than the C−terminus. In addition, the ESEEM data fitting yielded a (2)H layer density that was much lower than expected. The DEER experiments revealed the presence of oligomers, the presence of which was attributable to the negative mismatch and the electrostatic dipole of the peptide. A discussion of a possible arrangement of the individual helices in the oligomers that is consistent with the ESEEM and DEER data is presented

ATP-dependent binding of the chaperonin GroEL to its cofactor GroES forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution. It has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement, interactions with the cavity walls, and differences between the properties of cavity-confined and bulk water. However, experimental data regarding the cavity-confined water are lacking. Here, we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the Tyr71 → Cys GroES mutant obtained using two magnetic resonance techniques: electron-spin echo envelope modulation and Overhauser dynamic nuclear polarization. Residue 71 in GroES is fully exposed to bulk water in free GroES and to confined water within the cavity of the GroEL-GroES complex. Our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation, thus indicating that bulk water-exposed and cavity-confined GroES surface water share similar properties. Interestingly, the diffusion dynamics of water near the GroES surface are found to be unusually fast relative to other protein surfaces studied. The implications of these findings for chaperonin-assisted folding mechanisms are discussed.

SummaryThe structural organization of the functionally relevant, hexameric oligomer of green-absorbing proteorhodopsin (G-PR) was obtained from double electron-electron resonance (DEER) spectroscopy utilizing conventional nitroxide spin labels and recently developed Gd3+-based spin labels. G-PR with nitroxide or Gd3+ labels was prepared using cysteine mutations at residues Trp58 and Thr177. By combining reliable measurements of multiple interprotein distances in the G-PR hexamer with computer modeling, we obtained a structural model that agrees with the recent crystal structure of the homologous blue-absorbing PR (B-PR) hexamer. These DEER results provide specific distance information in a membrane-mimetic environment and across loop regions that are unresolved in the crystal structure. In addition, the X-band DEER measurements using nitroxide spin labels suffered from multispin effects that, at times, compromised the detection of next-nearest neighbor distances. Performing measurements at high magnetic fields with Gd3+ spin labels increased the sensitivity considerably and alleviated the difficulties caused by multispin interactions.

The rumen bacterium Ruminococcus albus binds to and degrades crystalline cellulosic substrates via a unique cellulose degradation system. A unique family of carbohydrate-binding modules (CBM37), located at the C terminus of different glycoside hydrolases, appears to be responsible both for anchoring these enzymes to the bacterial cell surface and for substrate binding.

Micellar to gel phase transition in the aqueous triblock copolymer F127 solutions occurs at higher temperatures in the presence of cyclodextrin and the temperature range corresponding to the gel phase is contracted. These features were observed by dynamic rheometry and the tube inversion method. We investigated further the behaviours of various molecular probes in Pluronic F127 solutions in the absence and in the presence of hydroxypropyl-β-cyclodextrin (HPB) as a function of temperature by continuous wave electron paramagnetic resonance (cw-EPR), Electron Spin Echo Envelope Modulation (ESEEM) and fluorescence spectroscopy, to correlate the observed macroscopic and microscopic changes. The use of molecular probes with different structural particularities allowed the targeting of different regions of F127 micelles which are inhomogeneous at the nanoscale level and the elucidation of the mode in which HPB influences the phase transition in such systems. Dansyl-TEMPO (DT), with a dual character (paramagnetic and fluorescent), spin labelled cyclodextrin (MTCYC), spin labelled Pluronics – L62NO and F127NO, dansyl β-CD (D-β-CD) and pyrene were employed as molecular probes. Both cw-EPR and fluorescence measurements revealed that molecular probes with a more hydrophobic character are more sensitive to phase transition occurring in the studied systems.

Background A widely accepted in vivo model for studying leukemia and its treatment is the highly immune-deficient mice NOD/SCID (b2M-/- or rag-/-). While this model is powerful and recapitulates the phenotypes of blood malignancies in vivo. it is costly and complex, requiring 1-2 months to establish engraftment and the mice are susceptible to spontaneous neoplasms. For these and other reasons the testing of new drugs on leukemia is primarily performed in vitro. The development of antileukemia therapies could be facilitated by a rapid and cost-effective in vivo system for evaluating human leukemia growth and its response to new drugs. Additionally, the treatment of relapsed or refractory disease could be individually tailored by this rapid and cost-effective in vivo system by evaluating patient's cells response to new agents. Turkey embryos are inexpensive, require no maintenance, are larger than chicks are more easily manipulated and have a more robust engraftment (Grinberg I, et al, Leuk Res, 2009; 33:1417-26). We recently described this new in-vivo system for studying multiple myeloma in the pre-immune turkey embryo (Farnoushi, Y., et al.,Br J Cancer, 2011; 105:1708-18). We now demonstrate application of this rapid alternative xenograft system for the preclinical assessment of leukemia growth and therapy. Methods BCR/Abl+ human leukemia lines K562 or LAMA-84 c-Kit+ CHRF 4288 and fresh patient cells were injected into turkey egg chorioallantoic membrane (CAM) veins. Cell injections were performed on day embryonic day E11as previously optimized (Farnoushi, Y., et al.,as above). To determine the engraftment of human AML cells on E19-23, in hematopoietic tissue, the engraftment of human AML cells in the BM was detected in BM by flow cytometry (FC) using anti-human CD71 for LAMA and K562, anti- human CD33 for CHRF and fresh leukemia samples. Engraftment in bone marrow (BM) and other organs was also monitored using Quantitive real time PCR (Q-PCR) comparing the amount of genomic human to the amount of avian DNA and number of human cells / avian cells in BM. Drug response was tested by IV injection of therapeutic range doses of Imatinib (Glivec ®) and Doxorubicin, 48H after cell grafting, at drug levels precalibrated to be non-toxic to the developing embryo by LD50 and BM cell viability compared to control. Six days later (E19) the embryos were sacrificed and the BM collected for FC and hematopoietic and non-hematopoietic tissues for molecular analysis. Results The optimal treatment and readout times were resolved by injecting cells on E11 and determining the kinetics of leukemia cell engraftment in the BM on E15, E18, and E23 in BM and liver. The highest engraftment level in the BM bone marrow (BM) and liver of lines tested was detected at E18 by Q-PCR, and FC in more than 90% of the injected embryos. The average engraftment (±s.d.) in the BM after one week was 4.6%+0.75 K562, 5.16%+2.15 LAMA-84, 7.65%+1.15 CHRF-4288 ( n=7-12 per group) and 2.5% fresh leukemia cells was detected by FC. Q-PCR results were similar to those of FC. Imatinib toxicity testing revealed 100% survival of embryos with no BM toxicity on embryos treated on E13 with doses similar to a human therapeutic dose, up to 0.75 mg/egg. Treatment of embryos with 100 ug Doxorubicin was previously shown to be not toxic to the embryos (Taizi M et al. Exp Hematol 2006; 34:1698–708). A single dose of 0.75 mg Imatinib/embryo dramatically reduced engraftment in BM and several other organs of all 3 AML cell lines or fresh patient leukemia cells. A similar effect was also obtained by a single dose Rx 100ug Doxorubicin. Treatment of a single dose of 0.75 Imatinib mg/embryo 48H after injecting ARH-77 (multiple myeloma) had no effect on cell engraftment. Treatment with a single non toxic dose of Revlimid as previously described (Farnoushi, Y., et al. as above) eliminated engraftment of ARH77 cells, clearly demonstrating the specificity of the drug treatments. Conclusions The results presented demonstrate the potential utility of a practical avian embryo model for testing drug activity in vivo. With further work the turkey embryo may provide a new xenograft in vivo method for studying the biology of leukemia engraftment, and for rapidly and affordably testing leukemia therapies. This system may provide a new platform for developing individualized patient screening for response or resistance to particular therapeutic agents. Disclosures: No relevant conflicts of interest to declare.