Protein nanocrystallography : Growth mechanism and atomic structure of crystals induced by nanotemplates

Protein nanocrystallography, a new technology for crystal growth based on protein nanotemplates, has recently been shown to produce diffracting, stable and radiation-resistant lysozyme crystals. This article, by computing these lysozyme crystals' atomic structures, obtained by the diffraction patterns of microfocused synchrotron radiation, provides a possible mechanism for this increased stability, namely a significant decrease in water content accompanied by a minor but significant \u3b1-helix increase. These data are shown to be compatible with the circular dichroism and two-dimensional Fourier transform spectra of high-resolution H NMR of proteins dissolved from the same nanotemplate-based crystal versus those from a classical crystal. Finally, evidence for protein direct transfer from the nanotemplate to the drop and the participation of the template proteins in crystal nucleation and growth is provided by high-resolution NMR spectrometry and mass spectrometry. Furthermore, the lysozyme nanotemplate appears stable up to 523 K, as confirmed by a thermal denaturation study using spectropolarimetry. The overall data suggest that heat-proof lysozyme presence in the crystal provides a possible explanation of the crystal's resistance to synchrotron radiation

Mesoscale Ordering of Phycocyanin Molecules in Langmuir-Blodgett Multilayers

Phycocyanin molecules, which are part of light-harvesting complexes in cyanobacteria, can be assembled into mesoscale multilayer nanofilms by the Langmuir-Blodgett technique. Results obtained by quartz crystal microbalance and atomic force microscopy confirm the homogeneity and reproducibility of phycocyanin Langmuir-Blodgett multilayer deposition. We show by cryo-electron microdiffraction that amorphous phycocyanin Langmuir-Blodgett multilayers form, after annealing at 150 °C and cooling to room temperature, a layered nanofibrillar lattice with rotational disorder. Scanning X-ray nanodiffraction suggests that structural transformation is not homogeneous through the film but limited to patches of up to about 10 μm diameter

Langmuir-Blodgett (LB)-based Nanobiocrystallography at the Frontiers of Cancer Proteomics.

BACKGROUND/AIM: Langmuir-Blodgett (LB) films used as templates for crystallization lead to marked changes in protein stability and water dehydration, despite slight changes in protein atomic structure. Herein, we discuss the importance of LB-based nanocrystallography at the frontiers of cancer proteomics focusing on two model proteins with important biological roles in cancer, namely CK2alpha and RNase A. MATERIALS AND METHODS: Computational mutagenesis using the KINARI Mutagen webserver exhibits different behaviors in terms of stability and robustness, as well as in terms of water dynamics. CONCLUSION: Introduction of LB film leads to the appearance of water molecules close to the protein surface with larger volume, causing changes in crystal stability against radiation and appearing replicated in mutant proteins. Implications for drug design, drug delivery and cancer-causing protein variants are herein presented, along with a review of the most recent findings in LB-based nanobiocrystallography

Detection and analysis of HLA class I specific alloantibodies in the sera of sensitised dialysis recipients waiting for kidney retransplantation.

Background. Almost all patients who rejected a kidney graft displayed anti-HLA antibodies (Abs), and de novo development of anti-HLA Abs in transplanted patients has been identified as a risk factor for both acute and chronic rejection. The aim of this study was to evaluate the specificities of anti-HLA class I Abs detected in the sera of alloimmunised patients waiting for a kidney retransplantation. Methods. Kidney recipients (n = 62; male/female: 42/20; age: 43 \ub1 18 years) on waiting list for kidney retransplantation (52 for a second and 10 for a third transplant) were enrolled during 2002-2004 for HLA Abs screening. Among these kidney recipients, 50 who displayed persistently a panel reactive antibody (PRA) values >10% were selected and analysed for Abs specificity. Abs detection was performed by using complement dependent cytotoxicity technique and subsequently by ELISA to confirm or define better the % PRA and anti-HLA class I specificity. The specificities of anti-HLA Abs were classified as private, public, or multispecific. Results. In 35 patients (70%) only Abs directed against private HLA class I specificities were found. These Abs were expressed by graft donors in 33 cases. In this group, PRA ranged from 20% to 60%. In 12 patients (24%), Abs directed against public epitopes belonging to cross reactive groups (CREG) or an association of anti-private and anti-public Abs occurred, with a PRA ranged from 25% to 90%. Three patients showed multispecific Abs with %PRA >80%. Conclusions. The results of these study indicate that in the majority of donor-recipient pairs the immunogenic determinants were private specificities of mismatched HLA-A and B antigens, whereas in a lesser extent public CREG epitopes were found. Only in three patients no anti-HLA class I specificities were determined, as they displayed multispecific Abs. These findings may lead to improve donor-recipient matching in dialysis recipients waiting for kidney retransplantation

Peripheral blood microchimerism in long-term survival female recipients after kidney transplantation from male donors.

Donor-type microchimerism is a phenomenon which occurs in some recipients after kidney transplantation (KT) and has been considered to play an important role to the induction or maintenance of allograft tolerance. In order to study the relevance of microchimerism to the long-term outcome of renal allograft, we retrospectively analyzed the presence of microchimerism in peripheral blood (PB) of 17 male-to-female kidney transplant recipients who had a stable graft function for 10 years or longer post-transplantation. Donor-type microchimerism, tested using nested\u2013PCR amplification for the SRY region of the Y-chromosome, was detected in 10 of 17 cases (58.8%) and in 7 of 17 cases (41.1%) with graft survival within 10 years and 15 years, respectively. Our results suggest that PB-DNA chimerism is present in a significant percentage of the long-term survival female kidney recipients from male donors, making useful the monitoring of chimeric status in these patients