Luca Belmonte

Oxygen-bound Hell's gate globin I by classical versus LB nanotemplate method.

X‐ray atomic structure of recombinant Hells gate globin I (HGbI) from Methylacidophilum infernorum was calculated from the X‐ray diffraction data of two different types of crystals: obtained by classical hanging drop and by LB nanotemplate method under the same crystallization conditions. After the accurate comparison of crystallographic parameters and electron density maps of two structures they appears to be quite similar, while the quality of the crystals grown by LB nanotemplate method was higher then of those grown by classical method. Indeed, the resolution of the LB crystal structure was 1.65 Å, while classical crystals showed only 3.2 Å resolution. Moreover, the reproducibility of this result in the case of LB crystals was much better—nine crystals from 10 gave the same structural results, while only two of 10 classical crystals were appropriate for the X‐ray structure resolution. J. Cell. Biochem. 113: 2543–2548, 2012.

Mass Spectrometry and Florescence Analysis of Snap-Nappa Arrays Expressed Using E. coli Cell_Free Expression System

We present the analysis of an innovative kind of self-assembling protein microarray, the “Nucleic Acid Programmable Protein Array” (NAPPA), express with the SNAP tag in E.coli coupled self free expression system. The goal is to develop a standardize procedure to analyze the protein protein interaction occurred on NAPPA array combining label free Mass Spectrometry (MS) and fluorescence technology for protein microarray. We employ in the process “Protein synthesis Using Recombinant Elements” (PURE) system. For the first time an improved version of NAPPA, that allows for functional proteins to be synthesized in situ with a SNAP tag directly from printed cDNAs just in time for assay, has been expressed with a novel cell-free transcription/translation system reconstituted from the purified components necessary for Escherichia coli translation the PURE system – and analyzed both in fluorescence and in a label free manner by four different mass spectrometers, namely three Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), a Voyager, a Bru ker Autoflex and a Bruker Ultraflex, and Liquid Chromatography-Electrospray Ionization Mass Spectrometry (LC-ESI-MS/MS). Due to the high complexity of the system, an ad hoc bioinformatic tool has been needed to develop for their successful analysis. The contemporary fluorescence analysis of NAPPA, expressed by means of PURE system, has been performed to confirm the improved characterization of this new NAPPA-SNAP system. complement to labeling methods [7,8]. Among all these techniques, that usually cannot reveal the identity of interaction proteins, MS is powerful to provide chemical and structural information that is difficult to obtain through other means. Combining MS and other surface techniques could offer new dimensions in protein analysis [5,8]. The integration of microarrays with MS has generated a powerful new tool to deal with the problems in protein analysis and identification area. The most successful example is the ProteinChip® System of Ciphergen Biosystems Inc. The design of the ProteinChip® array was originally derived from chromatography and is divided into two groups according to its surface characteristics; the array reader is a SELDI-TOF-MS instrument equipped with a pulsed UV nitrogen laser source [8]. Also Nedelkov et al. [9] coupled BIACORE with MS to demonstrate its feasibility in detecting multiple protein-protein interactions.In our research we employed two different mass spectrometry (MS) techniques, the Matrix Assisted Laser Desorption Ionization Time-ofFlight (MALDI-TOF) MS and Liquid Chromatography-Electrospray Ionization MS (LC-ESI-MS) (Figure 1). In a previous research we carried out a feasibility study of MALDIJournal of Nanomedicine & Nanotechnology J o u r n a l o f N an om edicine & Natechn o l o g y ISSN: 2157-7439 Citation: Nicolini C, et al. (2013) Mass Spectrometry and Florescence Analysis of Snap-Nappa Arrays Expressed Using E. coli Cell_Free Expression System. J Nanomed Nanotechnol 4: 181. doi:10.4172/2157-7439.1000181

In situ Monitoring By Raman Spectroscopy of Lysozyme Conformation during "Nanotemplate" Induced Crystallization

Using Raman spectroscopy and lysozyme, this latter as model protein, we investigate the differences in protein conformation before and after LB nanotemplate-induced crystal nucleation and growth. It was found that the main difference in lysozyme conformation is associated to the higher amount of S-S bonds in lysozyme of LB crystals, probably in C-end of protein, resulting in the higher stiffness of the lysozyme molecules and LB crystal in a whole. Growth in size of LB crystal over time is also accompanied by the formation of S-S bonds. Atomic structure, determined by X-ray diffraction, correlates Raman spectroscopy results confirm the main differences between LB and classical crystals are in terms of water molecules environment previously associated to the increased radiation stability of LB crystals.

Laser-microdissection of protein crystals down to submicron dimensions

We studied laser-microdissection of standard and Langmuir-Blodgett (LB) nanotemplate protein crystals in glycerol solution. The time required for microdissection was significantly longer for LB-crystals as compared to standardcrystals which also more rapidly dissolve. Microfragmentation of lysozyme crystals was observed after extended solvent exposure. Synchrotron radiation nanobeam mapping allowed localizing and aligning cryofrozen lysozyme microfragments. 3D data-sets obtained from two microfragments were refined to atomic resolution. The well-defined electron density maps showed no evidence for damage of radiation of sensitive side-groups. Our results suggest applications of laser-microdissection techniques in structural studies on crystals with a high mosaicity. They also provide a new window for the characterization of protein crystal organization down to the submicron scale, pointing to a new emerging biophysical technique.