Structure and growth of ultrasmall protein microcrystals by synchrotron radiation: I. µGISAXS and µdiffraction of P450scc

Abstract

Ultrasmall P450scc cytochrome microcrystals are grown by classical hanging vapor diffusion and by its modification using homologous protein thin-film template displaying a long-range order. The nucleation and growth mechanisms of P450scc microcrystals are studied at the thin cytochrome film surface by a new microbeam grazing incidence small angle X-ray scattering (microGISAXS) technique developed at the microfocus beamline of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. P450scc cytochrome crystals of about 5 microm are also investigated by synchrotron radiation diffraction in order to attempt a preliminary analysis of the atomic structure of this unique protein system yet unsolved.

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

“…Raman-based measurements of biological samples have already been exploited for the identification of molecular specific markers for disease detection and monitoring [1]. The use of fiber optic technology coupled with Raman spectroscopy is ideal for application to aqueous solutions, either with or without Langmuir-Blodgett (LB) nanotemplate [2,3]. Raman is expected to evaluate the lysozyme concentration changes in vapour diffusion hanging drop method with and without LangmuirBlodgett nanotemplate.…”

LB Crystallization and Preliminary X-ray Diffraction Analysis of L-Asparaginase from Rhodospirillum rubrum

“…Several protein non easily crystallizable by classical methods were successfully crystallized by LB nanotemplate method, as human CK2α, ribosomal SsIF2α and SsIF2β, phage GroEL, bovine cytochrome P450scc, oxygen-bound Hell's Gate Globin I [25][26][27][28][29]. While in the classical methods extensive search of the crystallization conditions should be performed, using large screening sets and robotic systems, LB method allows to reduce the number of trials, being the protein molecules already organized in the ordered bi-dimensional structure with the protein molecules from the solution, helping to overcome the energy barrier of crystallization by forming the specific aggregates inside the LB film.…”

Ab Initio Semi-Quantitative Analysis of Micro-Beam Grazing-Incidence Small-Angle X-Ray Scattering (Μ-GISAXS) during Protein Crystal Nucleation and Growth

“…The potential for studying thin protein films by µ-GISAXS during nanotemplate-assisted crystallization experiments was previously demonstrated in a study about the effect of temperature on long-range order [19] and during both ex situ and in situ experiments [32][33][34][35][36]. Ex situ data obtained using both Cytochrome P450scc and Lysozyme were extremely complex to interpret and needed to be studied in parallel with microscopy characterization, using either the classical method or the nanotemplate hanging-drop method [32,33], as well as in situ data [34][35][36].…”