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

Abstract

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 -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.

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

“…Raman spectroscopy probes the vibrational energy levels of the bonds within the protein and the water; as previously shown [17][18][19][20][21][22] unique water environment in LB crystals really differ from classical one. A 6 microliter protein drop is deposited on a microscope cover glass.…”

“…In this paper for the first time we present results of RS study of the mechanism of the crystallization with and without the nanotemplate, and estimate the influence of the LB nanotemplate to the protein nucleation and growth. Langmuir-Blodgett (LB) thin films used as template for protein crystallization develop crystal with improved radiation stability in presence of third generation synchrotron facility due to their different arrangements [10][11][12][13][14][15][16][17][18][19][20][21][22]. One of the main reasons of this success is apparently due to water molecules distribution around protein backbone [18].…”

Raman Spectroscopy of Protein Crystal Nucleation and Growth

“…In this study for the first time we present results of RS study of the mechanism of the crystallization with and without the nanotemplate and estimate the influence of the LB nanotemplate to the protein nucleation and growth. Langmuir-Blodgett (LB-this abbreviation should be explained earlier) thin films used as template for protein crystallization develop crystal with improved radiation stability in presence of third generation synchrotron facility (Pechkova and Nicolini, 2001;Nicolini, 1997;Pechkova et al, 2007;Pechkova and Nicolini, 2010;Pechkova et al, 2005a;2005b;2005c). One of the main reason of this success is apparently due to water molecules distribution around protein backbone .…”

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

“…Protein crystal nucleation and growth are facilitated and enhanced through a Langmuir-Blodgett (LB)-based nanobiotemplate [8][9][10][11][12][13][14].…”