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

Claudio Nicolini

doi:10.4172/jpb.1000303

Abstract

Micro-beam Grazing-Incidence Small-Angle X-ray scattering (μ-GISAXS), exploiting both the advantages of elastic X-ray scattering and the highly focused third-generation synchrotron radiation micro-beams, is an advanced scattering technique that enables scientists to unravel the details of crystal growth processes and to investigate large-scale structures in thin films, including nanobiofilms or other different kinds of surfaces, such as surface gradients or confined surfaces. In this study, we analyze semi-quantitatively and we simulate our previously acquired μ-GISAXS experiments of Thaumatin and Lysozyme Langmuir-Blodgett (LB)-film, shedding light on nucleation and crystal growth processes. Here, we show that, during LB-thin film facilitated nucleation, the particle radius of Thaumatin and of Lysozyme crystal increases while the film thickness reduces. Structural re-organization inside and within the LB-thin film are likely to lead to the crystal nucleation and growth. These semi-quantitative findings are in agreement with the model previously hypothesized. New insights and implications for protein nanocrystallography are also discussed.

Highlights

Protein crystallization is a challenging issue for solving and determining the crystallographic structures of molecules of crucial medical importance: it is a time-consuming, highly demanding task, characterized by many rate-limiting steps and bottlenecks [1,2,3,4,5].Problems and difficulties due to the identification of the precise crystallization conditions and parameters [6] are often encountered in classical crystallography, despite its advancements and achievements [7].In order to overcome these issues, in the last years we developed and proposed an approach termed as “protein nanocrystallography” or better “protein nanobiocrystallography”, in which nanobiotechnologies play a major role
Taken together the data of Lysozyme and of Thaumatin, we have proved that LB-thin film acts a transferring nanobiotemplate and can really enhance and facilitate crystallization growth, which is often difficult and demanding
In addition the lysozyme experimentation appear compatible with a model where the LB crystals keep growing at a constant rate while the classic crystals interrupt their growth in the same time interval

Summary

Introduction

Protein crystallization is a challenging issue for solving and determining the crystallographic structures of molecules of crucial medical importance: it is a time-consuming, highly demanding task, characterized by many rate-limiting steps and bottlenecks [1,2,3,4,5].Problems and difficulties due to the identification of the precise crystallization conditions and parameters [6] are often encountered in classical crystallography, despite its advancements and achievements [7].In order to overcome these issues, in the last years we developed and proposed an approach termed as “protein nanocrystallography” or better “protein nanobiocrystallography”, in which nanobiotechnologies play a major role. Protein crystallization is a challenging issue for solving and determining the crystallographic structures of molecules of crucial medical importance: it is a time-consuming, highly demanding task, characterized by many rate-limiting steps and bottlenecks [1,2,3,4,5]. LB nanobiotemplate enables scientists to finely manipulate molecules and proteins, and allows the development and design of highly ordered nanobiopatterns and sensitive nanobiosensors [15,16,17]. We found that protein crystals grown on nanobiotemplate surfaces exhibit very interesting features, such as thermostability [18,19], enhanced radiation resistance [20,21,22], unique water structure [23,24] and submicron domains [25]

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