Mesophase-based approaches for on-chip membrane protein crystallization and structure determination

Khvostichenko, Daria

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https://hdl.handle.net/2142/90063 Copy

Description

Title

Mesophase-based approaches for on-chip membrane protein crystallization and structure determination

Author(s)

Khvostichenko, Daria

Issue Date

2012-12-06

Director of Research (if dissertation) or Advisor (if thesis)

Kenis, Paul J

Doctoral Committee Chair(s)

Kenis, Paul J

Committee Member(s)

• Sligar, Stephen G
• Zukoski, Charles F
• Harley, Brendan A
• Schroeder, Charles M

Department of Study

Chemical & Biomolecular Engr

Discipline

Chemical Engineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• on-chip analysis
• membrane proteins
• LCP crystallization
• in meso crystallization
• microfluidics
• X-ray diffraction
• small-angle X-ray scattering
• phase behavior
• lipidic mesophases
• monoolein

Abstract

Transmembrane proteins traverse the lipid bilayers of cell membranes and play a highly important role in many processes in vivo. Malfunctions of membrane proteins have been shown to cause a variety of disease states, for example, cystic fibrosis, hereditary hearing loss, and hypothyroidism. Furthermore, membrane proteins are targets of over 60% of all drugs available on the market. Thus, information on membrane protein function is of immense importance for the understanding of processes associated with disease states and for the development of new and improved therapeutics. Detailed spatial structures of proteins are typically obtained from crystal X-ray diffraction data. Crystallizing membrane proteins, however, is extremely difficult due to their amphiphilic properties that affect their stability in aqueous solutions. The trial-and-error nature of the crystallization process requires large-scale screening efforts, which is often hampered by the limited availability of membrane protein samples. In meso crystallization is a powerful alternative to the traditional crystallization of membrane proteins directly from aqueous solutions. The method involves reconstitution of the proteins into so-called lipidic mesophases that are comprised of lipid bilayers, providing a native-like environment for the proteins. Lipidic mesophases form spontaneously upon mixing of the aqueous solution of a protein with a lipid. The microstructural properties of the mesophase play a highly important role in crystallogenesis in meso and depend on the composition of the overall mixture in a complex manner. Understanding the effect of mesophase microstructure on the outcome of crystallization trials is necessary for improving the success rate of the process. Mesophases, however, are difficult to handle due to their high viscosity, and require special mixing and dispensing tools both for protein crystallization and for microstructural studies. The properties of lipidic mesophases also hampered miniaturization using microfluidic technologies, commonly used for screening of protein crystallization from solutions. Microfluidic platforms developed in this dissertation are the only examples of microfluidic devices that combine mesophase-handling capabilities and X-ray transparency as required for in situ analysis of mesophases and of protein crystals. The platforms provide a route to reduce the preparative scale, automate sample formulation, and eliminate manual handling in two distinct aspects of mesophase-based technologies: (i) screening of the structure/composition relationships of the properties of lipidic mesophases, and (ii) in meso crystallization of membrane proteins. Macroscale studies of the phase behavior of lipidic mesophases with additives typically present in membrane protein crystallization highlight the complexity of structure/composition relationships in these systems, as discussed in Chapter 2. These studies are highly laborious and require the preparation of a large number of samples with systematically varying compositions. Microfluidic platforms developed and validated in Chapters 3 and 4 allow for automated simultaneous formulation of multiple samples and scale down the amount of material per sample at least 300-fold compared to the standard macroscale method. X-ray transparency of the platforms enables small-angle X-ray scattering analysis on-chip, as required to establish the microstructure of the mesophases. The platforms address two types of structural studies of mesophases. The microfluidic system presented in Chapter 3 is designed for the studies of the effect of additives on the microstructure of mesophases in multicomponent crystallization mixtures. The chip developed in Chapter 4 is applicable for studies of phase behavior in binary lipid/water mixtures, which is necessary for the understanding of fundamental principles of self-assembly in lipidic systems and for assessing suitability of novel lipids for in meso crystallization. Chapter 5 describes a microfluidic platform for in meso crystallization of membrane proteins. The platform reduces the amount of material per trial 7-fold compared to similar macroscale methods. Platform architecture has been validated by crystallizing a membrane protein Photosynthetic Reaction Center (RC) from Rhodobacter Sphaeroides. Furthermore, crystal structure of RC was solved using X-ray diffraction data collected on-chip. Thus, the platform fully eliminates manual crystal harvesting and is a highly promising tool for structural biology. The platform is uniquely positioned for the simultaneous analysis of the protein crystals and the surrounding mesophase, which is not possible with existing macroscale approaches. This information is invaluable for unraveling the factors defining the outcome of crystallization trials and for improving the success rate in membrane protein crystallization.

Graduation Semester

2012-12

Type of Resource

text

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Copyright and License Information

Copyright 2012 Daria Sergeyevna Khvostichenko

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