Reconstitution of Membrane Proteins into Liposomes

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Publisher Summary

The reconstitution of membrane proteins into liposomes is a powerful tool that can be used to identify the mechanism of the action of membrane proteins. The prospects of achieving optimal proteoliposome reconstitution are good when reliable methods and systematic experimental analysis are used. This chapter deals with the various strategies commonly used to reconstitute proteoliposomes and focuses on approaches that have led to the production of highly functional proteoliposomes. Four basic strategies are outlined—mechanical means, freeze-thawing, organic solvents, and detergents. The chapter also introduces a new method for membrane protein reconstitution. The new reconstitution strategy proceeds in four stages: (1) preparation of large, homogeneous, and unilamellar liposomes, (2) addition of detergent to the preformed liposomes, through all the range of the solubilization process, (3) addition of solubilized protein at each well-defined step of the solubilization process, and (4) detergent removal and characterization of the reconstituted products. Besides the need for measuring the activity of the protein, any method of membrane-protein reconstitution should fulfill a number of important criteria that must be analyzed to characterize unequivocally the efficiency of the reconstitution. The first parameter to analyze to check the efficiency of a reconstitution trial is the activity of the protein after reconstitution. The most accurate method to analyze the efficiency of membrane-protein incorporation is a density gradient.

Introduction

Screening of the genomes of various organisms demonstrated that about 25% of the sequenced genes encoded strongly hydrophobic proteins, which are integrated into cell membranes.1 This observation emphasizes the importance of membrane proteins in many biological processes essential for life. However, the complexity of most biological membranes makes it difficult to study these membrane proteins in situ. Therefore, purification from the native membrane and further reincorporation of a purified membrane protein into an artificial membrane continue to be crucial steps in studying the function and structure of these molecules. The necessity for reconstitution arises because many membrane proteins express their full activity only when correctly oriented and inserted in a lipid bilayer. In particular, reconstitution has played a central role in identifying and characterizing the mechanisms of action of membrane proteins with a vectorial transport function.2, 3, 3, 4 More generally, through biochemical and biophysical approaches, it has led to important information about lipid–protein and protein–protein interactions as well as topological and topographical features of different classes of membrane proteins. The reconstitution of membrane proteins to form two-dimensional crystals confined in a membrane has led to important high-resolution structural information by electron crystallography.5, 6

In many instances the ability to investigate membrane proteins through the use of reconstituted systems has long been limited by the fact that methods for producing high-quality proteoliposomes have not advanced in step with biochemical, biophysical, and molecular biology techniques. Thus, one of the limiting factors in obtaining molecular information is related to the lack of reproducible methods of reconstitution. Therefore, enormous efforts have been required to understand the mechanisms of reconstitution and for new approaches to be evaluated, refined, and applied to available proteins in order to make reconstitution even more important as a tool for further structure–function relationship studies on membrane proteins.

This chapter deals with the various strategies commonly used to reconstitute proteoliposomes and focuses on approaches that have led to the production of highly functional proteoliposomes. General guidelines and rules are proposed in this area of study, which has long been viewed as more art than science.

Section snippets

Strategies for Reconstitution of Membrane Proteins into Liposomes

From the abundant literature concerning the insertion of membrane proteins into liposomes, four basic strategies can be outlined: mechanical means, freeze-thawing, organic solvents, and detergents. Although these reconstitution strategies have proved useful to prepare pure phospholipidic vesicles,7 the additional insertion of a membrane protein during the reconstitution process has imposed many constraints that have hampered seriously their efficiency and applicability for proteoliposome

Mechanisms of Proteoliposome Formation and Efficiency of Reconstitution

Despite extensive studies and diverse applications of proteoliposomes, the mechanism of their formation has long been surprisingly ill defined. Reconstitutions from detergent micellar mixtures yielded proteoliposomes of various compositions depending on the nature of the detergent, the particular procedure used to remove it, as well as the nature of the protein and the lipid composition. Therefore, not surprisingly, each membrane protein responded differently to the various reconstitution

New Method for Membrane Protein Reconstitution: Step-by-Step Procedure

The new reconstitution strategy proceeds in four stages (Fig. 2): (1) preparation of large, homogeneous, and unilamellar liposomes, (2) addition of detergent to the preformed liposomes, through all the range of the solubilization process, (3) addition of solubilized protein at each well-defined step of the solubilization process, and (4) detergent removal and characterization of the reconstituted products.

Characterization of Reconstituted Proteoliposomes

Besides the need for measuring the activity of the protein, any method of membrane protein reconstitution should fulfill a number of important criteria that must be analyzed to characterize unequivocally the efficiency of the reconstitution (Fig. 4).

Conclusion

Reconstitution of membrane proteins into liposomes is a powerful tool that can be used to identify the mechanism of action of membrane proteins. As shown in this chapter, it appears that reconstitution is no longer “black magic,” and the prospects of achieving optimal proteoliposome reconstitution are obviously good when using reliable methods and systematic experimental analysis.

The future of membrane protein reconstitution appears bright in the light of the steadily expanding number of

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