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Despite a wide variety of biological functions, alpha-helical membrane proteins display a rather simple transmembrane architecture. Although not many high resolution structures of transmembrane proteins are available today, our understanding of membrane protein folding has emerged in the recent years. Now we begin to develop a basic understanding of the forces that guide folding and interaction of alpha-helical membrane proteins. Some structural requirements for transmembrane helix interactions are defined, and common motifs have been discovered in the recent years which can drive helix-helix interactions. Nevertheless, many open questions remain to be addressed in future studies. One general problem with investigating transmembrane helix interactions is the limited number of appropriate tools, which can be applied to investigate membrane protein folding. Only recently several new techniques have been developed and established, including genetic systems, which allow measuring transmembrane helix interactions in vitro and in vivo. In the first part of this review, we summarize several aspects of the current understanding of membrane protein folding and assembly. In the second part, we discuss genetic systems, which were developed in the recent years to measure interaction of transmembrane helices in the inner membrane of E. coli.

In vivo and in vitro requirements for the formation of cytochrome b 6 were examined to analyze the mechanisms of transmembrane b-type cytochrome formation. After heterologous expression of spinach cytochrome b 6 , formation of the holo-cytochrome was observed within the E. coli inner membrane. The transmembrane orientation of cytochrome b 6 appeared not to be critical for heme binding and holo-cytochrome formation. Furthermore, in vitro reconstitution of cytochrome b 6 was possible under oxidizing as well as under reducing conditions. Taken together these observations strongly indicate that transmembrane b-type cytochromes can spontaneously assemble in vitro as well as in a membrane.

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