The tetrameric α-helical membrane protein GlpF unfolds via a dimeric folding intermediate.
Many membrane proteins appear to be present and functional in higher-order oligomeric states. While few studies have analyzed the thermodynamic stability of α-helical transmembrane (TM) proteins under equilibrium conditions in the past, oligomerization of larger polytopic monomers has essentially not yet been studied. However, it is vital to study the folding of oligomeric membrane proteins to improve our understanding of the general mechanisms and pathways of TM protein folding. To investigate the folding and stability of the aquaglyceroporin GlpF from Escherichia coli, unfolding of the protein in mixed micelles was monitored by steady-state fluorescence and circular dichroism spectroscopy…
Unfolding a transmembrane helix dimer: A FRET study in mixed micelles
The exact nature of membrane protein folding and assembly is not understood in detail yet. Addition of SDS to a membrane protein dissolved in mild, non-polar detergent results in formation of mixed micelles and in subsequent denaturation of higher ordered membrane protein structures. The exact nature of this denaturation event is, however, enigmatic, and separation of an individual helix pair in mixed micelles has also not been reported yet. Here we followed unfolding of the human glycophorin A transmembrane helix dimer in mixed micelles by fluorescence spectroscopy. Energy transfer between differently labelled glycophorin A transmembrane helices decreased with increasing SDS mole fractions…
Oligomerization of polytopic α-helical membrane proteins: causes and consequences
Abstract Several polytopic α-helical membrane-integrated proteins appear to be organized in higher-ordered oligomeric complexes. While many aspects are still enigmatic, in recent years, the physiological impact of membrane protein oligomerization has been analyzed to some extent. In the present article, oligomerization of structurally well-defined membrane proteins is discussed. The available experimental information indicates the causes and physiological consequences of membrane protein oligomerization, including stabilization, cooperative functions, and control of specific activities. Based on the currently available observations, we aim to derive some general principles and discuss open …
Analyzing Oligomerization of Individual Transmembrane Helices and of Entire Membrane Proteins in E. coli: A Hitchhiker’s Guide to GALLEX
Genetic systems, which allow monitoring interactions of individual transmembrane α-helices within the cytoplasmic membrane of the bacterium Escherichia coli, are now widely used to probe the structural biology and energetics of helix-helix interactions and the consequences of mutations. In contrast to other systems, the GALLEX system allows studying homo- as well as heterooligomerization of individual transmembrane α-helices, and even enables estimation of the energetics of helix-helix interactions within a biological membrane. Given that many polytopic membrane proteins form oligomers within membranes, the GALLEX system represents a unique and powerful approach to monitor formation and sta…
Small Residues Inhibit Homo-Dimerization of the Human Carbonic Anhydrase XII Transmembrane Domain
Amino acids with small side chains and motifs of small residues in a distance of four are rather abundant in human single-span transmembrane helices. While interaction of such helices appears to be common, the role of the small residues in mediating and/or stabilizing transmembrane helix oligomers remains mostly elusive. Yet, the mere existence of (small)xxx(small) motifs in transmembrane helices is frequently used to model dimeric TM helix structures. The single transmembrane helix of the human carbonic anhydrases XII contains a large number of amino acids with small side chains, and critical involvement of these small amino acids in dimerization of the transmembrane domain has been sugges…
Transmembrane helix–helix interactions are modulated by the sequence context and by lipid bilayer properties
Abstract Folding of polytopic transmembrane proteins involves interactions of individual transmembrane helices, and multiple TM helix–helix interactions need to be controlled and aligned to result in the final TM protein structure. While defined interaction motifs, such as the GxxxG motif, might be critically involved in transmembrane helix–helix interactions, the sequence context as well as lipid bilayer properties significantly modulate the strength of a sequence specific transmembrane helix–helix interaction. Structures of 11 transmembrane helix dimers have been described today, and the influence of the sequence context as well as of the detergent and lipid environment on a sequence spec…