Helical membrane proteins generally have a hydrophobic nature, with apolar side chains comprising the majority of the transmembrane (TM) helices. However, whenever polar side chains are present in the TM domain, they often exert a crucial role in structural interactions with other polar residues, such as TM helix associations and oligomerization. Moreover, polar residues in the TM region also often participate in protein functions, such as the Schiff base bonding between Lys residues and retinal in rhodopsin-like membrane proteins. Although many studies have focused on these functional polar residues, our understanding of stand-alone polar residues that are energetically unfavored in TM helixes is limited. Here, we adopted bacteriorhodopsin (bR) as a model system and systematically mutated 17 of its apolar Leu or Phe residues to polar Asn. Stability measurements of the resulting mutants revealed that all of these polar substitutions reduced bR stability to various extents, and the extent of destabilization of each mutant bR is also correlated to different structural factors, such as the relative accessible surface area and membrane depth of the mutation site. Structural analyses of these Asn residues revealed that they form sidechain-to-backbone hydrogen bonds that alleviate the unfavorable energetics in hydrophobic and apolar surroundings. Our results indicate that membrane proteins are able to accommodate certain stand-alone polar residues in the TM region without disrupting overall structures.
Keywords: Membrane protein folding; Membrane protein stability; Membrane protein structure; Polar side chain mutation.
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