Effects of stand-alone polar residue on membrane protein stability and structure

• Published in: Biochimica et biophysica acta. Biomembranes 2024-06, Vol.1866 (5), p.184325, Article 184325
• Main Authors: Chang, Yu-Chu, Cao, Zheng, Chen, Wai-Ting, Huang, Wei-Chun
• Format: Article
• Language: English
• Subjects: Bacteriorhodopsins - chemistry; Bacteriorhodopsins - genetics; Bacteriorhodopsins - metabolism; Halobacterium salinarum - chemistry; Halobacterium salinarum - genetics; Halobacterium salinarum - metabolism; Hydrophobic and Hydrophilic Interactions; Membrane protein folding and stability; Membrane protein structure; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Models, Molecular; Mutation; Polar side chain mutation; Protein Stability; Protein Structure, Secondary
• ISSN: 0005-2736; 1879-2642; 1879-2642
• DOI: 10.1016/j.bbamem.2024.184325

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. [Display omitted] •Polar residue Asn is substituted into bacteriorhodopsin (bR) to assess the effect on the protein stability.•Folding stability of all the polar Asn mutant bRs are destabilized to different extents.•Extent of destabilization is correlated with the positions and local environments of mutant sites.•Structural analyses of these mutant bRs show minimal disruption of the structure.•Membrane proteins can tolerate newly introduced polar residue in the TM regions.