Membrane Transport Proteins with Complete Replacement of Transmembrane Helices with Polyalanine Sequences Remain Functional

Xiaoyong Bao,Yongyue Chen,Sung Haeng Lee,Sung Chang Lee,Luis Reuss,Guillermo A Altenberg

doi:10.1074/jbc.m413536200

Abstract

Approximately 25% of all genome coding sequences correspond to membrane proteins, which perform varied and essential functions in cells. Eukaryotic integral membrane proteins are predominantly alpha-helical proteins that span the membrane several times. The most frequent approach to identifying transmembrane-helix amino acids essential for function is to substitute native residues, one at a time, with Cys or Ala (Cys- and Ala-scanning mutagenesis). Here, we present a new approach, in which complete transmembrane-helix native sequences are substituted with poly-Ala sequences. We show that the basic functional features of two dissimilar membrane proteins, which function as a channel and a pump, respectively, are maintained when certain individual alpha-helices are replaced with poly-Ala sequences. This approach ("helix-scanning mutagenesis") allows for rapid identification of helices containing residues essential for function and can be used as a primary helix-screening tool, followed by individual amino acid substitutions when specific helix poly-Ala replacements cause functional changes in the protein.

Highlights

The hydrophobic amino acid residues Leu and Ala are the most frequently found in transmembrane helices [1]
We show that the basic functional features of two dissimilar membrane proteins, which function as a channel and a pump, respectively, are maintained when certain individual ␣-helices are replaced with poly-Ala sequences
The docking of hemichannels to form the gapjunctional channels depends on the structure of the two extracellular Cx loops, which are believed to form interdigitated ␤-sheets that insulate the channel from the extracellular solution [12]

Summary

Introduction

The hydrophobic amino acid residues Leu and Ala are the most frequently found in transmembrane helices [1]. Ala-scanning mutagenesis takes advantage of these properties of Ala and has been used to identify the so-called functional epitopes, i.e. “hot spots,” in the structural epitope identified in high-resolution structures, which contribute most binding energy in protein-ligand interactions [3]. If some ␣-helices in membrane proteins do not contain residues required for proper helix packing, it may be possible to replace them completely while preserving function. We present a simplified approach of multiple simultaneous mutagenesis, i.e. the replacement of complete transmembrane helices of membrane proteins with poly-Ala sequences (helix-scanning mutagenesis), and its application to two dissimilar membrane transport proteins, a channel (connexin 43 (Cx43)) and a pump (multidrug-resistance protein MRP1)

Methods

Results

Conclusion