The role of rare codons in determining correct folding of membrane proteins

Abstract

The folding of newly synthesised proteins to their functional states is considered as cracking the second half of the genetic code. The degeneracy of the genetic code means that amino acids are coded by more than one codon. The nature of codon usage is unclear. The use of rare codons has been suggested to play important roles in co-translational protein folding, as the protein folds whilst emerging from the ribosome. This project will focus on rare codons in the understudied area of membrane protein folding. Mutations of rare codons may lead to membrane protein misfolding and disease.
Project

Biological systems assemble themselves with precision. Protein folding has been intensely investigated but is heavily dominated by studies on water-soluble proteins. Membrane proteins have been scarcely probed by comparison, yet account for about a third of cellular proteins and dominate drug targets. The majority of membrane proteins insert and fold in the membrane co-translationally - as their mRNA is being translated by the ribosome, the elongating polypeptide chain inserts into the membrane via the translocon machinery. This project will test the hypothesis that the choice of codon affects translation and in turn folding. Codon optimisation seems to influence the amount of a membrane protein during over-expressed in the cell. Key to this investigation will be the combination of computational and wet laboratory work, where theoretical predictions will be tested in biophysical and cellular studies of co-translational folding. The project will identify rare codons in certain membrane proteins and investigate how they affect translation as well as the amount of functional protein that is expressed. Rare codons will also be used to manipulate folding for uses of membrane proteins in Synthetic Biology systems developed by Booth.

A bioinformatics approach will be used to identify unusual codon biases in membrane proteins of the E. coli inner membrane. We will investigate whether rare codons are overrepresented in membrane protein classes across different organisms. The influence of the identified rare codons on peptide elongation will be practically tested. These codons will be substituted to other more common codons of the identical amino acid and the peptide elongation determined by radiolabelling using cell and membrane extracts.

Methods:

Molecular biology: Standard methods to alter codons in E. coli (PCR, primer design, cloning and expression)
Genetics: Bioinformatics analyses of genome sequences
Biochemistry/Biophysics: Membrane protein purification, folding and reconstitution methods. Protein and cell extract preparations. Biochemical analysis, radiolabelling and protein quantification. Circular dichroism, isothermal titration calorimetry, transport assays.
Chemistry: Measurement and interpretation of kinetics rates associated with folding. Thermodynamic stability studies.
Microscopy/Electrophysiology: optical microscopy to locate protein in cell during over-expression
Simulation Modelling: minor - some modelling of proteins in bilayers
Bioinformatics: evolutionary analyses, genome analyses, structure prediction
Maths Statistics: statistical analyses of codon bias