Abstract
Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.
Highlights
Most proteins exist as multimeric complexes, in bacteria often as symmetric homomers with identical subunits derived from the same gene, whereas in eukaryotes, complex heteromers or proteins with domains that fused and evolved independently are relatively more common [1,2]
OpuA is a homodimeric membrane protein composed of two membrane subunits (OpuABC) and two nucleotide-binding subunits (OpuAA)
To increase the yield of heterodimeric OpuA, we switched from pILopuAS to more efficient protein purification, as we showed for the homodimeric complex (Figure 2B)
Summary
Most proteins exist as multimeric complexes, in bacteria often as symmetric homomers with identical subunits derived from the same gene (as is evident for e.g., ATP-binding cassette transporters), whereas in (higher) eukaryotes, complex heteromers or proteins with domains that fused and evolved independently are relatively more common [1,2]. OpuA is a homodimeric membrane protein composed of two membrane subunits (OpuABC) and two nucleotide-binding subunits (OpuAA). The. OpuA complex in the detergent-solubilized state disassembles into two OpuABC and two OpuAA subunits when the glycerol concentration falls below 15–20% (v/v), but the dissociation of the complex is reversible. OpuA complex in the detergent-solubilized state disassembles into two OpuABC and two OpuAA subunits when the glycerol concentration falls below 15–20% (v/v), but the dissociation of the complex is reversible In this way, we were able to create apparent heterodimeric complexes in which one of the transmembrane subunits was engineered and the other one not. Lactis andThey evaluated the aexpression native-likeand phospholipid bilayer environment, allowing the stability and functionality purification of homodimeric OpuA variants with three different affinity of membrane proteins [21,22]. OpuAA-opuABC, and they are cloned under the control of the tightly regulated nisin-inducible pNisA promoter, using the medium-copy number vectors pNZ8048
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call