Structure and function of membrane proteins in detergent-free systems

Main area:Protein chemistry
Target group:Biochemistry, Molecular Biomedicine
Educational level:Masters
Project description:

Overall aim

This project aims at developing a framework for detergent-free isolation of integral membrane proteins from their cell membranes for customized isolation and characterization in their native environments.

Background

Isolation and characterization of integral membrane proteins remains a major bottleneck in structural biology due to difficulties in extraction, while maintaining their structural and functional integrity. Extraction of membrane proteins from membranes is conventionally achieved with detergents stabilizing their transmembrane domains. However, the complexity of the native membrane environment is lost, and this discrepancy may disrupt and inactivate the proteins.

Detergent solubilized integral membrane proteins can be reconstituted into discoidal lipid bilayers, created by membrane scaffold protein (MSP) or Saposin A. While these approaches provide a stabilizing membrane environment, and enable information on protein-lipid interactions, endogenous cofactors are likely lost. Reconstitution into these systems is also time-consuming, requiring significant optimization.

An alternative route is extracting the integral membrane proteins with amphipathic copolymers, an approach which enables cofactors to be preserved by extracting the proteins in discs with lipids from the native membrane. The approach avoids detergents and allows a shorter total preparation time. However, the "native nanodisc approach" is still in its infancy, and current copolymers are too inefficient and harsh for most mammalian membrane proteins. This warrants development of more versatile copolymers, and a characterization of the underpinning biophysics and biochemistry before the polymers can be used routinely.

Approaches

Practically, the project entails screening a test set of membrane proteins expressed in bacteria and mammalian (HEK293) cells with copolymer libraries using fluorescent size-exclusion (FSEC). The best-behaved targets will be further purified and characterized with biophysical and biochemical techniques, including activity assays and cryo-electron microscopy (cryo-EM).

Info on the Autzen Lab

 

Info on working in the Autzen Lab

 

Methods used:Protein purification, mammalian, and bacterial protein expression, FSEC, cryo-EM
Keywords:Biochemistry, Structural biology, Membrane proteins, Native nanodiscs
Project home page: https://www.hautzen.com/native-nanodiscs
Supervisor(s): Henriette Elisabeth Autzen
Email:henriette.autzen[at]bio.ku.dk