5J9P

KcsA in vitro

Summary for 5J9P

• Entry DOI: 10.2210/pdb5j9p/pdb
• Descriptor: Fab, pH-gated potassium channel KcsA, POTASSIUM ION, ... (5 entities in total)
• Functional Keywords: membrane protein, ion channel, in vitro, metal transport
• Biological source: Mus musculus (house mouse); More
• Total number of polymer chains: 3
• Total formula weight: 57174.38

Authors

Matulef, K.,Valiyaveetil, F.I. (deposition date: 2016-04-10, release date: 2016-07-20, Last modification date: 2024-10-23)

Primary citation

Focke, P.J.,Hein, C.,Hoffmann, B.,Matulef, K.,Bernhard, F.,Dotsch, V.,Valiyaveetil, F.I.
Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.
Biochemistry, 55:4212-4219, 2016

PubMed Abstract: Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence.

PubMed: 27384110
DOI: 10.1021/acs.biochem.6b00488

Experimental method

X-RAY DIFFRACTION (2.85 Å)