In theory yes, you can fuse the termini to restrain the protein. You could use T4 lysozyme and insert in the loop which they use for crystallizing GPCRs (many references, including https://www.ncbi.nlm.nih.gov/pubmed/25450769). The potential problems are that you have no idea where the termini are distance-wise to each other in your receptor. Maybe trying some modelling with Phyre or Rosetta) You would probably have to make several constructs with varying lengths of flexible linkers and then there is no guarantee the fusion protein will actually express or crystallize.
You say you have had no luck crystallize the construct you have at the moment. Is it His-tagged? Can you remove it? If C-terminal you could use Carboxypeptidase A (1:100 w/w ratio) or limited proteolysis in general? You said it is a receptor. Have you tried with the ligand/binding protein? Lysine methylation? Thermal Shift Assay to identify a storage buffer which increases the Tm of the protein? You could make new constructs with trimmed termini. Surface entropy reduction of the protein (use the SER server, http://services.mbi.ucla.edu/SER/). You could try fusing it to MBP or surface entropy reduced MBP (https://www.ncbi.nlm.nih.gov/pubmed/20196072). Hope something helps and it crystallizes. Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrew Lovering Sent: Wednesday, March 06, 2019 9:28 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic: Membrane protein "into" soluble protein Dear ccp4bb members, A quick off topic question. I have a protein whose domain structure runs N-TM-receptor-TM-enzyme-C, i.e. is a two transmembrane-helix containing signalling protein. One would suspect that the receptor domain has ends that cluster, and that the enzyme (via homology with known systems) is activated when the two helices self-interact in a dimeric protein as a four helix bundle. So...my question - I have tried expressing the receptor domain as a soluble protein, it works well, folds, purifies, but doesn't crystallize. Does anyone know of an example of taking an exposed membrane protein loop/domain and making a chimera with a soluble 4 helix bundle such that the ends of the loop are tethered and restrained nicely? I was thinking that this must've been attempted for transmembrane alpha-helical signalling proteins, perhaps even "PDB output" successfully! Best Andy ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
