Re: [ccp4bb] how to change a membrane protein into a water soluble protein?
Another option is refolding which can increase soluble protein content and is used routinely to achieve soluble protein such as the TIMPs http://peds.oxfordjournals.org/cgi/content/abstract/7/8/1035 http://www.proteinscience.org/cgi/reprint/11/10/2493.pdf?ck=nck that said, this is not true of all membrane proteins. Addition of a fusion partner, MBP, to the normally membrane associated FMO3 has been shown to generate stable, soluble protein and the addition of a fusion protein allows purification downstream more easily. Here is a paper where they did as the original poster suggested and tried mutagenesis of hydrophobic regions, including a truncation of a membrane anchor. They achieved increased solubility with this in combination with use of detergents. Krueger SK, Siddens LK, Henderson MC, VanDyke JE, Karplus PA, Pereira CB, Williams DE. Abstract C-Terminal truncation of rabbit flavin-containing monooxygenase isoform 2 enhances solubility. Arch Biochem Biophys. 2006 Jun 15;450(2):149-56. Epub 2006 Mar 29. cheers Quoting Bil Clemons [EMAIL PROTECTED]: There is also the soluble KcsA. Computational design of water-soluble analogues of the potassium channel KcsA. A. M. Slovic, H. Kono, J. D. Lear, J. G. Saven, and W. F. DeGrado (2004) PNAS 101, 1828-1833 Bil Bil Clemons, PhD Assistant Professor of Biochemistry Caltech 157 Broad Center MC 114-96 Pasadena, CA 91125 (626) 395-1796 [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] From: Thomas J Magliery PhD [EMAIL PROTECTED] Reply-To: Thomas J Magliery PhD [EMAIL PROTECTED] Date: Mon, 3 Dec 2007 16:50:03 -0500 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to change a membrane protein into a water solub= le protein? =20 It's hard. See: =20 J Mol Biol. 2005 May 6;348(3):777-87. X-ray structure of a water-soluble analog of the membrane protein phospholamban:=20 sequence determinants defining the topology of tetrameric and pentameric coiled coils. Slovic AM, Stayrook SE, North B, Degrado WF. =20 Slovic, A. M., Summa, C. M., Lear, J. D. DeGrado, W. F. (2002). Computational design of a water-soluble analog of phospholamban. Protein Sci. 12, 337=AD348. =20 Li, H., Cocco, M. J., Steitz, T. A. Engelman, D. E. (2001). Conversion of phospholamban into a soluble pentameric helical bundle. Biochemistry, 40, 6636=AD6645. =20 Frank, S., Kammerer, R. A., Hellstern, S., Pegoraro, S., Stetefeld, J., Lustig, A. et al. (2000). Toward a high resolution structure of phospholamban: design of soluble transmembrane domain mutants. Biochemistry, 39, 6825=AD6831. =20 Tom =20 =20 Daniel Jin wrote: Hi, I am wondering whether there is a way to turn a membrane protein with known crystal structure into a water soluble protein by systematic mutagenesis. I guess it should be doable if we introduce enough hydrophilic residues on the surface. Has anyone tested this crazy idea before? Thank you for your help. Best, Chen =20 Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.=20 http://us.rd.yahoo.com/evt=3D51733/*http://mobile.yahoo.com/;_ylt=3DAhu06i6= 2sR8H DtDypao8Wcj9tAcJ%20 =20 =20 --=20 Thomas J. Magliery, Ph.D. Assistant Professor Department of Chemistry Department of Biochemistry The Ohio State University 1043 Evans Laboratory 100 West 18th Ave. Columbus, OH 43210-1185 =20 (614) 247-8425 office (614) 292-1685 fax [EMAIL PROTECTED] http://www.chemistry.ohio-state.edu/~magliery =20
Re: [ccp4bb] how to change a membrane protein into a water soluble protein?
Don't know if anyone has mentioned this paper but its an exact example how to make a K channel soluble. Roosild TP, Choe S. Redesigning an integral membrane K+ channel into a soluble protein. Protein Eng Des Sel. 2005 Feb;18(2):79-84. Epub 2005 Mar 23. PMID: 15788421 [PubMed - indexed for MEDLINE] Scott On Tue, December 4, 2007 4:04 am, Brenda Patterson wrote: Another option is refolding which can increase soluble protein content and is used routinely to achieve soluble protein such as the TIMPs http://peds.oxfordjournals.org/cgi/content/abstract/7/8/1035 http://www.proteinscience.org/cgi/reprint/11/10/2493.pdf?ck=nck that said, this is not true of all membrane proteins. Addition of a fusion partner, MBP, to the normally membrane associated FMO3 has been shown to generate stable, soluble protein and the addition of a fusion protein allows purification downstream more easily. Here is a paper where they did as the original poster suggested and tried mutagenesis of hydrophobic regions, including a truncation of a membrane anchor. They achieved increased solubility with this in combination with use of detergents. Krueger SK, Siddens LK, Henderson MC, VanDyke JE, Karplus PA, Pereira CB, Williams DE. Abstract C-Terminal truncation of rabbit flavin-containing monooxygenase isoform 2 enhances solubility. Arch Biochem Biophys. 2006 Jun 15;450(2):149-56. Epub 2006 Mar 29. cheers Quoting Bil Clemons [EMAIL PROTECTED]: There is also the soluble KcsA. Computational design of water-soluble analogues of the potassium channel KcsA. A. M. Slovic, H. Kono, J. D. Lear, J. G. Saven, and W. F. DeGrado (2004) PNAS 101, 1828-1833 Bil Bil Clemons, PhD Assistant Professor of Biochemistry Caltech 157 Broad Center MC 114-96 Pasadena, CA 91125 (626) 395-1796 [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] From: Thomas J Magliery PhD [EMAIL PROTECTED] Reply-To: Thomas J Magliery PhD [EMAIL PROTECTED] Date: Mon, 3 Dec 2007 16:50:03 -0500 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to change a membrane protein into a water solub= le protein? =20 It's hard. See: =20 J Mol Biol. 2005 May 6;348(3):777-87. X-ray structure of a water-soluble analog of the membrane protein phospholamban:=20 sequence determinants defining the topology of tetrameric and pentameric coiled coils. Slovic AM, Stayrook SE, North B, Degrado WF. =20 Slovic, A. M., Summa, C. M., Lear, J. D. DeGrado, W. F. (2002). Computational design of a water-soluble analog of phospholamban. Protein Sci. 12, 337=AD348. =20 Li, H., Cocco, M. J., Steitz, T. A. Engelman, D. E. (2001). Conversion of phospholamban into a soluble pentameric helical bundle. Biochemistry, 40, 6636=AD6645. =20 Frank, S., Kammerer, R. A., Hellstern, S., Pegoraro, S., Stetefeld, J., Lustig, A. et al. (2000). Toward a high resolution structure of phospholamban: design of soluble transmembrane domain mutants. Biochemistry, 39, 6825=AD6831. =20 Tom =20 =20 Daniel Jin wrote: Hi, I am wondering whether there is a way to turn a membrane protein with known crystal structure into a water soluble protein by systematic mutagenesis. I guess it should be doable if we introduce enough hydrophilic residues on the surface. Has anyone tested this crazy idea before? Thank you for your help. Best, Chen =20 Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.=20 http://us.rd.yahoo.com/evt=3D51733/*http://mobile.yahoo.com/;_ylt=3DAhu06i6= 2sR8H DtDypao8Wcj9tAcJ%20 =20 =20 --=20 Thomas J. Magliery, Ph.D. Assistant Professor Department of Chemistry Department of Biochemistry The Ohio State University 1043 Evans Laboratory 100 West 18th Ave. Columbus, OH 43210-1185 =20 (614) 247-8425 office (614) 292-1685 fax [EMAIL PROTECTED] http://www.chemistry.ohio-state.edu/~magliery =20 -- Scott D. Pegan, Ph.D. Visiting Senior Research Specialist Center for Pharmaceutical Biotechnology University of Illinois at Chicago
Re: [ccp4bb] how to change a membrane protein into a water soluble protein?
Another reference: N. Sukumar, Y.Xu, D.L. Gatti, B.Mitra and F.S. Mathews Structure of an Active Soluble Mutant of the Membrane-Associated (S)-Mandelate Dehydrogenase Biochemistry 40,9870-9878 (2001). In this paper, the membrane protein is converted into the soluble protein by replacing an intra-chain integral membrane-binding portion (39 residues) by a segment from its closely related soluble homologue (20 residues) and crystallized without any detergents. Sukumar -- N. Sukumar NE-CAT, Building 436E Argonne National Laboratory 9700 South Cass Avenue Argonne, IL 60439 e-mail: [EMAIL PROTECTED] Tel: 630-252-0681 Fax: 630-252-0687 On Tue, 2007-12-04 at 08:06, Scott Pegan wrote: Don't know if anyone has mentioned this paper but its an exact example how to make a K channel soluble. Roosild TP, Choe S. Redesigning an integral membrane K+ channel into a soluble protein. Protein Eng Des Sel. 2005 Feb;18(2):79-84. Epub 2005 Mar 23. PMID: 15788421 [PubMed - indexed for MEDLINE] Scott On Tue, December 4, 2007 4:04 am, Brenda Patterson wrote: Another option is refolding which can increase soluble protein content and is used routinely to achieve soluble protein such as the TIMPs http://peds.oxfordjournals.org/cgi/content/abstract/7/8/1035 http://www.proteinscience.org/cgi/reprint/11/10/2493.pdf?ck=nck that said, this is not true of all membrane proteins. Addition of a fusion partner, MBP, to the normally membrane associated FMO3 has been shown to generate stable, soluble protein and the addition of a fusion protein allows purification downstream more easily. Here is a paper where they did as the original poster suggested and tried mutagenesis of hydrophobic regions, including a truncation of a membrane anchor. They achieved increased solubility with this in combination with use of detergents. Krueger SK, Siddens LK, Henderson MC, VanDyke JE, Karplus PA, Pereira CB, Williams DE. Abstract C-Terminal truncation of rabbit flavin-containing monooxygenase isoform 2 enhances solubility. Arch Biochem Biophys. 2006 Jun 15;450(2):149-56. Epub 2006 Mar 29. cheers Quoting Bil Clemons [EMAIL PROTECTED]: There is also the soluble KcsA. Computational design of water-soluble analogues of the potassium channel KcsA. A. M. Slovic, H. Kono, J. D. Lear, J. G. Saven, and W. F. DeGrado (2004) PNAS 101, 1828-1833 Bil Bil Clemons, PhD Assistant Professor of Biochemistry Caltech 157 Broad Center MC 114-96 Pasadena, CA 91125 (626) 395-1796 [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] From: Thomas J Magliery PhD [EMAIL PROTECTED] Reply-To: Thomas J Magliery PhD [EMAIL PROTECTED] Date: Mon, 3 Dec 2007 16:50:03 -0500 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to change a membrane protein into a water solub= le protein? =20 It's hard. See: =20 J Mol Biol. 2005 May 6;348(3):777-87. X-ray structure of a water-soluble analog of the membrane protein phospholamban:=20 sequence determinants defining the topology of tetrameric and pentameric coiled coils. Slovic AM, Stayrook SE, North B, Degrado WF. =20 Slovic, A. M., Summa, C. M., Lear, J. D. DeGrado, W. F. (2002). Computational design of a water-soluble analog of phospholamban. Protein Sci. 12, 337=AD348. =20 Li, H., Cocco, M. J., Steitz, T. A. Engelman, D. E. (2001). Conversion of phospholamban into a soluble pentameric helical bundle. Biochemistry, 40, 6636=AD6645. =20 Frank, S., Kammerer, R. A., Hellstern, S., Pegoraro, S., Stetefeld, J., Lustig, A. et al. (2000). Toward a high resolution structure of phospholamban: design of soluble transmembrane domain mutants. Biochemistry, 39, 6825=AD6831. =20 Tom =20 =20 Daniel Jin wrote: Hi, I am wondering whether there is a way to turn a membrane protein with known crystal structure into a water soluble protein by systematic mutagenesis. I guess it should be doable if we introduce enough hydrophilic residues on the surface. Has anyone tested this crazy idea before? Thank you for your help. Best, Chen =20 Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.=20 http://us.rd.yahoo.com/evt=3D51733/*http://mobile.yahoo.com/;_ylt=3DAhu06i6= 2sR8H DtDypao8Wcj9tAcJ%20 =20 =20 --=20 Thomas J. Magliery, Ph.D. Assistant Professor Department of Chemistry Department of Biochemistry The Ohio State University 1043 Evans Laboratory 100 West 18th Ave. Columbus, OH 43210-1185 =20 (614) 247-8425 office (614) 292-1685 fax [EMAIL PROTECTED] http://www.chemistry.ohio-state.edu/~magliery =20
Re: [ccp4bb] how to change a membrane protein into a water soluble protein?
It's hard. See: J Mol Biol. 2005 May 6;348(3):777-87. X-ray structure of a water-soluble analog of the membrane protein phospholamban: sequence determinants defining the topology of tetrameric and pentameric coiled coils. Slovic AM, Stayrook SE, North B, Degrado WF. Slovic, A. M., Summa, C. M., Lear, J. D. DeGrado, W. F. (2002). Computational design of a water-soluble analog of phospholamban. Protein Sci. 12, 337–348. Li, H., Cocco, M. J., Steitz, T. A. Engelman, D. E. (2001). Conversion of phospholamban into a soluble pentameric helical bundle. Biochemistry, 40, 6636–6645. Frank, S., Kammerer, R. A., Hellstern, S., Pegoraro, S., Stetefeld, J., Lustig, A. et al. (2000). Toward a high resolution structure of phospholamban: design of soluble transmembrane domain mutants. Biochemistry, 39, 6825–6831. Tom Daniel Jin wrote: Hi, I am wondering whether there is a way to turn a membrane protein with known crystal structure into a water soluble protein by systematic mutagenesis. I guess it should be doable if we introduce enough hydrophilic residues on the surface. Has anyone tested this crazy idea before? Thank you for your help. Best, Chen Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. http://us.rd.yahoo.com/evt=51733/*http://mobile.yahoo.com/;_ylt=Ahu06i62sR8HDtDypao8Wcj9tAcJ%20 -- Thomas J. Magliery, Ph.D. Assistant Professor Department of Chemistry Department of Biochemistry The Ohio State University 1043 Evans Laboratory 100 West 18th Ave. Columbus, OH 43210-1185 (614) 247-8425 office (614) 292-1685 fax [EMAIL PROTECTED] http://www.chemistry.ohio-state.edu/~magliery
Re: [ccp4bb] how to change a membrane protein into a water soluble protein?
There is also the soluble KcsA. Computational design of water-soluble analogues of the potassium channel KcsA. A. M. Slovic, H. Kono, J. D. Lear, J. G. Saven, and W. F. DeGrado (2004) PNAS 101, 1828-1833 Bil Bil Clemons, PhD Assistant Professor of Biochemistry Caltech 157 Broad Center MC 114-96 Pasadena, CA 91125 (626) 395-1796 [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] From: Thomas J Magliery PhD [EMAIL PROTECTED] Reply-To: Thomas J Magliery PhD [EMAIL PROTECTED] Date: Mon, 3 Dec 2007 16:50:03 -0500 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to change a membrane protein into a water soluble protein? It's hard. See: J Mol Biol. 2005 May 6;348(3):777-87. X-ray structure of a water-soluble analog of the membrane protein phospholamban: sequence determinants defining the topology of tetrameric and pentameric coiled coils. Slovic AM, Stayrook SE, North B, Degrado WF. Slovic, A. M., Summa, C. M., Lear, J. D. DeGrado, W. F. (2002). Computational design of a water-soluble analog of phospholamban. Protein Sci. 12, 337348. Li, H., Cocco, M. J., Steitz, T. A. Engelman, D. E. (2001). Conversion of phospholamban into a soluble pentameric helical bundle. Biochemistry, 40, 66366645. Frank, S., Kammerer, R. A., Hellstern, S., Pegoraro, S., Stetefeld, J., Lustig, A. et al. (2000). Toward a high resolution structure of phospholamban: design of soluble transmembrane domain mutants. Biochemistry, 39, 68256831. Tom Daniel Jin wrote: Hi, I am wondering whether there is a way to turn a membrane protein with known crystal structure into a water soluble protein by systematic mutagenesis. I guess it should be doable if we introduce enough hydrophilic residues on the surface. Has anyone tested this crazy idea before? Thank you for your help. Best, Chen Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. http://us.rd.yahoo.com/evt=51733/*http://mobile.yahoo.com/;_ylt=Ahu06i62sR8H DtDypao8Wcj9tAcJ%20 -- Thomas J. Magliery, Ph.D. Assistant Professor Department of Chemistry Department of Biochemistry The Ohio State University 1043 Evans Laboratory 100 West 18th Ave. Columbus, OH 43210-1185 (614) 247-8425 office (614) 292-1685 fax [EMAIL PROTECTED] http://www.chemistry.ohio-state.edu/~magliery
