The de novo design of a biocompatible and functional integral membrane protein using minimal sequence complexity.

Publisher: Nature Publishing Group Country of Publication: England NLM ID: 101563288 Publication Model: Electronic Cited Medium: Internet ISSN: 2045-2322 (Electronic) Linking ISSN: 20452322 NLM ISO Abbreviation: Sci Rep Subsets: MEDLINE

Original Publication: London : Nature Publishing Group, copyright 2011-

Gene Expression*; Escherichia coli/*chemistry ; Membrane Proteins/*genetics ; Membrane Proteins/*metabolism ; Recombinant Proteins/*genetics ; Recombinant Proteins/*metabolism; Cell Membrane/chemistry ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Heme/metabolism ; Membrane Proteins/chemistry ; Protein Binding ; Protein Conformation ; Protein Stability ; Recombinant Proteins/chemistry ; Recombinant Proteins/isolation & purification

The de novo design of integral membrane proteins remains a major challenge in protein chemistry. Here, we describe the bottom-up design of a genetically-encoded synthetic membrane protein comprising only four amino acids (L, S, G and W) in the transmembrane domains. This artificial sequence, which we call REAMP for recombinantly expressed artificial membrane protein, is a single chain of 133 residues arranged into four antiparallel membrane-spanning α-helices. REAMP was overexpressed in Escherichia coli and localized to the cytoplasmic membrane with the intended transmembrane topology. Recombinant REAMP could be extracted from the cell membrane in detergent micelles and was robust and stable in vitro, containing helical secondary structure consistent with the original design. Engineered mono- and bis-histidine residues in the membrane domain of REAMP were able to coordinate heme in vitro, in a manner reminiscent of natural b-type cytochromes. This binding shifted the electrochemical potential of the cofactor, producing a synthetic hemoprotein capable of nascent redox catalysis. These results show that a highly reduced set of amino acids is sufficient to mimic some key properties of natural proteins, and that cellular biosynthesis is a viable route for the production of minimal de novo membrane sequences.

Biochim Biophys Acta. 2008 Sep;1778(9):1814-38. (PMID: 17942072)
J Am Chem Soc. 2010 Nov 10;132(44):15516-8. (PMID: 20945900)
J Mol Biol. 1999 Nov 5;293(4):807-14. (PMID: 10543969)
Science. 1988 May 27;240(4856):1177-81. (PMID: 2453923)
Biochim Biophys Acta. 2016 May;1857(5):503-512. (PMID: 26672896)
Chem Rev. 2014 Apr 9;114(7):3495-578. (PMID: 24661096)
Sci Rep. 2015 Oct 14;5:15086. (PMID: 26462555)
Nat Struct Biol. 1994 Dec;1(12):858-62. (PMID: 7773774)
J Mol Biol. 2014 Jun 26;426(13):2529-38. (PMID: 24793448)
Proteins. 2010 Aug 1;78(10):2349-68. (PMID: 20544970)
Chem Sci. 2014 Feb 1;5(2):507-514. (PMID: 24634717)
J Biomol NMR. 1995 Nov;6(3):277-93. (PMID: 8520220)
Cell. 1990 Sep 21;62(6):1135-41. (PMID: 2119256)
EMBO J. 1986 Nov;5(11):3021-7. (PMID: 16453726)
Protein Sci. 2009 Jul;18(7):1388-400. (PMID: 19544578)
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2250-5. (PMID: 11226225)
BMC Struct Biol. 2011 Mar 03;11:13. (PMID: 21371326)
Nat Struct Mol Biol. 2016 Jun 7;23(6):475-80. (PMID: 27273630)
J Biomol NMR. 2001 May;20(1):71-5. (PMID: 11430757)
Nat Chem Biol. 2013 Dec;9(12):826-833. (PMID: 24121554)
Proc Natl Acad Sci U S A. 2006 Sep 12;103(37):13658-63. (PMID: 16954199)
J Comput Chem. 2004 Oct;25(13):1605-12. (PMID: 15264254)
Nature. 2007 Dec 13;450(7172):1026-30. (PMID: 18075582)
Nat Struct Biol. 2000 Feb;7(2):154-60. (PMID: 10655619)
Proc Natl Acad Sci U S A. 2011 Aug 2;108(31):E359-64. (PMID: 21606334)
Nat Methods. 2012 Jul;9(7):671-5. (PMID: 22930834)
EMBO Rep. 2006 Nov;7(11):1111-6. (PMID: 17008929)
Methods. 2008 Oct;46(2):62-72. (PMID: 18952172)
Structure. 2015 Mar 3;23(3):527-541. (PMID: 25703378)
J Mol Biol. 1998 Dec 11;284(4):1185-9. (PMID: 9837736)
Nat Protoc. 2007;2(6):1528-35. (PMID: 17571060)
Biochim Biophys Acta. 2016 May;1857(5):493-502. (PMID: 26556173)
Proc Natl Acad Sci U S A. 2015 Aug 25;112(34):E4717-25. (PMID: 26261320)
Nature. 2005 Jan 27;433(7024):377-81. (PMID: 15674282)
J Mol Biol. 2001 Nov 9;313(5):1171-9. (PMID: 11700072)
Protein Sci. 2004 Jul;13(7):1711-23. (PMID: 15215517)
Science. 2008 Jul 11;321(5886):243-6. (PMID: 18556516)
Biochim Biophys Acta. 2001 May 2;1512(1):1-14. (PMID: 11334619)
Nat Chem. 2010 Jan;2(1):15-24. (PMID: 21124375)
Proteins. 2009 Dec;77(4):778-95. (PMID: 19603484)
J Biol Inorg Chem. 2012 Apr;17(4):557-64. (PMID: 22307279)
J Am Chem Soc. 2007 Jan 24;129(3):512-8. (PMID: 17227013)
Chembiochem. 2014 Jun 16;15(9):1257-62. (PMID: 24829076)
Proc Natl Acad Sci U S A. 2004 Aug 31;101(35):12860-4. (PMID: 15322276)
Sci Rep. 2016 Mar 18;6:23397. (PMID: 26987712)
J Biol Chem. 1997 Mar 7;272(10):6119-27. (PMID: 9045622)
Biol Proced Online. 2009 May 15;11:32-51. (PMID: 19495910)
J Mol Biol. 2010 Mar 19;397(1):190-201. (PMID: 20109468)
J Mol Biol. 2016 Jan 29;428(2 Pt A):399-411. (PMID: 26707197)
Biochemistry. 2005 Sep 20;44(37):12329-43. (PMID: 16156646)
Science. 2018 Mar 2;359(6379):1042-1046. (PMID: 29496880)
Curr Opin Struct Biol. 2017 Aug;45:45-52. (PMID: 27894013)
Nat Prod Rep. 2007 Jun;24(3):621-30. (PMID: 17534534)
Proteins. 2005 Jun 1;59(4):687-96. (PMID: 15815974)
Science. 2014 Dec 19;346(6216):1520-4. (PMID: 25525248)
Biopolymers. 2008 May;89(5):392-400. (PMID: 17896349)
Proc Natl Acad Sci U S A. 2000 May 23;97(11):5796-801. (PMID: 10823938)
Nature. 2009 Aug 13;460(7257):855-62. (PMID: 19675646)
Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4127-32. (PMID: 18326626)

BB/L01386X/1 United Kingdom BB_ Biotechnology and Biological Sciences Research Council; BB/L01386X/1 International Biotechnology and Biological Sciences Research Council (BBSRC)

0 (Membrane Proteins)
0 (Recombinant Proteins)
42VZT0U6YR (Heme)

Date Created: 20181003 Date Completed: 20191023 Latest Revision: 20210109

20221213

PMC6167376

10.1038/s41598-018-31964-8

30275547