I.C. Gunsalus Professor
Dept of Biochemistry
Dept of Chemistry
College of Medicine
Center for Biophysics and Computational Biology
Director, School of Molecular and Cellular Biology
| Sligar Lab | Stephen G. Sligar I.C. Gunsalus Professor Dept of Biochemistry Dept of Chemistry College of Medicine Center for Biophysics and Computational Biology Director, School of Molecular and Cellular Biology |
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In this document we compile the protein and nucleic acid sequences for some of the various membrane scaffold protein (MSP) constructs we have made. Note that currently we primarily use two versions, MSP1D1 (earlier called MSP1T2) and MSP1E3D1 and are the only ones for which we have stockpiled protein. The original MSP1 series generates the ~9.7 nm diameter Nanodiscs and had a Factor X protease site. However Factor X also cleaves MSP non-specifically which makes it difficult to remove the His-tag easily. The current versions of scaffold proteins have a TEV protease site (which was engineered by us to give complete and specific cleavage) and also has an additional Δ(1-11) truncation from MSP1 to give more stable discs, since we showed that not all of the first helix was involved in lipid binding (JACS 126, 3477). To make larger discs we most often use the MSP1E3D1 construct which has an additional three helices (Helix 4, 5, and 6 – see below) inserted into the sequence between Helix 3 and Helix 4 which generates the ~12.1 nm bilayer discs. For our most recent constructs, we have used gene synthesis from Integrated DNA Technologies. Note that all sequences have been published, often in the supplementary material. See for instance JACS 126, 3477 and NanoLetters 2, 853. Our laboratory web site provides a lists all our Nanodisc and Membrane Scaffold Protein related publications.
MSP1D1 (MSP1T2):
Protein:
GHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEM
SKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQE
KLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEY
HAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQDNA:
ACCATGGGTCATCATCATCATCATCATCACGATTATGATATTCCTACTACTGA
GAATTTGTATTTTCAGGGTTCTACCTTCAGTAAACTTCGCGAACAACTGGGCC
CCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGC
GTCAGGAAATGTCCAAAGATTTAGAAGAGGTGAAGGCCAAGGTTCAGCCATA
TCTCGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAA
AAGGTGGAACCGCTGCGTGCGGAACTGCAAGAGGGGGCACGCCAAAAACTC
CATGAGCTCCAAGAGAAGCTCAGCCCATTAGGCGAAGAAATGCGCGATCGCG
CCCGTGCACATGTTGATGCACTCCGGACTCATTTGGCGCCGTATTCGGATGAA
CTTCGCCAGCGTTTGGCCGCACGTCTCGAGGCGCTGAAAGAAAACGGGGGTG
CCCGCTTGGCTGAGTACCACGCGAAAGCgACAGAACACCTGAGCACCTTGAG
CGAAAAAGCGAAACCGGCGCTGGAAGATCTACGCCAGGGCTTATTGCCTGTT
CTTGAGAGCTTTAAAGTCAGTTTTCTGTCAGCTCTGGAAGAATATACTAAAAA
GCTGAATACCCAGTAAGCTTMSP1E3D1:
Protein:
MGHHHHHHHDYDIPTTENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQE
MSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHEL
QEKLSPLGEEMRDRARAHVDALRTHLAPYLDDFQKKWQEEMELYRQKVEPLRA
ELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAAR
LEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSA
LEEYTKKLNTQDNA:
ACCATGGGTCATCATCATCATcATCATCACGATTATGATATTCCTACTACTGA
GAATTTGTATTTTCAGGGTTCTACCTTCAGTAAACTTCGCGAACAACTGGGCC
CCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGC
GTCAGGAAATGTCCAAAGATTTAGAAGAGGTGAAGGCCAAGGTTCAGCCATA
TCTCGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAA
AAGGTGGAACCGCTGCGTGCGGAACTGCAAGAGGGGGCACGCCAAAAACTC
CATGAGCTCCAAGAGAAGCTCAGCCCATTAGGCGAAGAAATGCGCGATCGCG
CCCGTGCACATGTTGATGCACTCCGGACTCATTTGGCGCCATATCTCGATGAC
TTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAAAAGGTGGAA
CCGCTGCGTGCGGAACTGCAAGAGGGGGCACGCCAAAAACTCCATGAGCTCC
AAGAGAAGCTCAGCCCATTAGGCGAAGAAATGCGCGATCGCGCCCGTGCAC
ATGTTGATGCACTCCGGACTCATTTGGCGCCGTATTCGGATGAACTTCGCCAG
CGTTTGGCCGCACGTCTCGAGGCGCTGAAAGAAAACGGGGGTGCCCGCTTGG
CTGAGTACCACGCGAAAGCGACAGAACACCTGAGCACCTTGAGCGAAAAAG
CGAAACCGGCGCTGGAAGATCTACGCCAGGGCTTATTGCCTGTTCTTGAGAG
CTTTAAAGTCAGTTTTCTGTCAGCTCTGGAAGAATATACTAAAAAGCTGAATA
CCCAGTAAGCTTHuman Apo-AI:
For reference we are including the sequence for Human ApoA1 using the assignment of helices from C. G. Brouillette et al., BBA 1531 (2001) 4-46.
MODULAR PICTURE OF MEMBRANE SCAFFOLD PROTEINS
It may be useful to have a modular picture of the construction of various membrane scaffold proteins. To that end, consider the following schematic:
Building blocks: GLOB DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLN HisX MGHHHHHHIEGR HisTEV MGHHHHHHHDYDIPTTENLYFQG Helix 1 (H1): LKLLDNWDSVTSTFSKLREQLG Helix 2 (H2): PVTQEFWDNLEKETEGLRQEMS Helix 3 (H3): KDLEEVKAKVQ Helix 4 (H4): PYLDDFQKKWQEEMELYRQKVE Helix 5 (H5): PLRAELQEGARQKLHELQEKLS Helix 6 (H6): PLGEEMRDRARAHVDALRTHLA Helix 7 (H7): PYSDELRQRLAARLEALKENGG Helix 8 (H8): ARLAEYHAKATEHLSTLSEKAK Helix 9 (H9): PALEDLRQGLL Helix 10(H10): PVLESFKVSFLSALEEYTKKLNTQ Truncated Helices: Helix 0.5 (H0.5): STFSKLREQLG Helix 10.5(H10.5): SALEEYTKKLNTQ Helix 2S (H2): PVTQEFWDNLEKETEGLRQEMS Then: Apo A-I GLOB-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 MSP1D1 HisTev-H0.5 -H2-H3-H4-H5-H6-H7-H8-H9-H10 MSP1E3D1 HisTev-H0.5-H2-H3-H4-H5-H6-H4-H5-H6-H7-H8-H9-H10 Our roughly 25 other constructs can be described easily in these terms.
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