Supporting Information

Fluorescence study of the effect of cholesterol on spectrin -aminophospholipid interactions

Madhurima Mitra1, Malay Patra2 and AbhijitChakrabarti3*

1Biophysics & Structural Genomics Division, Saha Institute of Nuclear Physics Kolkata-700064, 2Chemistry Department, University of Calcutta, Kolkata 700009 and 3Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics Kolkata 700064, India.

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Table-S1: Amino acid sequence of lipid binding site of erythroidspectrin.

Domain / Amino acid sequence
-spectrin N-4 / MTSATEFENV GNQPPYSRIN ARWDAPDDEL DNDNSSARLF ERSRIKALAD EREVVQKKTF TKWVNSHLAR VSCRITDLYK DLRDGRMLIK LLEVLSGEML PKPTKGKMRI HCLENVDKAL QFLKEQRVHL ENMGSHDIVD GNHRLVLGLI WTIILRFQIQ DIVVQTQEGR ETRSAKDALL LWCQMKTAGY PHVNVTNFTS SWKDGLAFNA LIHKHRPDLI DFDKLKDSNA RHNLEHAFNV AERQLGIIPL LDPEDVFTEN PDEKSIITYV VAFYHYFSKM KVLAVEGKRV GKVIDHAIET EKMIEKYSGL ASDLLTWIEQ TITVLNSRKF ANSLTGVQQQ LQAFSTYRTV EKPPKFQEKG NLEVLLFTIQ SRMRANNQKV YTPHDGKLVS DINRAWESLE EAEYRRELAL RNELIRQEKL EQLARRFDRK AAMRETWLSE NQRLVAQDNF GYDLAAVEAA KKKHEAIETD TAAYEERVRA LEDLAQELEK ENYHDQKRIT ARKDNILRLW SYLQELLQSR RQRLETTLAL QKLFQDMLHSIDWMDEIKAH LLSAEFGKHL LEVEDLLQKH KLMEADIAIQ GDKVKAITAA TLKFTEGKGY QPCDPQVIQD RISHLEQCFE ELSNMAAGRK AQLEQSKRLW KFFWEMDEAE SWIKEKEQIY SSLDYGKDLT SVLILQRKHK AFEDELRGLD AHLEQIFQEA HGMVARKQFG HPQIEARIKE VSAQWDQLKD LAAFCKKNLQ DAE
-spectrin 10-14 domain / LELQNFL QNCQELTLWI NDKLLTSQDV SYDEARNLHN KWLKHQAFVA ELASHEGWLE NIDAEGKQLM DEKPQFTALV SQKLEALHRL WDELQATTKE KTQHLSAARS SDLRLQTHAD LNKWISAMED QLRSDDPGKD LTSVNRMLAK LKRVEDQVNV RKEELGELFA QVPSMGEEGG DADLSIEKRF LDLLEPLGRR KKQLESSRAK LQISRDLEDE TLWVEERLPL HLQSSWDRLR EAAAGRLQRL RDANEAQQYY LDADEAEAWI GEQELYVISD EIPKDEEGAI VMLKRHLRQQ RAVEDYGRNI KQLASRAQGL LSAGHPEGEQ IIRLQGQVDK HYAGLKDVAE ERKRKLENMY HLFQLKRETD DLEQWISEKE LVASSPEMGQ DFDHVTLLRD KFRDFARETG AIGQERVDNV NAFIERLIDA GHSEAATIAE WKDGLNEMWA DLLELIDTRM QLLAAS
-spectrin 6-11 domain / RRRLLKE SLLLQKLYED SDDLKNWINKKKKLADDEDY KDIQNLKSRV QKQQVFEKEL AVNKTQLENI QKTGQEMIEG GHYASDNVTT RLSEVASLWE ELLEATKQKG TQLHEANQQL QFENNAEDLQ RWLEDVEWQV TSEDYGKGLA EVQNRLRKHG LLESAVAARQ DQVDILTDLA AYFEEIGHPD SKDIRARQES LVCRFEALKE PLATRKKKLL DLLHLQLICR DTEDEEAWIQ ETEPSATSTY LGKDLIASKK LLNRHRVILE FQQYLADLHE AETWIREKEP IVDNTNYGAD EEAAGALLKK HEAFLLDLNS FGDSMKALRN QANACQQQQA APVEGVAGEQ RVMALYDFQA RSPREVTMKK GDVLTLLSSI NKDWWKVEAA DHQGIVPAVY VRRLAHDEFP MLPQRRREEP GNITQRQEQI ENQYRSLLDR AEERRRRLLQ RYNEFLLAYE AGDMLEWIQE KKAENTGVEL DDVWELQKKF DEFQKDLNTN EPRLRDINKV ADDLLFEGLL TPEGAQIRQE LNSRWGSLQR LADEQRQLLG

Table-S2 :NPL and number of spectrin molecules associated with DMPC/DMPE phospholipid SUVs at 15C and 50C. The data in parenthesis are those at 50C.

Membranes / Stoichiometry (NPL) / Spectrin/SUV
DMPC (100%) / 321 (355) / 7 (6)
DMPC/Cholesterol
(80:20) / 665 (811) / 4 (3)
DMPC/DMPE
(75:25) +
0% Cholesterol / 994 (1299) / 3 (3)
DMPC/DMPE
(75:25) +
10% Cholesterol / 690 (1270) / 5 (3)
DMPC/DMPE
(75:25) +
20% Cholesterol / 764 (1172) / 3 (3)

Table-S3 : Fluorescence lifetime of erythroid and nonerythroidspectrin in absence and presence of phospholipid SUVs.(The data in parenthesis are those for non-erythroidspectrin)

System / α1(%) / 1(ns) / α2(%) / 2(ns) / α3(%) / 3(ns) / Mean lifetime(ns) / χ 2
Spectrin / 25.0
(27.0) / 1.24
(1.27) / 67.0
(67.0) / 4.08
(4.08) / 57.0
(57.0) / 0.22
(0.23) / 3.65
(3.62) / 1.08
(1.06)
100% DMPC / 30.1
(28.5) / 1.27
(1.15) / 65.14
(67.0) / 4.00
(3.58) / 4.76
(3.9) / 0.14
(0.18) / 3.65
(3.28) / 1.03
(1.01)
DMPC/Chol
(80:20) / 19.61
(21.35) / 1.10
(1.14) / 50.85
(66.35) / 3.66
(3.64) / 29.54
(3.64) / 0.016
(0.027) / 3.39
(3.41) / 1.02
(1.04)
DMPC/DMPE (75:25)+ 0% Cholesterol / 18.82
(21.04) / 1.094
(1.248) / 74.11
(42.78) / 3.702
(3.85) / 7.07
(36.18) / 0.06
(0.008) / 3.51
(3.49) / 0.99
(1.07)
DMPC/DMPE (75:25)+ 10% Cholesterol / 30.14
(16.1) / 1.307
(0.941) / 5.64
(54.8) / 0.189
(3.419) / 64.22
(29.11) / 3.846
(0.017) / 3.22
(3.22) / 1.03
(1.00)
DMPC/DMPE (75:25)+20% Cholesterol / 18.71
(19.61) / 1.165
(1.102) / 68.69
(50.85) / 3.684
(3.663) / 12.6
(29.54) / 0.096
(0.016) / 3.47
(3.39) / 1.01
(1.02)
100% DOPC / 20.94
(20.24) / 1.20
(1.19) / 56.33
(55.63) / 4.05
(4.03) / 22.73
(24.13) / 0.008
(0.007) / 3.76
(3.75) / 1.00
(0.99)
DOPC/Chol
(80:20) / 21.51
(22.2) / 1.14
(1.22) / 66.37
(10.56) / 3.64
(0.037) / 12.12
(67.24) / 0.027
(3.34) / 3.40
(3.58) / 1.04
(1.02)
DOPC/DOPE (75:25) + 0% Cholesterol / 14.71
(21.5) / 1.003
(1.068) / 80.15
(70.99) / 3.503
(3.845) / 5.14
(7.5) / 0.123
(0.059) / 3.37
(3.62) / 1.01
(1.00)
DOPC/DOPE (75:25) + 10% Cholesterol / 13.88
(24.53) / 1.017
(1.174) / 9.49
(65.68) / 0.055
(4.002) / 76.63
(9.79) / 3.705
(0.094) / 3.57
(3.71) / 0.98
(1.01)
DOPC/DOPE (75:25) / 17.82
(23.82) / 1.272
(1.223) / 70.73
(64.87) / 3.715
(4.018) / 11.45
(11.31) / 0.118
(0.085) / 3.50
(3.72) / 0.99
(1.05)

Figure-S1: SDS-Polyacrylamide Gel Electrophoresis of erythroidspectrin on 7.5% gel.