Lti30+DOPC

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l.respo

nse/RU

SupplementalFigure1.BindingofL330andK‐segmenttoDOPC.

BiacoreexperimentshowingtheK6dehydrinL330(10µM)bindingtoDOPC at pH 6.3 and the K‐segment (EKKGIMDKIKEKLPG) binding toDOPCataconcentra3onof60µMwhichgivesthesametotalnumberofK‐segmentinbothruns.

0.003

0.002

0.001

Cp(

cal/°

C)

Temperature (°C)

20 22 24 26 28

Lipids Cor47+Lipids (P:L ,1:100)

Lti30+Lipids (P:L ,1:100)

Lti30+Lipids (P:L ,1:30)

0.000

Supplemental Figure 2. Differential scanning calorimetry (DSC) data of Lti30 and the dehydrin Cor47 in the presence of DMPC:DMPG LUVs (3:1 molar ratio) at P:L, 1:100 (Lti30 and Cor47) and 30:1 (Lti30).

DMPC:DMPG (3:1, molar ratio) vesicles alone (black) and in the presence of Lti30 (blue) (L:P, 100:1 molar ratio) or at P:L, 30:1 (molar ratio, red). Lti30 at P:L, 1:100 reduces the phase transition temperature of the lipid vesicles by approximate 2.5°C and Tm is further decreased as Lti30 concentration is increased. In comparison, the non lipid binding dehydrin Cor47 have no response on the lipid phase transition temperature.

500 510 520 530

vesicles+ triton X+Lti30

Inte

nsity

Wavelength (nm)

4 1046 1048 1041 105

1.2 1051.4 105

SupplementalFigure3.L330andcalceinvesicleleakageexperiments.

POPC:POPG(100μM)(3:1molarra3o)calcein(55mM)containingvesiclesalone()oraTer120mininthepresenceofL330(0.06mg/ml)()andin0.1%tritonX(),showingnochangeinthesignalinducedbyL330.Thecalceinfluorescencewasrecordedat25°C.AnincreaseinL330concentra3onto0.12mg/mldidnotchangethesignal(datanotshown).

LUVswithentrappedcalceinwerepreparedasdesciribedinmaterialsandmethodswiththeexcep3onthatthedriedlipidfilmwasdissolvedin55mMcalceinpresentinthebuffer(finalpHwasadjustedto7.2byaddi3onofNaOH).SolventcalceinnotentrappedwasseparatedfromtheLUVsondesal3ngPD‐10columns(GEhealthcare).FluorescencewasmeasuredonaHoribaJobinYvonFluorolog‐3spectrometer.Emissionwasscannedfrom500‐550nmandexcita3onwassetto495nm.L330(0.06mg/ml)wasaddedtoLUVscomposedof100μMphospholipidmixturesofPOPC:POPG(3:1molarra3o)content.ATer120minincuba3onat25°C,thereleaseofcalceinfromtheLUVswasdeterminedfromtheincreaseinthefluorescenceintensity.Themaximumfluorescenceintensity(100%leakage)wasdeterminedbylysingthevesicleswitha10%(w/v)solu3onofthedetergentTritonX‐100.

SupplementalFigure4.TheK‐segments,withorwithoutflankinghis3dinesabilitytoaggregatelipidvesicles.

Lightmicroscopicpicturesofshortpep3desofA.theK‐segment(0.7µM)(EKKGIMDKIKEKLPG)andB.thehis3dineflankedK‐segment(0.7µM)(HHEKKGMTEKVMEKIKEQLPGHH)in1.4mMPOPC:POPG(3:1molarra3o)vesiclesatpH4.3showingthecontribu3onofprotonatedhis3dineflankstovesicleaggrega3on.C.Aggrega3onofLUVsinthepresenceofK‐segmentorthehis3dineflankedK‐segment(HIS‐K‐segment)measuredbyabsorbanceat400nmatpH4.3,inthepresenceof1.4mMPOPC:POPG(3:1molarra3o)vesicles.

A. K‐segment+LUVatpH4.3

50 µM

B. His3dineflanked‐K‐segment+LUVatpH4.3

50 µM

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Protein (mg/ml)

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K‐segment+LUV

HIS‐K‐segment+LUV

SupplementalFigure5.CDspectraofL330(blackline)andphosphorylatedL330(L330P,greyline),indica3ngnostructuralchangesuponphosphoryla3onofL330.

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Lti30Lti30P

[∫] (d

eg c

m2 d

ecim

ol-1

)

Wavelength (nm)

0

4 104

3 104

2 104

1 104

00,20,40,60,81

1,21,4

0 0,05 0,1 0,15 0,2 0,25

Lti30PLti30

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rban

ce Lti30 (mg/ml)

D

SupplementalFigure6.Titra3onofL330andphosphorylatedL330(L330P)into1.4mMDOPC:DOPGLUVs(3:1molarra3o)atpH7.2.Aggrega3onmeasuredbyabsorbanceat400nm.

SupplementalFigure7.AminoacidsequencesofthevesiclebindingmaizeDHN1(YSK2)andthenonvesiclebindingsoybeanrGMDHN1(Y2K)withKsegmentsinblueandflankinghis3dinesinred.

Maize Dhn1

MEYGQQGQRGHGATGHVDQYGNPVGGVEHGTGGMRHGTGTGGMGQLGEHGGAGMGGGQFQ PAREEHKTGGILHRSGSSSS SSSEDDGMGG RRKKGIKEKI KEKLPGGHKDDQHATATTGGAYGQ QGHTGSAYGQQGHTGGAYATGTEGTGEKKGIMDKIKEKLPGQH

Glycine max rGmDHN

MASYQKHYDD QGRKVDEYGN VERQTDEYGN PVHATSVTYV ATKSVGGND DANKQHDITG VYPEKDTGRH HFGRGYDGDT NKQHDATGVY PGIDIGRDGH TTGVYGLNTD RHHGSTGVNP GIDTHNQQHT TTGGYAGDTG RQHGNTGGLY YGTDTADTGA GPRSGNTGGT GYGGTGGTDY GTAGGTGYGS GTGYGINTGG AHTEAGYGKE HRQHEQSHGG QHEKKGILDK IKEKLPGGHS DK

Supplemental Figure 8. Light microscopic picture of DOPC:DOPG (1.4mM, 3:1 Molar ratio) LUVs at different pH showing no aggregation at any pH.

Light microscopic pictures of DOPC:DOPG (3:1 molar ratio) LUVs at A. pH 4.3, B. pH 6.3, C. pH 7.2 and D. pH 9.0, showing that the vesicles do not spontaneously aggregate in the absence of Lti30.

B. LUVs pH 6.3 A. LUVs pH 4.3

D. LUVs pH 9.0 C. LUVs pH 7.2

50µM

50µM

50µM

50µM

Supplementary Table 1. The 99 diferent K-segments of the dehydrins in manuscript Table 1, grouped according to the pattern of their His flanks. In groups G6-G18 , the His are separated from the K-segment by 1-3 linker residues, most frequently Gln or Gly. The K-segments from the vaccinum dehydrins AAB84258 and AAF34606 are all deviant by having His within their sequences (G12, G19 and G20). Only 9 % of the K-segments lack His flanks altogether (G21), but are accompanied by His-flanked K-segments in the full-length proteins (Supplementary Table 2)

G1. HH-EKKGMTEKVMEQLPG-HH HH-EKKGMTEKVMEQLPG-HH HH-EKKSVTEKVMEKLPG-HH HH-EKKGIAEKIKEQLPG-HH HH-ENKSTMDKIKEKLPG-GHH

G2. HH-EKKGVIGQVKDKLPG- HH-EKKGMMDKIKEKLPG-

G3. -QKKGITEKIMEKLPG-HH -EKKGAMDKIKEKLPG-HH -RKKGITQKIKEKLPG-HH -DKKGLVEKIKEKLPG-HH -EKKGLVEKIKEKLPG-HH

G4. H-EKKGIIGQVKDKLPG- H-EKKGLIEKIKDKLPG-

G5. -EKKGFMDKIKEKLPG-H -EKKGFMEKLKEKLPG-H

G6. HHH-QKEGLTEKIKENIPG-VGH

G7. -EKKGIMEKIKEKLPG-GH -EKKGLMEKIKDKLPG-GH -EKKGVMENIKDKLPG-GH -EKKSLMDKIKDKLPG-QH -EKKSLMENIKEKLPG-GH -GKKGVMENIKDKLPG-GH -EKKSLMDKIKDKLPG-QH -EKKSLMDKIKDKLPG-QH -EKKGIMENIKEKLPG-GH -EKKSLMDKVKEKLPG-QH -EKKGVVENIKEKLPG-GH -ENKSIMDKIKDKLPG-QH -EKKGVIENIKEKLPG-GH -ENKSIMDKIKDKLPG-QH -NKDSVLDKIKDKLPG-QH -KKGGVMDKIKDKLSG-QH -NKDSVLDKIKDKLPG-QH -KKDGFLEKIKDKIPG-LH -ERKGVGEKVKEKLPG-GH -EKKGIMDKIKEKLPG-QH -EKKGIVDKIKEKLPG-QH -EKKGIMDKIKEKLPG-QH -RKKGIKEKIKEKLPG-GH -EKKGIMDKIKEKLPG-GH -EKKGFLEKIKEKLPG-GH -EKKGLLGKIMDKLPG-YH -EKKGFLDKIKEKLPG-YH -EKKGILEKIKEKLPG-YH -EKKGFMDKIKEKLPG-YH -KKKGLKEKIKEKISG-DH -EKKGLMEKIKEKLPG-YH -EKKGILEKIKEKLPG-YH -EKKGILEKLKEKLPG-YH -EKKGIIEKIKEKLPG-YH

G8. HHY-QKKGVMEKNQEKIPG-LH HHD-NKGGVFGKIKEKLPG-QH

G9. HHHQEE-KKDSVLDKIKDKMSG-QH HHHQEE-KKGGVLDKIKDKLPG-QH

G10. -EKKGVMENIKEKLPG-GHGDH -EKKGVMENIKDKLPG-GHGDH -EKKGVMENIKEKLPG-GHSDH -EKKGIMEKIKEKLPG-GHGDH -EKKGVMENIKDKLPG-GHADH -EKKGVMENIKDKLPG-GHDDH -EKKGVMENIKDKLPG-GHDDH -EKKGIMENIKEKLPG-GHDDH -EKKGVMENIKEKLPG-GHGDH -EKKGVMENIKEKLPG-GHSDH

G11. HAG-EKKGIMEKIKEKLPG-GHGDH HGG-EKKGLMENIKDKLPG-GHGDH HGG-EKKAVMENIKDKLPG-GHGDH HAG-EKKGVMENIKDKLPG-GHQDH

G12. HQ-EKKGALDKIKDKLPG- HK-EGGGLIYKVKDKIHG- HQ-EKKGLLGQLQDKLPG- HQ-EKKGIIGQVKDKLPG-

G13. HHQEE-NKGGVLDKIKDKLPG-

G14. HED-KKDSVLDKIKDKLPG-QH HQD-KKDNILDKIKDKLPG-KH HPV-EKKGILEKIKEKLPG-YH

G15. -EKKGMMEKIKEKLPG-GGHH -EKKGFLEKIKEKLPG-GGH -EKKGFLEKIKEKLPG-GGH

G16. HEPE-EKKGFLDKIKEKLPG-H

G17. HPE-EKKGLMEKIKEKLPG-H

G18. HPE-EKKGFLNKIKEKLPG-

G19. -HNEGLADKVKDKIHG- -HNEGLADKVKDKIHG-

G20. -EGGGLMDKVKDTIHG- -EGGGLIYKVKDKIHG- -EGGGLMDKVKDKIHG- -EGGGLMDKVKDTIHG-

G21. -EKKGFLDKIKEKLPG- -EKKGVMDKIKEKLPG- -EGGGLMDKVKDKIPG- -EGGGLMDKVKDKIPG- -EGGWLMDKVKDKIPG- -QKKGLVEKIKEKIPG- -EKKGIIGQVKDKLPG- -EKKGIIDQVKDKLPG- -RKKGIKEKIKEKLPG- -ENQGVMDRIKEKFPL-

Supplementary Table 2. Composition of K-segments in the dehydrins in manuscript Table 1. The dehydrins are organized according to their accession number in the order they appear in manuscript Table 1. The most deviant proteins are AAB84258 and AAF34606 from Vaccinium, which contains K-segments with internal His.

AAF01693 -EKKGIMEKIKEKLPG-GH -EKKGLMEKIKDKLPG-GH -EKKGVMENIKDKLPG-GH -EKKGVMENIKDKLPG-GHDDH -EKKGVMENIKDKLPG-GHDDH -EKKGIMENIKEKLPG-GHDDH -EKKGVMENIKEKLPG-GHGDH -EKKGVMENIKEKLPG-GHSDH -EKKSLMDKIKDKLPG-QH

P46526 HAG-EKKGIMEKIKEKLPG-GHGDH -EKKGVMENIKDKLPG-GHADH -EKKSLMENIKEKLPG-GH HGG-EKKGLMENIKDKLPG-GHGH -GKKGVMENIKDKLPG-GH HGG-EKKAVMENIKDKLPG-GHGDH -EKKSLMDKIKDKLPG-QH

NP190666 -QKKGITEKIMEKLPG-HH HH-EKKGMTEKVMEQLPG-HH HH-EKKGMTEKVMEQLPG-HH HH-EKKSVTEKVMEKLPG-HH HH-EKKGIAEKIKEQLPG-HH HH-ENKSTMDKIKEKLPG-GHH

P46525 -EKKGIMEKIKEKLPG-GHGDH HAG-EKKGVMENIKDKLPG-GHQDH -EKKGVMENIKEKLPG-GHGDH -EKKGVMENIKDKLPG-GHGDH -EKKGVMENIKEKLPG-GHSDH -EKKSLMDKIKDKLPG-QH

AAB84258 -EGGGLMDKVKDKIPG- -EGGGLMDKVKDTIHG- -EGGGLMDKVKDKIPG- -EGGGLIYKVKDKIHG- -EGGGLMDKVKDKIHG-

AAF34606 -HNEGLADKVKDKIHG- -HNEGLADKVKDKIHG- HK-EGGGLIYKVKDKIHG- -EGGWLMDKVKDKIPG- -EGGGLMDKVKDTIHG-

CAM98306 -QKKGLVEKIKEKIPG- HHH-QKEGLTEKIKENIPG-VGH HHY-QKKGVMEKNQEKIPG-LH

AAB18203 -EKKGIMENIKEKLPG-GH -EKKSLMDKVKEKLPG-QH

AAC05923 -EKKGVVENIKEKLPG-GH -ENKSIMDKIKDKLPG-QH

AAB18204 -EKKGVIENIKEKLPG-GH -ENKSIMDKIKDKLPG-QH

AAB88628 HHD-ENKGGVFGKIKEKLPG-QH HQD-EKKDNILDKIKDKLPG-KH HQE-EKKGGALDKIKDKLPG

-ENKDSVLDKIKDKLPG-QH HHHQEE-EKKDSVLDKIKDKMSG-QH HHHQEE-EKKGGVLDKIKDKLPG-QH HHQEE-ENKGGVLDKIKDKLPG HED-KKKDSVLDKIKDKLPG-QH -EKKGGVMDKIKDKLSG-QH -ENKDSVLDKIKDKLPG-QH -EKKDGFLEKIKDKIPG-LH

CAC00637 HQ-EKKGLLGQLQDKLPG- HH-EKKGVIGQVKDKLPG- H-EKKGIIGQVKDKLPG- HQ-EKKGIIGQVKDKLPG- -EKKGIIGQVKDKLPG- -EKKGIIDQVKDKLPG- -ERKGVGEKVKEKLPG-GH H-EKKGLIEKIKDKLPG-

AAB03330 -EKKGIMDKIKEKLPG-QH

AAX96132 -EKKGIVDKIKEKLPG-QH -RKKGIKEKIKEKLPG-

CAA33364 -EKKGIMDKIKEKLPG-QH -RKKGIKEKIKEKLPG-GH

CAA48178 -RKKGITQKIKEKLPG-HH HH-EKKGMMDKIKEKLPG-

ABA93397 -EKKGMMEKIKEKLPG-GGHH

CAE47768 H-EKKGIMDKIKEKLPG-GH

P46524 -EKKGFLEKIKEKLPG-GH -EKKGLLGKIMDKLPG-YH

AAP44575 -EKKGFLEKIKEKLP-GGGH -EKKGFLDKIKEKLP-GYH

BAA23547 -EDKKGLVEKIKEKLP-GHH -EKKGLVEKIKEKLPG-HH -EKKGILEKIKEKLPG-YH

CAA62448 -ENQGVMDRIKEKFPL- -EKKGFMDKIKEKLPG-H -EKKGFMDKIKEKLPG-YH

AAP44575 -KKKGLKEKIKEKISG-DH -EKKGFLEKIKEKLPG-GGH -EKKGFLDKIKEKLPG-

AAY84736 -EKKGAMDKIKEKLPG-HH -EKKGVMDKIKEKLPG- -EKKGIIEKIKEKLPG-YH

ABD95986 HPE-EKKGLMEKIKEKLPG-H -EKKGLMEKIKEKLPG-YH

ABS12340 HEPE-EKKGFLDKIKEKLPG-H -EKKGILEKIKEKLPG-YH

AAN78125 HPE-EKKGFLNKIKEKLPG- -EKKGILEKLKEKLPG-YH

BAA04569 -EKKGFMEKLKEKLPG-H HPV-EKKGILEKIKEKLPG-YH