SUPPLEMENTAL DATA. Experimental Procedures

Transcription

1 SUPPLEMENTAL DATA Experimental Procedures For surface plasmon resonance (SPR) measurements, the setup was similar to a previous study (1). In brief, a gold surface (Au SIA-Kit, GE Healthcare) was carboxylated with 11-mercaptoundecanoic acid and subsequently functionalized via amine coupling (Amine coupling Kit, GE Healthcare) of Neutravidin (Sigma) in a Biacore2000 (GE Healtcare) instrument with 10 mm sodium acetate buffer, ph 4.7, as running buffer. Running Buffer was switched to a buffer identical to the liposomes (50 mm Tris/HCl ph 8.0, 50 mm NaCl) and liposomes were immobilized on the surface using a Biotin/DNA/cholesterol-linker (memlayer, Layerlab). 250 ul of 300 mm sugar alcohol solutions in 50 mm Tris/HCl ph 8.0, 50 mm NaCl were injected onto the liposome-chip to observe the release from the vesicles after switching back to running buffer at a flow rate of 100 ul/s and a data collection rate of 10 Hz. To estimate the magnitude of the mixing effect, a negative control using sucrose was used. The first 0.5 seconds of the traces were truncated to avoid observing the mixing effect. The remaining data were fitted to a single and double exponential function respectively to estimate rate constants using Kaleidagraph 3.6 (Synergy Software). Figures Fig. S1. Collision induced dissociation (CID) fragmentation spectra of A) the unmodified peptide TGGNLTVTGPKETASIF sequence annotated is the b-ion series. B) Fragment spectra of the peptide TGGN(Man 9 GlcNac 2 )LTVTGPKETASIF with a N-linked glycan at the asparagine. The main annotated ion series consist of the release of all mannose on the triply charged precursor ion resulting in the remaining peptide TGGN(GlcNac 2 )LTVTGPKETASIF, in the high m/z region of the spectra. The same release was observed on the double charged ion series. Green circle, mannose (Man); Blue square, N- acetylglucosamine (GlcNac). Fig. S2. Functionality and activity measurements of haqp10 containing proteoliposomes using surface plasmon resonance (SPR) measurements using biacore. Traces for proteoliposomes containing wild type haqp10 are marked in blue, haqp10-n133q in orange and control liposomes lacking protein in gray. The inserts represent a zoomed in version of the fast phase in the plot for A) Erythritol and B) Xylitol transport. Fig. S3. CD analysis of various haqp10 forms and the resulting derived melting curves. Traces for wild type haqp10 are marked in blue, haqp10-δglyc in green, haqp10-n133q in orange. To reduce the noise, the raw data (presented in Fig 4B) has been derived using the formula f (x)=(f(x+2h) f(x-2h))/4h resulting in a clear peak, corresponding to the melting temperature T m. A single lorentzian function was fitted to the data and shown as a dotted black curve.

2 Table S1. Protein Accession Pos Sequon Score Location haqp0 P NFT ++ Outside haqp1 P NQT + Outside 205 NFS + Outside haqp2 P NST + Outside haqp3 Q NGT + Outside haqp4 P NLT ++ Outside 206 NYT ++ TM-helix 283 NRS + Inside haqp5 P NNT + Outside 125 NTT + Outside haqp8 O NGT ++ TM-helix 85 NIS ++ TM-helix 139 NAS + Outside haqp9 O NAT + Outside haqp10 Q96PS8 75 NVS ++ Inside 128 NYT + Outside 133 NLT +++ Outside haqp12 Q8IXF9 5 NVS +++ Inside haqp6 Q13520 No sites predicted in this sequence haqp7 O14520 No sites predicted in this sequence haqp11 Q8NBQ7 No sites predicted in this sequence Prediction of all N-linked glycosylation sequons in human aquaporins. All protein sequences are from Uniprot ( and the accession number is given for easy reference. Positions (Pos), Sequon and Score are all prediction from (2). The score is the averaged output of nine neural networks and can range from + to ++++ for a glycosylation site. Locations are predictions from (3). Only sequons with a score of ++ or higher are considered to be of high specificity and only location outside is biologically reasonable. The score is based on the output of nine neural networks as described on the homepage for the prediction server.

3 Table S2. m/z Mass (Da) Score 1 Start End Miss Peptide sequence VFTQAPAEIMGH TQAPAEIMGHL ARQCLAEF AVTSGETKGNFF TSGETKGNFF VGGNVSGAHLNPAF AMCIVGRLPW HDALQNY TGGNLTVTGPKETASIF NKGVPAGLEPVVVGML NPARDLGPRLF WWVPVVAPL VALHHPEGPEPAQDL HHPEGPEPAQDL KASELETPASAQML All peptides identified by LC-MS/MS 1 Identification of peptides was accepted with a peptide score above The asparagine (underlined) was only found to be glycosylated in the high molecular weight band from the SDS-PAGE. The mass given is for the non-glycosylated form. The glycosylated fragment had a mass of 3557 Da giving a difference of 1.9 kda. A comparison between the nonglycosylated band shown in the table and the glycosylated is shown in Figure S1.

4 Table S3. Erythritol Transport Single exp Double exp Protein Rate const (s -1 ) R 2 Rate const (s -1 ) Rate const (s -1 ) R 2 Control Liposomes ± haqp ± ± ± haqp10-n133q ± ± ± Xylitol Transport Single exp Double exp Protein Rate const (s -1 ) R 2 Rate const (s -1 ) Rate const (s -1 ) R 2 Control Liposomes ± haqp ± ± ± haqp10-n133q ± ± ± Rate constants ± standard error for transport of erythritol and xylitol acquired through surface plasmon resonance (SPR) measurements using biacore. A single exponential curve was fitted to the slow phase (16s-100s) giving rate constants for the passive diffusion and a double exponential curve to the fast phase (insert) giving the constants for both the fast (first value) and the slow phase (second value) (see Fig. S3). The fast phase corresponds to facilitated transport through the protein and the slow phase is the passive diffusion and should be similar as the value for the single exponential fit. The goodness of the curve fit is given as the coefficient of determination, R 2.

5 Table S4. Aquaporins Protein Accession C-loop haqp0 P haqp1 P haqp2 P haqp4 P haqp5 P haqp6 Q haqp8 O Aquaglyceroporins Protein Accession C-loop haqp3 Q haqp7 O haqp9 O haqp10 Q96PS8 37 Bacterial aquaporins Protein Accession C-loop aqpz P glpf P0AER0 38 Length of loop C in number of amino acids based on predictions (41). Orthodox aquaporins, aquaglyceroporins and two family members from the bacteria E. coli are listed separately for clarity. Running a student s t-test with p<0.01 for the two groups containing human aquaporins, aquaporins and aquaglyceroporins, there is a significant difference in length where the glycerol transporting ones have a longer C-loop.

6 References 1. Branden, M., Tabaei, S. R., Fischer, G., Neutze, R., and Hook, F. (2010) Biophysical journal 99, CBS Prediction Servers. (2007) NetNGlyc 1.0 Server. 3. CBS Prediction Servers. (2009) TMHMM Server v. 2.0.

7 Figure S1.

8 Figure S2.

9 Figure S3.