IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ weers-2011-2 in vitro In vitro crosslink cross-linking study crosslink weight identificat confirmation by molecular weight weight identificat Weers PM., Patel AB., Wan LC., Guigard E., Kay CM., Hafiane A., McPherson R., Marcel YL., Kiss RS. cross-linker: dimethyl suberimidate (DMS) mutation Ser36Ala was remapped to Ser60Ala, matching to the following authors' statement:"Interestingly, there have been very few reported mutations in this linker region: 34STOP (functional), A37T (variant-nonfunctional), G39E (variant-nonfunctional) (taken from the Human Gene Mutation Database; www.hgmd.cf.ac.uk) (19). We have recently identified a novel heterozygous mutation (S36A) in a 63-year-old male with HDL deficiency " robert.kiss@mcgill.ca Journal of Lipid Research 2011 Only protein-protein interactions imex curation imex curation weers-2011-1 in vitro In vitro comig non denat gel comigration in non denaturing gel electrophoresis comig non denat gel weight by comassie molecular weight estimation by coomasie staining weight by comassie Weers PM., Patel AB., Wan LC., Guigard E., Kay CM., Hafiane A., McPherson R., Marcel YL., Kiss RS. mutation Ser36Ala was remapped to Ser60Ala, matching to the following authors' statement:"Interestingly, there have been very few reported mutations in this linker region: 34STOP (functional), A37T (variant-nonfunctional), G39E (variant-nonfunctional) (taken from the Human Gene Mutation Database; www.hgmd.cf.ac.uk) (19). We have recently identified a novel heterozygous mutation (S36A) in a 63-year-old male with HDL deficiency " robert.kiss@mcgill.ca Journal of Lipid Research 2011 Only protein-protein interactions imex curation imex curation weers-2011-3 in vitro In vitro solution sedimentati cosedimentation in solution solution sedimentati predetermined predetermined participant predetermined Weers PM., Patel AB., Wan LC., Guigard E., Kay CM., Hafiane A., McPherson R., Marcel YL., Kiss RS. mutation Ser36Ala was remapped to Ser60Ala, matching to the following authors' statement:"Interestingly, there have been very few reported mutations in this linker region: 34STOP (functional), A37T (variant-nonfunctional), G39E (variant-nonfunctional) (taken from the Human Gene Mutation Database; www.hgmd.cf.ac.uk) (19). We have recently identified a novel heterozygous mutation (S36A) in a 63-year-old male with HDL deficiency " robert.kiss@mcgill.ca Journal of Lipid Research 2011 Only protein-protein interactions imex curation imex curation apoa1_human Apolipoprotein A-I APOA1 Apolipoprotein A1 protein protein human Homo sapiens Human MKAAVLTLAVLFLTGSQARHFWQQDEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ apo1_dimer-1 3604291 N/A 3604294 unspecified role unspecified role neutral component neutral component purified purified P02647:p.Ser60Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false human-plasma homo sapiens blood plasma plasma blood plasma Stoichiometry: 2.0 direct interaction direct interaction false false false Fig. 7B ApoA-I was then crosslinked with DMS and analyzed by SDS-PAGE. This revealed that the majority of the S36A protein was in a monomeric state. However, WT apoA-I showed multiple cross-linked species of dimer, tetramer, and the appearance of a prominent band in the top portion of the gel representing apoA-I octomers (Fig. 7B). apoa1-16 3604291 N/A 3604294 unspecified role unspecified role neutral component neutral component purified purified P02647:p.Ser60Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false human-plasma homo sapiens blood plasma plasma blood plasma Stoichiometry: 4.0 direct interaction direct interaction false false false Fig. 7B ApoA-I was then crosslinked with DMS and analyzed by SDS-PAGE. This revealed that the majority of the S36A protein was in a monomeric state. However, WT apoA-I showed multiple cross-linked species of dimer, tetramer, and the appearance of a prominent band in the top portion of the gel representing apoA-I octomers (Fig. 7B). apoa1-17 3604291 N/A 3604294 unspecified role unspecified role neutral component neutral component purified purified P02647:p.Ser60Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false human-plasma homo sapiens blood plasma plasma blood plasma Stoichiometry: 8.0 direct interaction direct interaction false false false Fig. 7B ApoA-I was then crosslinked with DMS and analyzed by SDS-PAGE. This revealed that the majority of the S36A protein was in a monomeric state. However, WT apoA-I showed multiple cross-linked species of dimer, tetramer, and the appearance of a prominent band in the top portion of the gel representing apoA-I octomers (Fig. 7B). apoa1-15 3604292 N/A 3604294 unspecified role unspecified role neutral component neutral component purified purified P02647:p.Ser60Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false human-plasma homo sapiens blood plasma plasma blood plasma Stoichiometry: 2.0 direct interaction direct interaction false false false Fig. 7A To gain insight into the oligomerization state of S36A apoA-I, the proteins were analyzed by nondenaturing-PAGE. The gel showed that WT apoA-I was present as multiple bands, indicative of protein-protein interactions resulting in the formation of dimers and tetramers (Fig. 7A). The migration pattern of an identical concentration of S36A apoA-I was distinctively different, showing a more homogeneous apoA-I population. apoa1-14 3604292 N/A 3604294 unspecified role unspecified role neutral component neutral component purified purified P02647:p.Ser60Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false human-plasma homo sapiens blood plasma plasma blood plasma Stoichiometry: 4.0 direct interaction direct interaction false false false Fig. 7A To gain insight into the oligomerization state of S36A apoA-I, the proteins were analyzed by nondenaturing-PAGE. The gel showed that WT apoA-I was present as multiple bands, indicative of protein-protein interactions resulting in the formation of dimers and tetramers (Fig. 7A). The migration pattern of an identical concentration of S36A apoA-I was distinctively different, showing a more homogeneous apoA-I population. apoa1-18 3604293 N/A 3604294 unspecified role unspecified role neutral component neutral component purified purified P02647:p.Ser60Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false human-plasma homo sapiens blood plasma plasma blood plasma Stoichiometry: 4.0 direct interaction direct interaction false false false Fig. 8 The modeling of both systems showed evidence of monomer-tetramer with the monomer being predominant, more so in the case of the S36A apoA-I. As well, the calculated association constant (Ka) values were 8.561 × 1014 M−1for WT apoA-I and 6.119 × 1014 M−1for S36A apoA-I (Fig. 8). The global molecular average results suggested that S36A apoA-I clearly had less aggregation than WT apoA-I.