Journal: bioRxiv

Article Title: Design principles of the common Gly-X6-Gly membrane protein building block

doi: 10.1101/2025.06.25.661420

Figure Lengend Snippet: ( a ) N-terminal SUMO fusion constructs for synthetic G-X 6 -G TM domains aligned by heptad position. Glycines red, bolded. Interface positions, bold. TM domain length listed right. Left, cartoon of SUMO (purple) and TM domain (red). ( b ) Size exclusion chromatography (SEC) trace of absorbance 280 nm (normalized to peak max) by column volume of 2 mg/mL SUMO-TM Design-2 (representative of n=3) on superdex200i 10/300 column, mobile phase: 50 mM Na Phosphate pH 7.8, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, and 1 mM DDM. Red spheres, assigned oligomer. Orange box is expected elution range for a dimer. ( c ) SEC of SUMO-Design-2 (representative of n=3) in 1 mM DDM, major peak falls into dimer range ( d ) SEC of SUMO-Design-3a (representative of n=3) in 1 mM DDM, major peak falls into dimer range ( e ) Left, SDS-PAGE of 1 mg/mL Design-2 in DDM at increasing loading volumes in Tris-Glycine-SDS or the point mutant with central Gly mutated to Phe (G16ΔF). Middle, Design-1a gel. Right, Design-2 after mixing with 4% w/v lithium dodecyl sulfate (LDS) or 0.2% SDS and heating to 95 ° C for 30 minutes. ( f ) SEC of SUMO-Design-4 incubated in 30 mM C14B or 40 mM DDM and run in mobile phase of 3 mM C14B or 1 mM DDM. C14B-incubated protein in C14B mobile phase results in a likely trimer (11.8 mL). DDM-incubated protein in DDM mobile phase results in a dimer. C14B-incubated protein in DDM mobile phase results in trimer-dimer mixture.

Article Snippet: Purified proteins were concentrated to 2-3 mg/mL and injected to a Superdex200i 10/300 column (Cytiva) at room temperature using a mobile phase of 25 mM NaPi at pH 8, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA and detergent (DDM, Anatrace; C14B; LDAO, Cayman).

Techniques: Construct, Size-exclusion Chromatography, SDS Page, Mutagenesis, Incubation

Journal: bioRxiv

Article Title: Design principles of the common Gly-X6-Gly membrane protein building block

doi: 10.1101/2025.06.25.661420

Figure Lengend Snippet: ( a ) Design model of de novo A-X 6 -A TM domain forming antiparallel fold (white), and overlayed with ESMfold structure prediction (purple, 1.5 Å Cα RMSD). Motif alanines, orange sticks. ( b ) Designed A-X6-A sequence. Motif Ala residues underlined in red, aligned with 7-residue heptad repeat. ( c ) Thiol-disulfide equilibrium exchange LC-MS UV maxplot chromatogram of A-X 6 -A nCys and cCys peptide mixture reconstituted in 10 mM LDAO, with major peaks nCys monomer and N-C heterodimer annotated by cartoon as in , representative of n=3 trials. ( d ) Size exclusion chromatography (SEC) trace of absorbance 280 nm (normalized to peak max) by column volume of 2 mg/mL SUMO-TM A-X 6 -A design (representative of n=3) on superdex200i 10/300 column, mobile phase: 50 mM Na Phosphate pH 7.8, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, and detergent (6 mM LDAO, 3 mM C14B, and 1 mM DDM). Red spheres denote monomer or dimer assignment of peak. Orange box is expected elution range for a dimer in that detergent based on known TM domain reference proteins.

Article Snippet: Purified proteins were concentrated to 2-3 mg/mL and injected to a Superdex200i 10/300 column (Cytiva) at room temperature using a mobile phase of 25 mM NaPi at pH 8, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA and detergent (DDM, Anatrace; C14B; LDAO, Cayman).

Techniques: Sequencing, Residue, Liquid Chromatography with Mass Spectroscopy, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Design principles of the common Gly-X6-Gly membrane protein building block

doi: 10.1101/2025.06.25.661420

Figure Lengend Snippet: (a) Design-1a and 1b in LDAO (n=3) or DDM (n=2) mobile phases, respectively . (b) Reference TM domain trimer, MS3 in LDAO (n=1). (c) Reference TM domain trimer, MS3, and two dimers, MS1-GX6G and GpA, in C14B, representative of n=3 experiments. (d) Design-2 in DDM and LDAO (each n=3), including in LDAO with 15-minute pre-incubation at 85° C (n=1). (e) Design-3a in C14B and LDAO (each n=3), including in LDAO with 15-minute pre-incubation at 85° C (n=1). (f) Left, Design-3b in DDM and C14B (each n=3). Right, Design-3c in C14B elutes as two peaks (n=1). Chromatograms are 280 nm absorbance (normalized to peak max) from 2-3 mg/ml protein in superdex200i 10/300 column, mobile phase: 50 mM Na Phosphate pH 7.8, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, and detergent (6 mM LDAO, 1 mM DDM, or 3 mM C14B). Red spheres, assigned oligomer.

Article Snippet: Purified proteins were concentrated to 2-3 mg/mL and injected to a Superdex200i 10/300 column (Cytiva) at room temperature using a mobile phase of 25 mM NaPi at pH 8, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA and detergent (DDM, Anatrace; C14B; LDAO, Cayman).

Techniques: Incubation

Journal: bioRxiv

Article Title: Design principles of the common Gly-X6-Gly membrane protein building block

doi: 10.1101/2025.06.25.661420

Figure Lengend Snippet: ( a ) Design-2 at 2 mg/mL after 30-minute incubation at 90° C run in a superdex200i 10/300 column at 25° C, mobile phase: 50 mM Na Phosphate pH 7.8, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, and 1 mM DDM. Chromatograms is 280 nm absorbance (normalized to peak max), representative of n=3 trials. Single and double red circles denote monomer (∼15.5 mL) and dimer (12.8 mL) peaks. Orange rectangle denotes expected dimer range for a single-span SUMO-TM fusion in DDM. ( b ) Purified SUMO fusion TM domain constructs for Designs 1a, 1b, 3a, 3b, and 3c in NuPage Bis-Tris gel ran with MES-SDS running buffer, representative of n=3 trials. ( c ) Purified SUMO fusion TM domain constructs for the A-X 6 -A design (left), mutant G-X 6 -G Design-2 GΔF (middle), and G-X 6 -G Design-3a in BioRad anyKd gel ran with Tris-Glycine-SDS running buffer. All proteins run as predominantly monomers under these conditions, representative of n=3 trials.

Article Snippet: Purified proteins were concentrated to 2-3 mg/mL and injected to a Superdex200i 10/300 column (Cytiva) at room temperature using a mobile phase of 25 mM NaPi at pH 8, 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA and detergent (DDM, Anatrace; C14B; LDAO, Cayman).

Techniques: Incubation, Purification, Construct, Mutagenesis