Table 3.1.
Hydrodynamic properties of empty- and NTS1f-nanodiscs.
| Nanodisc | DLS | SV | ||||
|---|---|---|---|---|---|---|
|
| ||||||
| Rh,1 (nm)a | s20,w (S), f/fob | M (kDa)b | % load, c (μM)c | Lipidd (εJ/ε280) | Rh (nm)e | |
| Empty-MSP1D1 | ||||||
| POPC | 4.7 | 2.88 ± 0.01 (1.25) | 158 ± 8 (127.696) | 91 (20) | 114 | 4.2 |
| POPC/POPG | 5.2 | 3.21± 0.01 (1.23) | 169 ± 8 (128.297) | 88 (17) | 139 | 3.9 |
| POPG | 5.1 ± 0.3 | 3.64 ± 0.02 (1.20) | 185 ± 12 (128.897) | 91 (20) | 102 | 3.6 |
| Empty H7-MSP1D1 | ||||||
| POPC | 5.4 | 3.29 ± 0.01 (1.20) | 160 ± 11 (133.194) | 87 (16) | 113 | 4.1 |
| POPC/POPG | 5.3 | 3.60 ± 0.01 (1.16) | 166 ± 12 (133.794) | 89 (20) | 107 | 3.9 |
| POPG | 5.4 | 4.05 ± 0.01 (1.08) | 169 ± 14 (134.394) | 87 (19) | 95 | 3.6 |
|
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| Average | 112 ± 15 | |||||
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|
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| NTS1f MSP1D1 | ||||||
| POPC | 6.2 ± 0.5 | 6.50 ± 0.02 (1.40) | 205 ± 8 (189.893) | 81 (3.7) | 63 | 5.4 |
| POPC/POPG | 6.4 ± 0.5 | 6.66 ± 0.02 (1.40) | 210 ± 8 (190.221) | 79 (3.6) | 105 | 5.3 |
| POPG | 6.6 ± 0.8 | 6.84 ± 0.02 (1.34) | 205 ± 6 (190.548) | 64 (2.7) | 52 | 5.2 |
|
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| Average | 73 ± 23 | |||||
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Data were analyzed in terms of two discrete species (empty-MSP1D1 nanodiscs) or a second moment cumulant (empty-H7-MSP1D1 nanodiscs, NTS1f-nanodiscs). The hydrodynamic radii (Rh,1) values were calculated from the averages of independent experiments (± SD) (empty-nanodiscs with MSP1D1: POPC, n=1; POPC/POPG, n=2; POPG, n=2; empty-nanodiscs with H7-MSP1D1: POPC, n=1; POPC/POPG, n=1; POPG, n=1; NTS1f-nanodiscs: POPC, n=3; POPC/POPG, n=2; POPG, n=2). In addition to monodisperse species, aggregates (Rh,2 > 100 nm) were found in empty-nanodisc MSP1D1 preparations.
Sedimentation coefficients (s20,w) and experimental molar masses (M) represent average values for the major species observed in the continuous c(s) distribution in a single experiment for each nanodisc preparation. The frictional ratios f/fo indicated in brackets represent an average of the best-fit values obtained from the absorbance and interference continuous c(s) distributions. Averages in s20,w and M were obtained from both the absorbance and interference data analyzed using a continuous c(s) distribution in SEDFIT, as well as various hybrid local continuous distributions and global discrete species models in SEDPHAT. Sedimentation coefficient corrections to s20,w are carried out using revised values of the partial specific volumes at 10.0 and 20.0°C, as are estimates of the molar mass. Molar masses in brackets are the calculated values based on the expected stoichiometries of 110:2 lipid:MSP1D1 and 60:2:1 lipid:MSP1D1:NTS1f for the empty and receptor nanodiscs, respectively.
Percent of the loading absorbance that represents the major species of interest, based on the best-fit continuous c(s) distribution observed with SEDFIT. The observed concentration c, based on the absorbance data, of the major nanodisc species is indicated in brackets.
Lipid stoichiometries per single nanodisc based on the presence of 2 MSP1D1 molecules, and in the case of the receptor-nanodiscs, 1 NTS1f. Stoichiometries are based on signal contributions of the major species to the absorbance (protein alone) and interference (protein and lipid) data.
Hydrodynamic radii based on the observed sedimentation coefficient, calculated molar masses and experimental partial specific volumes obtained from the sedimentation equilibrium data.