SASDRP5 – SEC-SAXS analysis of recombinant survival motor neuron protein (flSMN) conjugated to the protein transduction domain of HIV-1 Tat protein (Tat-flSMN)
Synchrotron SAXS data from solutions Tat-flSMN protein fusion in 50 mM Tris-HCl, 500 mM NaCl, pH 8 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.9 m and at a wavelength of λ = 0.09918 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 50.00 μl sample at 3 mg/ml was injected at a 0.07 ml/min flow rate onto a GE Superdex 75 Increase 3.2/300 column at 20°C, connected to an online Nexera HPLC system (Shimadzu). SAXS data were recorded as a set of 1 second data frames throughout the SEC elution, where 54 successive 1 second frames were collected through the respective sample elution peak. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.
Recombinant TAT-flSMN was concentrated using VIVACON centrifugal filters (3 kDa cutoff, Sartorius), reaching a final concentration of 3 mg/mL immediately prior to SEC-injection. The SEC-SAXS data were processed with CHROMIXS for the evaluation of peak regions and buffer subtraction. Kratky analysis, extrapolation of radii of gyration and molecular masses were performed using PRIMUS.
s, nm-1
