Shape Determination of Proteins in Dilute Solutions

Small angle X-ray scattering (SAXS) is becoming extremely popular as a method for studying biological systems, and specifically for protein solutions. By envelope reconstruction of the studied protein, SAXS measurements enable determination of macromolecular shape conformation.

The collection of SAXS data was performed for the standard protein Lysozyme, using the Xenocs Nano-inXider. The radius of gyration (Rg) and the pair distance distribution function (PDDF) were determined from this data.

Measurements and Results

Measurements were done on 1.5, 3.0 and 5.0mg/ml concentration solutions with the Xenocs Capillary Flow Cell, using the buffer 40mM acetic acid 50mM NaCIpH 4.0.

Using the PRIMUS1 software, the structural parameter Rg results were taken. The obtained values for each concentration with a range of exposure times are given in Table 1. The values are consistent with the synchrotron data2 of Rg = 1.43nm. 10 min short exposure times are enough to determine these essential structural parameters.

Table 1. Radius of gyration of Lysozyme depending on the concentration and exposure time

c (mg/ml) Exposure time Guinier Rg (error) AutoRg1
1.5 mg/ml 10 min
30 min
1.36 nm (0.34)
1.41 nm (0.04)
3.0 mg/ml 10 min
30 min
1.43 nm (0.22)
1.40 nm (0.15)
5.0 mg/ml 10 min
30 min
1.38 nm (0.14)
1.39 nm (0.02)

The GNOM1 software was used to calculate the PPDF p(r). In Figure 1, it can be observed that the curves obtained from different concentrations overlap. Consistent data can thus be collected from low concentration measurements.

Pair-distance distribution function for c = 1.5, 3.0 and 5.0mg/ml. Exposure time = 30min.

Figure 1. Pair-distance distribution function for c = 1.5, 3.0 and 5.0mg/ml. Exposure time = 30min.

Figure 2 shows the comparison between two exposure times for 5mg/ml concentration. The curves almost overlap, which is an indication of how a short exposure time of 10min is enough to provide relevant data.

Pair-distance distribution function for c = 5mg/ml. Exposure times = 10 and 30min.

Figure 2. Pair-distance distribution function for c = 5mg/ml. Exposure times = 10 and 30min.

Conclusion

Xenocs' clean-beam technology3 is completely integrated in the Nano-inXider, enabling precise bio-macromolecular studies of highly diluted systems. In addition, the use of the Xenocs Low Noise Flow Cell brings down the container scattering to push further the limits of BioSAXS measurements in the lab.

References

  1. Petoukhov et al., J. Appl. Cryst., 2007,40, s223-s2282
  2. Svergun et al, J. Appl. Cryst., 2005, 38, 555–558
  3. Xeuss Technical Note – Demonstration of High Signal-to-Noise Ratio

This information has been sourced, reviewed and adapted from materials provided by Xenocs.

For more information on this source, please visit Xenocs.

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