Shape Determination of Proteins in Dilute Solutions

Table of Contents

Introduction
Measurements and Results
Conclusion

Introduction

Small angle X-ray scattering (SAXS) is gaining a lot of popularity these days as a method to study biological systems - protein solutions in particular. With the help of envelope reconstruction of the examined protein, the determination of macromolecular shape conformation can be done through SAXS measurements.

The collection of SAXS data was carried out for the standard protein Lysozyme, with the help of the Xenocs Nano-inXider. The pair distance distribution function (PDDF) and the radius of gyration (Rg) were evaluated from this data.

Measurements and Results

Solutions of concentration 1.5, 3.0 and 5.0 mg/ml were measured using the Xenocs Capillary Flow Cell, and the buffer 40 mM acetic acid 50 mM NaCI pH 4.0.

By using the PRIMUS1 software, the structural parameter Rg results were taken. The obtained values for each concentration with a range of exposure times are tabulated (Table 1). The values match with the synchrotron data2 of Rg = 1.43 nm. 10 minute short exposure times are sufficient for determining these important 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 PPDF p(r) was computed by the GNOM1 software. From Figure 1, it can be studied that the curves obtained from varied concentrations overlap. Hence, consistent data can be collected from low concentration measurements.

Pair-distance distribution function for c = 1.5, 3.0 and 5.0 mg/ml

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

In Figure 2, a comparison has been drawn between two exposure times for 5mg/ml concentration. The curves nearly overlap, which indicates that a very short exposure time of 10 minutes is enough to offer relevant data.

Pair-distance distribution function for c = 5 mg/ml

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

Conclusion

The clean-beam technology3 offered by Xenocs is fully integrated in the Nano-inXider, allowing precise bio-macromolecular studies of highly diluted systems. Furthermore, the use of the Xenocs Low Noise Flow Cell brings down the container scattering in order to exceed 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

Xenocs

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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