Seminar on Graduate Institute of Food Science & Biotechnology
National Chung Hsing University (2015 S/S)
Title: The Statistical Conformation
of a Highly Flexible Protein: Small-Angle X-Ray Scattering of S.
aureus Protein A
Author: Jo A. Capp, Andrew Hagarman, David C. Richardson and Terrence G. Oas
Journal: Structure (Impact factor: 6.794)
Speaker: 鄭慎禕
Moderator: 郭雅軒
Advisor: 葉娟美 教授
Date: 2015/5/22 (7th)
I.
Introduction:
Staphylococcus protein A (SpA) functions as a crucial S. aureus virulence factor through a wide
array of intermolecular interactions. It has been shown to bind to the Fc fragment of antibodies to
inhibit host immune response. A description of SpA’s structural flexibility will facilitate a better
understanding of the role this property plays in the protein’s diverse functions. Within the definition
of flexibility, there are two extremes of conformational flexibility to consider. SpA is a multi-domain
protein consisting of an N-terminal domain containing a signal sequence and five IgG binding domains,
and a C-terminal region used to target the protein to the cell surface. The N-terminal half of this
protein (SpA-N) interacts with the host-cell proteins, mediating the immune response. The five IgG
binding domains in the N-terminal half are the functional portion of the protein and have a high
degree of sequence identity. It is unknown how the individual domains are structurally related to each
other and how the presence of repeated domains structurally constrains the protein. It is these
structural constraints that constrain the flexibility of the statistical conformation and affect the
thermodynamics of the statistical conformation. So, by determining the statistical conformation allow
to gain important insights into how the statistical conformation contributes to the function of SpA-N.
In this study, small-angle X-ray scattering (SAXS) can determine and describe the statistical
conformation of SpA-N.
II. Experimental Procedures
1. Plasmid constructs were transformed into BL21(DE3)
2. A single colony was inoculated to 1L LB media (containing 100mg Ampicillin)
3. The cells were induced by IPTG until an OD600 of 0.8~1.0 was reached
4. The cells were harvested 4~6hr post-induction and centrifuged
5. The cell pellet was suspended in Tris-HCl buffer, EDTA and protease inhibitors.
6. The cell lysed and insoluble material was cleared by centrifuged
7. The clear lysate was brought to GdmCl and TCEP
8. The protein solution was loaded on to SP sepharose column
9. The most pure solution was loaded onto DEAE column
10. The most pure fractions were pooled and dialyzed into deionized water. The protein was then
lyophilized and stored in a desiccator.
III. Results
1. SAXS data were obtained from five B domain protein fragments (BdpA, 2-BdpA, 3-BdpA,
4-BdpA, 5-BdpA) and the N-terminal region of SpA (SpA-N).
2. A Guinier analysis of the scattering data allows for direct estimation of the radius of gyration
(Rg) of each protein construct.
3. The Guinier plot is an algebraic transformation of the data that produce a linear q2
dependence in the Guinier region.
4. Excellent linear correlations within the Guinier regions are observed for 2-BdpA, 3-BdpA,
4-BdpA, 5-BdpA and SpA-N data.
5. The SpA-N Rg is 6.8% smaller than that of 5-BdpA, indicating that SpA-N is more compact than
5-BdpA, even though the two molecules have nearly identical molecular weights (1.8%
difference)
6. This difference may be the result of inter-domain interactions that are more favorable in
SpA-N or more unfavorable in 5-BdpA.
7. The systematic increase in Rg among the n-BdpA protein fragment suggests that the
relationship between monomer number and Rg can fit to a polymer model.
8. The statistical conformation of 5-BdpA can be described as excluded volume pearl necklace
polymer model.
IV. Discussion
1. The present SAXS data strongly support the conclusion that SpA-N and n-BdpA are highly
flexible chains of inflexible domains that lack any significant favorable inter-domain
interaction.
2. SpA-N was not described as a structure rather than the statistical conformation of domains
according to end-to-end distance distribution, persistence length, Flory coefficient and radius
of the identical spheres.
3. Its sequence has evolved to perform a wide array of functions that confer virulence to S. aureus
including binding to both Fc and Fab fragments of antibodies.
4. The statistical conformation includes a large ensemble with a variety of inter-domain
orientations.
V. Conclusion
Using SAXS to determine the statistical conformation of a highly flexible protein or domain is
more specific.
VI. Reference
1. Bernado ́ , P., and Svergun, D.I. (2012a). Analysis of intrinsically disordered proteins by
small-angle X-ray scattering. Methods Mol. Biol. 896, 107–12
2. Bernado ́ , P., and Svergun, D.I. (2012b). Structural analysis of intrinsically disordered
proteins by small-angle X-ray scattering. Mol. Biosyst. 8, 151–167.
3. Graille, M., Stura, E.A., Corper, A.L., Sutton, B.J., Taussig, M.J., Charbonnier, J.B., and Silverman,
G.J. (2000). Crystal structure of a Staphylococcus aureus protein A domain complexed with
the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell
receptors and superantigen activity. Proc. Natl. Acad. Sci. USA 97, 5399–5404.
4. Teilum, K., Olsen, J.G., and Kragelund, B.B. (2009). Functional aspects of protein flexibility.
Cell. Mol. Life Sci. 66, 2231–2247.
5. Flory, P.J. (1953). Principles of polymer chemistry. (Ithaca: Cornell University Press).
6. Chi, Q., Wang, G., and Jiang, J. (2013). The persistence length and length per base of
single-stranded DNA obtained from fluorescence correlation spectroscopy measurements
using mean field theory. Physica A 392, 1072–1079.