Secondary Structure Characterization of FlgM peptides
from Aquifex aeolicus
Ethan Paddock, April Rodriguez, and Matthew J. Gage, Ph.D.
Department of Chemistry and Biochemistry, Northern Arizona University, Flagstaff, AZ
Abstract
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Intrinsically disorder proteins (IDPs) are known to have a lack of
secondary and tertiary structure due to their low hydrophobic amino
acid content. The flexible structure of IDPs facilitates the wide range
of biological functions that they have been linked to and allows IDPs
to interact with multiple proteins. Our lab has been studying a model
IDP, FlgM, which functions as a negative regulator of flagella
synthesis by binding to the Sigma-28 transcription factor. A previous
study identified unbound FlgM derived from S. typhimurium to be
mostly unfolded in dilute solution conditions. In contrast, another
variant of FlgM from the thermophile A. aeolicus exhibits alpha helical
structure via CD at 20 °C, though this structure is lost as the
temperature increases to 85 °C. We have been studying the
structure and stability of peptides corresponding to various regions of
the A. aeolicus FlgM protein. A total of seven truncated fragments
corresponding to individual helical regions and combinations of
helices have been analyzed for their secondary structure
characteristics at 20 °C and 85 °. We also explored potential
transient structural in each peptide using 2,2,2-trofluoroethanol as a
stabilizing agent. Spectra were collected using a 0-50%
concentration gradient of TFE at 5% increments. All samples were
prepared using 50-100 μM of peptide, in 25-50 mM sodium
phosphate of pH 7.4. Our results suggest that the second and third
helices are the most stable helices and that these two helices provide
a core that the rest of the protein forms around.
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Figure 2: Far-UV CD spectra of H2 and H3 FlgM peptides Far-UV CD spectra of FlgM A. aeolicus of 50 μM H2 (Figure 2A) ran in 25 mM sodium phosphate, pH 7.4,
and shows alpha helical structure as the concentration of TFE 0-50% with 10% increments, starting from a random-coil configuration. Figure 2B Far-UV spectra shows
similar results for 50 μM H3 peptide in 25 mM sodium phosphate, pH 7.4; however, H3 peptide at 0% TFE concentration shows a prominent alpha helical structure
compared to H2 peptide.
Introduction
H4, H2H3, H3H4 20 C°Molar Ellipticity
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Intrinsically disordered proteins, or IDPs, belong to a special group of
proteins that do not require a defined secondary or tertiary structure to
function [2]. IDPs, such as FlgM, play important roles in the function of
biological life. FlgM is responsible for the regulation of length of the
flagellar hook [1]. Flagella function for the purpose of motion and are
also classified as a sensory organelle. Flagella are sensitive to
chemicals and fluctuation of temperatures outside of the functioning
cell [5]. FlgM functions as a negative regulator by binding to the RNA
transcription factor σ28 involved in expression of class 3 genes that are
involved in late-stage assembly after the basal body-hook is created
properly [4]. FlgM protein from the thermophile Aquifex aeolicus,
displays a more ordered confirmation at 20˚C though this structure is
lost as the temperature increases to 85 °C [3]. A total of seven
truncated fragments of FlgM peptides derived from A. aeolicus
corresponding to individual helical regions and combinations of helices
were analyzed for their secondary structure characteristics at 20 °C
and 85 °C. The results may provide insight of how the environment of
IDPs could affect their structure.
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Figure 3: Far-UV CD spectra of H4, H2H3, and H3H4 FlgM peptides Far-UV CD spectra of FlgM A. aeolicus of 50 μM H4 (Figure 3A) ran in 25 mM sodium
Figure 1: A rendering of FlgM IDP created through RaptorX software for
protein modeling and analysis. The 88 amino acid sequence of FlgM was
submitted and the 3D rendering was processed.
Conclusions
• Through data displayed in figure 3, H3 and H2H3
peptides have an increase in alpha helical secondary
structure signal as the TFE increased.
• H2 at 0% TFE, began with disordered random coil
structure.
• When the TFE concentration reached 25%, the H2
peptide transitioned to an alpha helical structure
(Figure 2)
phosphate, pH 7.4, and shows characteristics a beta-sheet structure. However, when a two peptide complex of 50 μM H2H3 (Figure 3B) in 25 mM sodium phosphate pH
7.4 Far-UV spectra shows an alpha helical structure when H2 and H4 are paired together. Figure 3C shows H3H4 FlgM peptide Far-UV CD spectra that indicates a
stabilization of a random coil structured to a alpha helical structure by 0-50% TFE gradient. Figure 3D is a rendered 3-dimensional predicted ribbon structure of the H2H3
peptide which was calculated by RaptorX program.
Future Directions
•H1 and H1H2 still need to be ran through the CD
and need to be analyzed
•All 7 truncated variations of FlgM will be further
studied at the temperature of 85 ˚C. This will help
substantiate the effects temperature has on
secondary and tertiary structure of IDPs
Acknowledgments
The authors would like to thank the NACP and
IMSD program for funding and other members of
the Gage lab for their support.
References
[1] Courtney CR, Cozy LM, Kearns DB (2012) Molecular characterization of the flagellar hook in Bacillus subtilis. J Bacteriol
194: 4619-4629.10.1128/JB.00444-12 PubMed: 22730131.
[2] Ma, WK, R Hendrix, C Stewart, EV Campbell, M Lavarias, K Morris, S Nichol, and MJ Gage . "FlgM proteins from
different bacteria exhibit different structural characteristics ." Biochim Biophys Acta.. 1834.4 (2013): 808-16. Web.
17 Jan.2014.
[3] Molloy, R. G., Ma, W. K., Allen, A. C., Greenwood, K., Bryan, L., Sacora, R., Williams, L., &
Gage, M. J. (2010). Aquifex aeolicus FlgM protein exhibits a temperature-dependent
disordered nature. Biochimica et Biophysica Acta, 1804, 1457–1466.doi:10.1016/j.bbapap.2010.03.002
[4] P. Tompa, Intrinsically unstructured proteins, Trends Biochem. Sci. 27 (2002) 527-533
[5] Wang, Qingfeng; Suzuki, Asaka; Mariconda, Susana; Porwollik, Steffen; Harshey, Rasika M
(2005). "Sensing wetness: A new role for the bacterial flagellum". The EMBO Journal 24
(11): 2034–42. doi:10.1038