Related Experiment Videos
Circular dichroism and molecular modeling of the E. coli TolA periplasmic domains
R Derouiche1, R Lloubès, S Sasso
1Laboratoire d'Ingénierie des Systèmes Macromoléculaires, CNRS UPR 9027, Institut de Biologie Structurale et Microbiologie, Marseille, France.
Abstract:
Colicins are killer proteins that use envelope proteins from the outer and the inner membranes to reach their cellular target in susceptible cells of Escherichia coli. Each group A colicin uses a combination of Tol proteins to cross the outer membrane of gram-negative bacteria and to exert their killing activity. The TolA protein, necessary for the import of all the group A colicins, is a 421-amino acid residue protein composed of three domains (TolAI, TolAII, and TolAIII). TolAIII interacts with the N-terminal domain of colicin A (AT1). Analytical ultracentrifugation reveals that TolAII and TolAIII are monomer structures, TolAII has an elongated structure, and TolAIII is rather globular. Circular dichroism (CD) spectra were done with TolAII-III, TolAII, TolAIII, AT1, and the AT1-TolAII-III complex. TolA CD spectra reveal the presence of alpha-helix structure in aqueous solution and the intensity of the a-helix signal is the highest with TolAII. Few structural changes are observed with the complex AT1-TolAII-III. Molecular modeling was done for TolAII-III, taking into account CD and ultracentrifugation data and show that domain II can adopt a barrel structure made of three twisted alpha-helices similar to coiled coil helices while domain III can adopt a globular structure.
Insights
Group A colicins utilize Tol proteins to penetrate Escherichia coli outer membranes. Structural analysis of TolA domains reveals alpha-helical structures, with TolAII being highly helical and TolAIII globular.
Area of Science:
- Bacteriology
- Structural Biology
- Molecular Biology
Background:
- Colicins are bacteriocins produced by bacteria, acting as toxic proteins to kill related strains.
- Group A colicins employ the Tol pathway, involving Tol proteins, to translocate across the Gram-negative bacterial envelope.
- The TolA protein is crucial for group A colicin import, consisting of three domains: TolAI, TolAII, and TolAIII.
Purpose of the Study:
- To investigate the structural properties of TolA domains (TolAII and TolAIII) and their interaction with colicin A (AT1).
- To elucidate the secondary structure and conformational changes of TolA domains upon complex formation.
Main Methods:
- Analytical ultracentrifugation was used to determine the quaternary structure and shape of TolA domains.
- Circular dichroism (CD) spectroscopy was employed to analyze the secondary structure of TolA domains and their complex with AT1.
- Molecular modeling was performed to predict the structural arrangement of TolAII-III based on experimental data.
Main Results:
- Analytical ultracentrifugation indicated that TolAII and TolAIII exist as monomers, with TolAII being elongated and TolAIII globular.
- CD spectra revealed significant alpha-helical content in TolAII and TolAIII in solution, with TolAII exhibiting the strongest signal.
- Complex formation between AT1 and TolAII-III resulted in minimal observed structural changes.
Conclusions:
- TolAII and TolAIII possess distinct structural characteristics, with TolAII being predominantly alpha-helical and TolAIII being globular.
- The TolAII domain may form a coiled-coil structure, while TolAIII adopts a globular fold.
- These structural insights contribute to understanding the mechanism of colicin import across the bacterial envelope.