Unravelling the Structure of SecA

Title: Unravelling the Structure of SecA: Analysis of WT- vs. Y134S-SecA

Author: Sabreen Alam

Academy: Southern California Academy of Sciences

High School: Portola High School

Mentor: Dr. Guillaume Roussel, Department of Physiology and Biophysics, UCI School of Medicine

Protein secretion controls all aspects of cellular function, from building and repairing tissues to cellular communication via enzymes. SecA and the integral membrane proteins SecYEG control the secretion of most proteins across the plasma membrane in bacteria. A functional copy of SecA is essential for the survival of E. coli bacteria. Understanding the structure and function of SecA mutants can provide insight into the function SecA as a whole. Y134S-SecA in particular is a crucial mutant of SecA because it can secrete proteins with a modified signalling peptide (attached to protein exports from the ribosome) more than WT-SecA. Understanding how this mutant affects the stability of SecA would begin the process of characterizing the differences between WT and Y134S-SecA, and contribute to the general understanding of how protein secretion occurs in bacteria. The objective of this project is to examine the difference in structure and stability between WT and Y134S-SecA.

Question: How does the conversion of WT-SecA to the Y134S-SecA mutant in E. coli affect the structure and stability of SecA?

Hypothesis: The amino acid in position 134 of SecA is tyrosine in WT-SecA and serine in Y134S-SecA. The location of this amino acid is critical because it lies between two domains of SecA that bind and control ATP hydrolysis in SecA. Tyrosine is a polar hydrophobic amino acid, and serine is a polar hydrophilic amino acid. The difference between these amino acids indicates that changing tyrosine to serine in Y134S-SecA would probably result in a significant change in the structure of SecA, destabilizing the two domains surrounding serine. This would be shown by the fact that WT-SecA and Y134S-SecA would have two distinct temperatures at which they start to unfold.

Variables:

Independent Variable: type of SecA mutant (WT and Y134S-SecA)

Dependent Variable: unfolding threshold of SecA protein

In my experiment, I incorporated the WT-secA- and Y134S-secA- gene using T5 plasmids in BL21 E. coli cells. The resulting SecA protein expressed (WT and Y134S-SecA) by these cells was purified using Immobilized-Metal Affinity Chromatography, concentrated with a desalting column, and analyzed via tryptophan fluorescence (thermal unfolding from 310 nm to 400 nm) at temperatures ranging from 20˚C to 60˚C. The difference in the unfolding threshold- the temperature at which each protein (WT and Y134S-SecA) converted from a folded to a fully unfolded state was recorded.

As stated in my hypothesis, I thought that there would be a significant difference in the temperature at which WT and Y134S-SecA converts from a folded to a fully unfolded state. The data collected from my investigation shows that my hypothesis is supported. The mutation of tyrosine (WT-SecA) to serine (Y134S-SecA) at position 134 resulted in a shift in the unfolding threshold of the protein of about 2.6˚C. This indicates that the overall structure of Y134S-SecA is less stable than WT-SecA. Tyrosine residues present in WT-SecA appear to play a role in protein stability- when tyrosine was replaced with serine, the temperature at which the protein unfolded was significantly lower. The distinction of this temperature between Y134S-SecA and WT-SecA was still apparent after the correction for temperature dependence of fluorescence intensity was applied. Clearly, the difference in stability is significant after external variables are removed from the data.

In the future, my experiment can be extended by examining the possible link between the Y134S-SecA mutation and cell metabolism. The 134 amino acid lies in a crucial position- in between two domains of SecA that catalyze ATP hydrolysis. My experiment indicated that this mutation significantly affects protein stability, but I have not yet investigated the ATPase activity of WT and Y134S-SecA. Extending my experiment by including this variable would allow more a more comprehensive understanding of how the Y134S-SecA mutation affects the structure, stability, and function of SecA. Future research includes investigating the effects of cytoplasmic salts found in E. coli (for example, KGlu or KCl) on Y134S-SecA to simulate an environment more similar to the interior of E. coli. Previous studies have shown that the presence of these salts stabilizes WT-SecA. It would be interesting to see a similar effect is seen in Y134S-SecA, despite the substitution of a serine for a tyrosine that I have already shown destabilizes the SecA protein.

This experiment models the fact that the structure and stability of SecA is significantly affected by the Y134S-SecA mutation. Identifying the significance of the 134 amino acid in SecA paves the way for more research into its role in facilitating the secretion of preproteins. Based on this data, scientists who study SecA or protein secretion in general gain analyze the stability and selective secretion of WT-SecA compared to Y134S-SecA. Researchers can better understand protein secretion and transport across the plasma membrane in both bacteria and humans (via homologs of the SecA complex) if the function of SecA is more thoroughly researched. The relationship between increased signalling peptide recognition by Y134S-SecA and the 134 amino acid can also be identified based on these results if further experimentation is done to investigate the effects of this mutation in vivo.

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I would like to thank Dr. Guillaume Roussel and Dr. Stephen White for all of their support and guidance throughout my project, and for allowing me to use the equipment and resources in their lab. I would also like to thank my parents for their moral support and encouragement, and my grandparents for driving me to UCI School of Medicine every day. Finally, I would like to thank Ms. Gloria Takahashi and Dr. Kimo Morris for giving me the opportunity to participate in the Southern California Academy of Sciences Research Training Program, where I received constructive feedback and support for my project.

I started this project last year (in my sophomore year of high school) at the Department of Physiology and Biophysics at UCI School of Medicine, where I worked for several months on developing the protocol and conducting my research. The Southern California Academy of Sciences Research Training Program provided me with the resources to develop a professional poster and research paper, and effectively communicate my results to the scientific community. Through this program, I also had the opportunity to submit my abstract to AJAS and participate in this conference.