Study of Fused Protein Complexes in Rhodobacter capsulatus

Vivian at her lab workstation

Funding Source




Professor of Biology

Project Summary

This summer I have had the opportunity to continue working within Prof. Fevzi Daldal’s lab under the guidance of a post-doctoral student. My research project is concerned with understanding the molecular basis of the biological electron transfer that occurs during cellular chemical energy transduction in metabolic processes such as photosynthesis and respiration. This electron transfer is possible through several multi-subunit protein complexes, which are vital components for photosynthesis and respiration, the end product of which are energy-rich molecules that are needed for important cellular processes. When these complexes cannot function correctly, photosynthesis and respiration cannot be carried out properly, leading to low crop yields in plants, and neurological and muscular diseases in humans.

As a model system, I use the bacterium Rhodobacter capsulatus. The protein complexes that concern my research project are the membrane-bound cytochrome c reductase (bc1) and the cytochrome c oxidase (cbb3), which are respiratory enzymes; their electron carriers, cytochrome c2 and cytochrome cy, are also of interest. The respiratory enzymes form protein supercomplexes. However, their isolation for further studies proves to be difficult due to low abundance and weak interactions. To overcome this issue, genetic fusions are created producing R. capsulatus strains that have covalent linkers of various lengths between the bc1 and cbb3 complexes. I have recently been working with a strain that contains covalent linkers between bc1, cbb3, and cy. The covalent linkers between the two complexes and electron carrier are usually partially cleaved by proteolytic activity within the R. capsulatus cells, and in order to analyze the structure and function of this fusion (bc1-cbb3-cy), it is desirable to optimize the amount of stable supercomplexes extracted from the R. capsulatus cells. The production of stable complexes can be influenced by the environment within which this R. capsulatus strain grows, and through previous research I have been able to identify an optimal growth condition yielding a greater amount of intact bc1-cbb3-cy fusion.

I spent this summer growing R. capsulatus cells in bulk using the optimal growth condition, extracting protein complexes from the cells and purifying them through anion exchange and affinity chromatography. After several trials of cell growth, I got the hang of the process of protein purification and I obtained samples of purified protein complexes. To ensure that the protein obtained was in fact the bc1-cbb3-cy complex, I continued to conduct experiments on the obtained protein samples to measure the redox activity of the complexes, which indicates electron transfer within the complexes. This activity was analyzed by UV/Vis spectroscopy and by monitoring oxygen consumption using an oxygen electrode, procedures that I was not familiar with before, but that I got to understand and carry out on my own. During this analysis, I carried out a number of trails analyzing the extent of redox activity within the complexes in the presence of several activity-enhancing substances, such as cytochrome c2 and E. coli membrane lipids.

Throughout the process of protein purification and analyzing redox activity, I was able to recognize chemical and biochemical principles that I had learned about through courses I took in the past two years. Seeing these principles in practice within my project further emphasized their importance and helped me understand their practical application. I’ve come to realize that learning these principles in the confines of a lecture hall and applying them to my research are two different experiences; having a practical approach is of great importance. And I couldn’t have had this great summer experience without the help of CURF funding and the Fevzi Daldal Lab.