Understanding bacterial growth (cell division) is important to be able to synthesize stronger antibiotics that stop the growth. When a bacterial cell divides, it pinches off at the central region leading to two new cells. This process is carried out by a concerted effort by various proteins that assemble in the cell membrane. We know that these proteins interact with each other, but aren't sure how exactly they interact and carry out division.
So I began with one of those proteins, and asked a QUESTION – What is the structure of this protein (called transmembrane helix peptide) in the membrane region? Does it contribute to division as a monomer (single peptide), or does it form a dimer (pair) with another copy of itself ? To answer this question I used a phenomenon called FRET.
Now what is FRET? In ROYGBIV, consider two colors, blue and green. Because blue is adjacent to indigo, a blue dye will absorb indigo light and emit part of that light as blue fluorescence. So under indigo light, isolated blue dyes will fluoresce, whereas isolated green dyes will stay 'dark'. However, if we bring the two dyes in CLOSE PROXIMITY, blue dye will TRANSFER its blue fluorescent energy to green dye, and we'll see green fluorescence instead of blue. This phenomenon is Forster Resonance Energy Transfer or FRET!
To use FRET to investigate whether the transmembrane peptide forms a dimer, I attached Blue and Green dyes to many copies of this peptide, mixed them, and excited them with indigo light. If the peptide tends to form a dimer, the dyes will be in close proximity, and we'll observe FRET in the form of green fluorescence. If it tends to stay a monomer, the dyes will be away from each other, so we'll see only blue fluorescence and the green labeled peptides will be 'dark'. If the dimer formed is very strong, we'll see more green than blue fluorescence in the mixture and vice versa.
Transmembrane peptide copies were chemically synthesized on a solid scaffold (Resin). Half of them were labeled with blue and half with green dyes. Labeled peptides were purified from unlabeled peptides using Liquid Chromatography. Peptides were added to artificial membranes called 'liposomes' and control experiments were performed by separately exciting isolated blue and green labeled peptides with indigo light. The FRET experiment was performed by mixing an equal amount of blue and green labeled peptides in liposomes and exciting them with indigo light.
Results and Discussion:
Transmembrane peptides clump together and resist labeling with dyes, so only ~50% got labeled. Thus purification was necessary to remove the unlabeled peptides. The experiments were performed in liposomes as they provide an artificial environment close to the cell membrane, which helps us study the transmembrane peptides in a near-natural set-up. Under indigo light, isolated blue labeled peptides emitted blue fluorescence, and isolated green labeled peptides were dark, ensuring the dyes were behaving well in the liposome environment. For the FRET experiment, appearance of alternating blue and green fluorescence indicated that FRET was occurring, suggesting that the peptide exists in equilibrium between a monomer-dimer state.
Since all the peptide molecules are same irrespective of their label, they'll form various dimer types: blue-blue and green-green apart from the blue-green (FRET) pair. Also, the mixture may contain some leftover unlabeled peptide due to imperfect purification which will also form dimers. All these 'No-FRET' dimers will contribute to the total amount of dimers, but we can't see them as they don't show any green fluorescence. These factors have to be added to the observed FRET in the final calculation to get the amount of dimer vs monomer.
Through this process we found that this transmembrane peptide tends to form a strong dimer, which might be functionally important during bacterial division. This finding brought us one step closer to targeting the mechanism of cell division in bacteria that will aid development of better inhibitors of bacterial growth.
I'd like to thank all my friends who participated in this project for me, without which this depiction would have been difficult. The wonderful team comprises current or former Masters and PhDs students from different disciplines ranging from the Chemical and Life Sciences, to Civil, Electrical and Chemical Engineering in the University of Wisconsin-Madison and an Economics faculty member in UW Whitewater. I also thank Union South at UW Madison for providing me the space and equipment to carry out this project. Finally, I'm grateful to Gonzo labs for creating this wonderful platform for us to share our research in the form of a performing art.
Khadria A and Senes A, Methods Mol. Biol. 2013 1063, 19-36
Khadria AS and Senes A, Biochemistry 2013, Articles ASAP, Publication Date: October 1, 2013