Cheng Chen (Molecular and Cell Biology major)
"Kinetic Constant Determination of Multidrug Efflux Pump"
Sponsor: Hiroshi Nikaido, Molecular and Cell Biology
Project Description
Multidrug efflux pump, which sometimes pumps out almost all of the commonly used antibiotics, plays a major role in bacterial resistance. The design of better antibiotics which will overcome this mechanism will require knowledge of the kinetic behavior of this pumping process. Recently, Cheng participated in a study that determined the kinetic constants for one class of antibiotics, cephalosporins. In this project Cheng will be using other antibiotics as the potential competitor of the cephalosporin flux to develop a more complete understanding of kinetic behavior of the pump. The knowledge of kinetic constants for various antibiotics will allow design of more suitable compounds that will evade the multidrug efflux process and will be effective in the treatment of human infections in the 21st century.
Scholar's Photo
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Cheng setting up his cuvettes for the spectrophotometric assay. |
Scholar's Journal
The project I worked on dealt with the kinetic constants of the multidrug efflux pump, AcrB. AcrB can pump out many different antibiotics and drugs and contributes to the antibiotic resistance in many Gram Negative bacteria. To really understand the behavior of this pump, we need to determine the protein structure and the kinetic constants, such as Vmax and Km. The structure will allow us to see the binding site of the protein, and the kinetic constants will enable us to gain insights on the catalytic mechanism of the enzyme. So far crystal structures have been taken of this protein, but no kinetic constants have been determined.
This summer I tried to measure the hydrolysis rate of nitrocefin to indirectly determine the Vmax and Km. At first this assay did not work at all. The data I collected made absolutely no sense. Even after several experiments, I was not far from where I started. I had to take a step back and examine each component of the experiments and see what the problem was. At first my suspicion was that the drug had already expired. However, through simple tests it was clear the drug had no problem. The next obvious question was whether I had the correct strain of bacteria. I plated the bacteria from the stab culture onto plates with specific antibiotics to isolate the strain I wanted. Lo and behold, the assay worked. I was able to generate a nice curve that showed AcrB indeed exhibited the classic Michaelis-Menton kinetics.
Over the next several weeks the assay would work on occasion. At first I thought that was simply because intact cells behave very strangely from time to time. But it bothered me that even under the same conditions the cells can have such huge physiological differences. So again, I was back to finding out the problem with the assay. Through many different trials, I finally figured out that the permeability of the cells changed as soon as they came off the shaker from the warm room; despite the fact they are suspended in the buffer. The best way to prevent the permeability change is to store it on ice. Even then that doesn't stop the permeability change, but only slows it down. Hopefully from now on, I will be able to collect my data quickly before there's noticeable change in the permeability.
