You may include any references to papers as in: the use of JSmol in Proteopedia [1] or to the article describing Jmol [2] to the rescue.
Overview
This project seeks to determine the function of a protein with an identified structure. The query protein, in this case, can be identified by the Protein Data Bank Identification (PDB ID) code 3H04. 3H04 is a alpha/beta hydrolase found in Escherichia Coli, a bacterium that resides in the lower intestines of warm-blooded mammals. The more specific purpose of this study is to characterize the function of 3H04. Plasmid purification is just one method for identifying an unidentified protein. Protein purification in bacterial cells requires the plasmids, a small circular double-stranded DNA molecule, to be purified and identified. The purification of the plasmid is meant to isolate and purify the plasmid DNA. The protein in the plasmid is identified through bacterial cells, and grows against ampicillin so the bacterial cells are growing against the antibiotic; the cells that grow have AmpR (Ampicillin Resistance gene). And in efforts to characterize our protein, 3H04, we are comparing it to other proteins with significant parts in common to hopefully help characterize the function of 3H04. The comparison of the different aspects of our protein to different aspects of other proteins also helped us determine our assay.
Hypothesis
In the beginning, we predicted that our protein was an esterase, because originally our most promising homologs were an aminopeptidase and an esterase. And even though the most hits from our Promol search were enzyme class 3.4 (aminopeptidases), no further information was found about the catalytic triads of these hits. We decided to use a homolog in E.C. 3.1 (esterase) because there was information about their catalytic triads. We knew that our protein is a hydrolase, and we believe that 3H04 is characterized as an esterase (3.1), instead of an aminopeptidase (3.4). Even though the 3.4 category proteins had the most motif structure similarity in Promol, we chose 1TAH (a lipase) as our target homolog. 1TAH is a characterized protein and has a similar catalytic triad to what we predict for our protein (3H04). When we aligned the two catalytic triads 1TAH and 3H04, they had an RMS of 0.016. In the beginning we were looking for a homolog with more protein sequence and structure similarity, but because neither protein sequence or structure determine the function of 3H04, we decided that those two factors matter much less than the catalytic triad of the protein. So, we used Promol to find what the catalytic triad of 3H04 is and we used 1TAH as our target homolog for the catalytic triad. We predict, because the RMS was so low, that 3H04 and 1TAH will be having very similar enzymatic functions.
Methods
We performed a Lipase Assay to determine what kind of esterase our protein is, because our target homolog (1TAH) is a lipase. The literature about 1TAH had no useful information about assays, but we used an assay from a paper regarding a different lipase.
Results
Figure 1: Lipase Assay Result. 3H04 was tested with two different concentrations of pNPB to test how the concentration altered the rate of the reaction.
Figure 2: Coomassie Gel. Coomassie Gel of 3H04 (Lanes 1-8). 1. Cell Extract, 2. Flow Through, 3. Wash 1, 4. Wash 3, 5. Elution 1, 6. Elution 2, 7. Elution 3, 8. Ladder. The protein is being expressed wherever there is a darker, thinker band in the same row as the 75 kDa.
Conclusion
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