User:Jennifer Taylor/Sandbox 2

From Proteopedia

Introduction

This protein (PDB ID 3h04) is an uncharacterized protein that was structurally solved in 2000 during a Protein Structure Initiative(PSI) by the National Institutes of Health. This initiative lasted fifteen years and scientists determined a large quanitity of structures of proteins. We selected this protein from their collection.While it's structure was solved and known, it's actual function was yet to be revealed. Through in Silico Analysis, Bacterial Transformations, Protein Expression, Protein Purification, Colorimetric Enzyme (Lipase) Assay, we attempt to confirm that 3H04 Is a Lipase. Prior to our research the only information on its characteristics was that it was potentially a hydrolase that could be a alpha/beta found in E.Coli.

Proteins are extremely important to current science and research in all aspects. The function that proteins carry can often be used in ground breaking research for all areas of medical treatments. While many proteins have been structurally solved, they still remain un characterized and therefore their function unknown. The issue with this is that while it is good to have the structural integrity of a protein derived, the function is what determines its applicability in certain fields. In this experiment we attempt aid in the efforts of characterization and characterize 3h04

Approach

Initially to isolate our proteins function we performed protein purifications. This allowed us to introduce our proteins function into new cells. This allows us to isolate our function and produce cells that strictly express our proteins function. This was proven when we ran our new cells through a gel electrophoresis and the cells revealed a band around 75 kDa which was the targeted band length for 3h04 ( see figure below). After we purified our protein and produced cells that strictly expressed 3h04 In our efforts to characterize 3h04 we used computer softwares such as PyMOL, ProMol, BLAST, Pfam as well as Dali. After using Blast Pfam and Dali we were able to analyze it's In silico enzymatic characteristics, however to further understand our protein, we utilized PyMol, ProMol, and Pfam. Through these programs we were able to take a closer look at it's active sites and catalytic triad. After loading our protein into these proteins, we were able to compare RMS levels with other similar proteins hoping that finding a protein with a similar structure would reveal similar functions. Through this analysis it revealed that 3h04 showed close similarities with 1AZW and 1YSC with a RMS. While the structure were similar we hoped for a lower RMS. As a result we began analyzing the active sites and Catalytic Triads. Reasoning for this was that the Active site and Catalytic are where the protein generally carries out its function-therefore it would have a more prevalent relationship towards it function. Through this approach it revealed an extremely low RMS with protein 1TAH of 0.016. We then decided to research 1TAH and it revealed that it was a Hydrolase; Esterase; Lipase. After this analysis we decided to run a Lipase Assay.

Hypothesis

After receiving information on its similarity to 1AZW and1YSC, our RMS levels were relatively low however after comparing Active sites it revealed 1TAH to be the new target homolog. After researching 1TAh it revealed that it was a Lipase. Through this knowledge we hoped to perform a Lipase Assay that used a spectrophotometry protocol. The idea was that we would expose our protein to a solution that would cause a color change (Change to yellow). If 3H04 truly is a Lipase we should expect to see a strong color change once introduced to the solution. (p-Nitrophenyl Butyrate (pNPB)). We would also test different concentrations of 3h04 against pNPB to see if our results were linear and therefore accurate.

Complications

Throughout our experiment, we encountered many setbacks that slowed down our efforts in characterizing 3h04. One issue was our time constraints. There were many times where our class period would end and leave us without time to fully finish a procedure. As a result some days we had to leave our cells in a freezer when we should've either plated them or ran them through a gel. These minute alterations in procedures could have caused us to yield skewed results. As a note in the future when conducting an assay, or plating more of your protein, make sure you have ample time so you don't have to stop mid procedure.

Relevance

Structural highlights

This is a sample scene created with SAT to by Group, and another to make of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

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References