User:Jennifer Taylor/Sandbox 1

Background

Proteins are an important type of macromolecule in biological systems and can be considered a sequence of subunits known as amino acids. The development of high-throughput genome squencing techniques allowed proteins to be sequenced more quickly than their structures could be solved. In an effort to close this gap, in 2000, the National Institutes of Health launched the 15-year Protein Structure Initiative. Many structures were deposited in the Protein Data Bank, but many of these proteins with solved structures, such as YxiM (PDB ID: 2O14), remain functionally uncharacterized. YxiM is transcribed by the yxiM gene from Bacillus subtilis, a ubiquitous bacterial species that dwells in soil and gastrointestinal tracts. YxiM is 375 amino acids in length and its molecular weight is 41.8 kDa. It appears to have two domains: one dominated by α-helices, and one by β-sheets.

In silico Analysis

A common theme in biology is that form follows function. Thus, we used computer programs to find which proteins were most homologous to YxiM in terms of sequence and structure, with the expectation that YxiM is likely to be functionally similar to those proteins that have similar sequences and structures.

We used BLAST and PFam to find characterized proteins whose sequences aligned best with YxiM. Sequence analysis suggests that YxiM is a GDSL-like lipase, a type of esterase. Esterases are molecules that hydrolyze (decompose) a class of organic molecules known as esters. GDSL-like lipases demonstrate broad substrate specificity due to their flexible structures. BLAST showed that the proteins 1J00, 1IVN, and 1JRL have the highest sequence homology to YxiM. These proteins are multifunctional hydrolases that show both esterase and protease activity.

Next, we used PyMOL to align the 3D structures of the BLAST hits with that of YxiM. The proteins 1J00, 1IVN, and 1JRL all align well with the α-helix domain of YxiM.

The Dali server finds the most similar proteins based on 3D structures, and the top 30 hits for YxiM were are all rhamnogalacturonan acetylesterases, GDSL lipases, LAE5s (hydrolases), or acetyl xylan esterases, which further suggests that YxiM is an esterase.

Finally, we used ProMOL to perform a structural alignment of active sites of other proteins with YxiM to predict the active site of YxiM. We found that YxiM aligns best with the active site of IBWR, which is an esterase. The of YxiM consists of amino acids S171, D339, and H342.

Based on these analyses, we predicted that YxiM is an esterase and proceeded to perform in vitro assays to confirm esterase activity.

Plasmid Purification

A plasmid is a type of circular bacterial DNA. By placing the gene that transcribes our protein in the plasmid and transforming, or placing, the plasmid into bacteria, we can use the bacteria to create more of the protein. First, we transformed DH5α Competent E. coli cells with the plasmid pET21-yxiM to create more of this plasmid. Then, we lysed the bacteria and collected the plasmid.

Bacterial Transformation

While DH5α E. coli are good for purifying plasmids, BL21(DE3) E. coli are more efficient at expressing protein. Thus, we transformed the plasmid into BL21(DE3). We plated the bacteria on agar with the antibiotic ampicillin. The plasmid we used for transformation encodes ampicillin resistance to the bacteria. Since bacteria are not typically resistant to ampicillin, this means that only bacteria that were successfully transformed with the plasmid will survive on the plates.

Protein Expression

After a day, colonies of transformed bacteria were visible on the agar plates. To express YxiM, we inoculated a single colony of bacteria into liquid culture. Then, we added IPTG, which induced expression of YxiM in the bacteria.

Protein Purification

The YxiM proteins are "tagged" with a chain of histidine amino acids. This allows us to separate YxiM proteins from the other types of proteins in the E. coli cells. When we put the mixture of cell extract through nickel columns, the tagged proteins stuck to the nickel column, while the other proteins flowed through the column. Finally, we added elution buffer to the columns, which caused the proteins to detach from the nickel columns, leaving us with a solution of just the YxiM proteins.

Esterase Activity Assay

To test the function of YxiM in vitro, we placed it in a test tube with 4-nitrophenyl butyrate, a type of ester. We expect esterases to cleave 4-nitrophenyl butyrate and form the products butyric acid and 4-nitrophenol. Since 4-nitrophenyl is a yellow color, we can use a colorimeter to measure the absorbance, or the "yellowness" of the solution, as a proxy for esterase activity. We found that the absorbance increases over time, which suggests that YxiM is indeed an esterase.

Discussion

YxiM is a previously uncharacterized protein whose crystal structure has been solved and deposited in the PDB. On the basis of protein sequence and structural analysis in silico and functional assays in vitro, we conclude that YxiM is an esterase. There is some disagreement regarding the definitions of esterases and lipases. We consider lipases to be a subclass of esterases; lipases specifically hydrolyze lipids, whereas esterases hydrolyze ester bonds in general.

PyMOL shows that these hits also align well with the 3D structure of the α-helix domain of YxiM. Almost all the top structural hits in Dali are esterases as well, and ProMOL shows that the active site of YxiM most resembles one of an esterase or protease. The same catalytic triad (S171, D339, H342) is implicated in both protease and esterase activity, suggesting YxiM could be a multifunctional hydrolase. The catalytic motif of the esterase 1BWR aligns particularly well with YxiM.

Given that all in silico tests suggest that YxiM is an esterase, we tested YxiM for esterase activity in vitro. Protein expression and purification were successful. YxiM showed esterase activity on 4-nitrophenyl butyrate, as absorbance increased during the assay. Our Lineweaver-Burk plot of YxiM esterase activity is linear, which is typical of enzymes.

Future Directions

We can perform mutagenesis on the catalytic triad by performing PCR on the plasmid DNA with specialized primers. If we mutate the catalytic triad, then we expect that the protein will not be able to perform its function anymore. Through another round of transformation and purification of this mutated plasmid, we would expect the protein to have no function in our esterase assay.

Our data also suggest a potential protease functionality for YxiM. To test this, we could perform protease assays as well.

To further study enzyme kinetics, we need to relate absorbance with concentration of protein. We can achieve this by performing a Bradford protein assay to compute the extinction coefficient.