Greg's ASBMB poster - Dowling College

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2 Οκτ 2013 (πριν από 3 χρόνια και 8 μήνες)

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Introduction


As of April 9
th
, 2012 there are 2,965 protein structures listed
with unknown function in the Protein
D
ata
B
ank (PDB).
ProMOL
,

a plugin for the molecular visualization software
PyMOL
, is a tool which may be useful in determining the
function of these proteins.
ProMOL

uses
PyMOL’s

built in
selection algebra to compare a queried protein structure
against a database of known active site templates, called
motifs. Should a motif be found in the query protein,
ProMOL

can align the active residues in both the motif and the query
(as well as the entire protein structure) to asses similarity
visually.

Figure 5. Comparison of predicted active sites on 3DS8 and 3FLE, two proteins of
unknown function.
ProMOL

assigned a probable
hydrolase

or lipase function. Note the
highly conserved secondary structure between the two proteins. 3DS8 was isolated
from
Listeria
innocua
, while 3FLE was isolated from
Staphylococcus epidermis
.

Improving a Software
S
ystem for Protein
A
ctive
S
ite
D
etermination

Greg James Dodge
1
, Cyprian Corwin
2
, Herbert J. Bernstein
3
, Paul A. Craig
1
.

1
Chemistry,
2
Computer Science, Rochester Institute of Technology, Rochester NY,

3
Mathematics and Computer Science, Dowling College, Shirley NY

After the summer of 2011, attempts to implement parallel
computing were scaled back in favor of confirming results
from
ProMOL

experimentally.
ProMOL’s

library of motifs had
been expanded to house 440 active sites, up from 142 at this
time last year. Upon testing this expanded library against
proteins of unknown function, several very good alignments
were produced. In particular, a protein with a PDB ID 3DS8
aligned very well with a member of the A/B
hydrolase

superfamily

PDB ID 3LIP.

Figure 2. BLAST Alignment between 3ds8 and the entire PDB. Unexpectedly, this protein was
extremely similar to another protein of unknown function; having 91% query coverage with PDB ID
3FLE.

Program No. 978.7


Abstract #A294

ProMol

is a
plugin

for the
PyMOL

molecular graphics system that

is designed to locate the active site on a query protein.
ProMol

does this by comparing the three dimensional distances of all of the

atoms in a protein to the three dimensional positions of a library of

known active site residues. This is a computationally intensive

process, and it would take months to run the entire PDB (Protein

Data Bank) through the program. Recently however, advances in

GPGPU (General Purpose Graphics Processing Unit) computing

and GPU design have allowed simple computations, such as those

used by
ProMol
, to be performed rapidly across hundreds of cores.

Utilizing GPGPU computing, the time needed to run proteins

through our program could be drastically reduced. Due to the rapid

turnover of authors on the project however,
ProMol's

codebase was

quite fragmented. Over the course of summer 2011, our group

worked to streamline
ProMol's

source in preparation for

implementing GPGPU computing. This work consisted of bug

hunting, re
-
implementing the PDB loading functionality, and

cleaning up the user interface, among other things. Ideally, with

the changes in place, we will be able to adapt
PyMOL

and

ProMol's

selection algebra to run in parallel across many GPU

Cores.

Methods

After the initial alignment between 3DS8 and a hydrolase, the 3DS8 plasmid was
obtained from the plasmid repository at Arizona State University. The protein was then
was over expressed in XL
-
21B
E.coli

cell having been grown in ampicillin containing
media. The protein was then purified via cobalt affinity column chromatography. Once
purified, the functionality of the protein was tested with a
QuantiCleave
TM

Protease
Assay kit (Thermo Scientific). The results from the assay suggested some protease
activity, but they were not entirely conclusive. The FASTA sequence of the protein was
then tested against the entire PDB using NCBI’s protein BLAST tool
1
. This alignment
showed putative conserved domains between 3DS8 and the esterase lipase superfamily.
In particular, there was 91% sequence coverage between 3DS8 and 3FLE, another
protein of unknown function.

Further analysis within
ProMOL

revealed a very good active site alignment
between these two proteins of unknown function with 1TAH, a lipase with a
known and well documented active site
3
.

Future Plan


Although the initial goal of implementing parallel computing
has not been met, it has been worthwhile to explore the
validity of
ProMOL’s

alignments
in vitro
. The plasmid for 3FLE
is currently on order, as well as the reagents to run two
separate lipase activity assays. Once all of the necessary
materials have been gathered, the same expression,
purification, and analysis pipeline from the earlier work on
3DS8 will be followed. Additionally, work is being done on
another protein with a predicted function of a
galactosyl
-

transferase

using the same approach. Biochemical
characterization of an enzyme is one true test of its function.
In the future, we plan to continue to compare functional assay
results with predicted behavior. This project combines classical
enzymology with bioinformatics; as such it may be suitable for
incorporation into the undergraduate biochemistry lab
curriculum
.

References

1.
Stephen
F.
Altschul
, Thomas L. Madden, Alejandro A.
Schäffer
,
Jinghui

Zhang,
Zheng

Zhang, Webb Miller, and David J.
Lipman

(1997),
Nucleic
Acids Res
.
25:3389
-
3402

2.

The
PyMOL

Molecular Graphics System, Version 1.5.0.1 Schrödinger, LLC.

3.
Noble, M.E.M., A.
Cleasby
, L.N. Johnson, M.R.
Egmond
, and L.G.J.
Frenken
.
FEBS Letters

331, no. 1

2 (September 27, 1993): 123

128.


Conclusions

Our goal is to determine the function of proteins listed with
unknown function in the PDB using
ProMOL
, a plug in for
PyMOL
. Using
ProMOL

and other bioinformatics tools, we
identified two proteins (3DS8, 3FLE) that may belong to the
A/B hydrolase superfamily, more specifically the lipase family.
Early lab results indicate that 3DS8 has
hydrolase

function, as
predicted. Based on these results, it appears that using
ProMOL

to determine active site motifs is a promising
bioinformatics tool.




Figure 1. The motif maker and motif finder tabs in
ProMOL
. Protein
analysis is as simple as entering the PDB ID into the search box under
the motif finder tab, selecting a set of motifs, and clicking search.
Creating a motif is accomplished by entering the PDB ID, the EC #, and
the active residue information under the Motif Maker tab.


Figure 3. Active site alignments of 1TAH (white)
with the predicted site on 3DS8 (red). The active
residues on 1TAH are ser 87, his 285, and asp 263.
ProMOL

predicts that these correspond to ser 102,
his 222, and asp 188 on 3DS8.

Figure 4. Active site alignments of 1TAH (white)
with the predicted sites on 3FLE (red). The active
residues on 1TAH are ser 87, his 285, and asp 263.
ProMOL

predicts that these correspond to ser 144,
his 269, and asp 235 on both the A and B chains of
3FLE.

Acknowledgments

The
authors gratefully acknowledge the assistance of current and former students who have worked at Dowling
College and at RIT on the SBEVSL project.

Funding
: This work has been supported in part by National Science
Foundation Division of Undergraduate Education grant 0402408, National Institute of General Medical Sciences
grants 2R15GM078077
-
02, 3R15GM078077
-
02S1. The content is solely the responsibility of the authors and
does not necessarily represent the official views of the funding agencies.