Download Characterization of the RNase A active site by Phage Panning

Characterization of the RNase A active
site by Phage Panning
Jeremy Kitchen, Neville Forlemu, and James Nolan
School of Science and Technology, Georgia Gwinnett College, Lawrenceville, GA 30043
ABSTRACT
We used the Phage Display technique to identify
peptide sequences that bind to a protein target. A
library of bacteriophages, each expressing a different
segment of seven random amino acids at the
beginning of the capsid protein on its exterior was
allowed to bind to the immobilized target protein; in
this case the RNAse A enzyme was immobilized on
small petri plates. The eluted phage were propagated
by replication in the host bacteria and the process was
repeated for three rounds to select for sequences that
bind the RNAse A active site with highest affinity.
Individual phage plaques from the last round of
purification were amplified and used to prepare DNA
samples for sequence analysis. We analyzed the
predicted amino acid sequences of active molecules
for common features in order to develop hypotheses
about how these proteins may interact with their
target. We used Autodock molecular docking software
to predict interaction affinities, identify the binding
domains and interaction mechanism between RNase
A and sample peptide sequences. This project was
performed as a lab module of the Advanced
Biochemistry course at Georgia Gwinnett College
during the Fall 2014 and Spring 2015 semesters.
INTRODUCTION
The Phage Display technique is a method to identify
peptides which can bind a target protein with high
affinity. (1) A library of M13 bacteriophage are
constructed with a randomized 7 amino acid
sequence attached to the N-terminal sequence of
the gpIII coat protein. These randomized phages are
then given a chance to bind with a given protein
(Figure 1A) with the unbound phage washed away.
The phage with the peptide sequence that binds
tightly to the protein will stay bound until it is outcompeted by a ligand known to bind to a specific
site. The target protein in this experiment is RNase A
and the ligand used to displace the phage from the
active site is a vanadyl ribonucleoside complex
(VRC) (2), a ligand known to bind to the active site.
This quality allows for specific selection of the phage
that best match to the active site. Because the
phage each contain the genetic code for their unique
protein sequence, they can be amplified in host
bacteria to allow for multiple rounds of binding
ensuring that the phage with the peptide sequence
that best fits the active site will be the ones
sequenced in the end.
REFERENCES
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Scott, JK and Smith GP. (1990) Science 249, 386-390.
Berger SL, Birkenmeier CS. Biochemistry. (1979) Nov
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New England Biolabs E8100 Manual (2012).
BigDye-Terminator v3.1 cycle sequencing kit protocol.
Waterhouse, A.M.et al. (2009) Bioinformatics 25 (9) 1189-1191
http://www.jalview.org
Mandava, S L. Makowski, J. Uzubell, S. Devarapalli and DJ Rodi
(2004). Proteomics 4; 1439-1460.
Morris, GM., Huey, R, Lindstrom, W, Sanner, MF, Belew, RK,
Goodsell, DS, and Olson, AJ (2009) J. Comput. Chem., 30: 27852791.
http://www.pdb.org/pdb/explore/explore.do?structureId=1RUV
1A
Phage population expressing different random
peptides incubate with immobilized RNaseA
1B
2A
Unbound phage washed away
VRC used to displace phage peptides bound
to active site
2B
Eluted phage amplified and selection
repeated twice more
Figure 1. A. Phage Panning Method. RNase A was bound to a
petri dish and a phage library expressing random 7-mer peptide
surface sequences was added. Unbound phage were then
washed away. The VRC enzyme inhibitor was then used to
displace the phage bound to the active site. These phage were
amplified and the process was repeated. The resulting phage
were then sequenced. B. Aligned and Clustered Sequences.
DNA sequence was determined from 24 random phage plaques.
The translated sequences were clustered into groups using
Jalview nearest-neighbor analysis. (5) The * and the **
sequences were found to each have three residue combinations
in common through the Motif 2 program. (6)
2C
Figure 2. A. RNase A with VRC Complex. The crystal structure
(8) of RNase A is shown. The protein is shown in surface
representation and the inhibitor VRC can be seen as a spacefilling model, which binds in the active site cleft of the enzyme.
B. RNase A with docked peptides. RNase A is shown as in A with peptide sequences from Group 2 bound near the
active site as directed by the Autodock modeling software. Peptides are represented in wireframe with different colors
representing each sequence. Other sequence groups showed similar distributions. C. Docking with a representative
peptide. Sequence SC5.1 from Group 2 shown in space-filling representation as in 2A. The peptide appears to bind in
the active site cleft as well as surrounding area.
MATERIALS AND METHODS
DISCUSSION
RNase A (25 ug/ml) was bound to petri dishes
overnight at 4°C. 1X1011 plaque forming units (pfu)
from the Ph.D-7 phage library (3) were incubated
with the dishes for 30 minutes, then washed ten
times with TBST. The phage bound to the active site
were then eluted with 1mL of 0.5mM VRC. These
eluted phage were then amplified back to the 1X1011
pfu/mL in ER2738 E. Coli for the next round of
selection. After 3 rounds of selection, individual
plaques were picked and sequenced (4), then
analyzed at the UGA Georgia Genomics Facility. The
translated sequences were then clustered by
similarities using the Jalview and Motif 2 programs
(5, 6) (Figure 1B). The sequences were then input
into the Autodock molecular docking software to
further characterize their possible interactions with
the active site (7), based upon previous descriptions
of the enzyme structure (8).
The output from Jalview (Figure 1A) showed five
different groups of similar peptides. Additional
sequence analysis with the RELIC motif search
programs identified two possible binding motifs: in
group 1, a SLxxQ motif was identified in two of the
four (*) suggesting a similar mode of binding to
the target protein. Group 2 also included two
members with a different motif, VRL, identified by
motif search. Groups 2 and 3 also share a
positively charged residue in the 5 position in
several of the sequences. Groups 3,4, and 5
share a proline, which might indicate a particular
bend is required for binding to the active site.
Autodock analysis of the binding of the peptides to
the known 3D structure of the RNase A enzyme
has just begun, but should help to identify how the
peptides interact with their target.
ACKNOWLEDGEMENT
This project was supported by the GGC School of
Science and Technology and by a STEM minigrant from
the GA BOR to GGC..