Transcript protein-protein interactions

Research
Methodology of
Biotechnology:
Protein-Protein
Interactions
Yao-Te Huang
Aug 16, 2011
Introduction
Protein interactions and
functions are intimately related.
The structure of a protein
influences its function by
determining the other molecules
with which it can interact and
the consequences of those
interactions.
Introduction (contd.)
Experimental
methods available
to detect protein
interactions vary in
their level of
resolution.
These observations
can be classified
into four levels: (a)
atomic scale, (b)
binary interactions,
(c) complex
interactions, and
(d) cellular scale.
Introduction (contd.)
Atomic-scale methods:
showing the precise structural
relationships between interacting
atoms and residues
The highest resolution methods: e.g.,
X-ray crystallography and NMR
Not yet applied to study protein
interactions in a high-throughput
manner.
Introduction (contd.)
Binary-interaction methods:
Methods to detect interactions
between pairs of proteins
Do not reveal the precise
chemical nature of the
interactions but simply report
such interactions take place
The major high-throughput
technology: the yeast two-hybrid
system
Introduction (contd.)
Complex-interaction methods:
Methods to detect interactions
between multiple proteins that form
complexes.
Do not reveal the precise chemical
nature of the interactions but simply
report that such interactions take
place.
The major high-throughput
technology: systematic affinity
purification followed by mass
spectrometry
Introduction (contd.)
Cellular-scale methods:
Methods to determine where
proteins are localized (e.g.,
immunofluorescence).
It may be possible to determine
the function of a protein directly
from its localization.
COIB (2001), 12:334-339
Principles of proteinprotein interaction analysis
These small-scale analysis methods
are also useful in proteomics because
the large-scale methods tend to
produce a significant number of false
positives.
They include (a) genetic methods, (b)
bioinformatic methods, (c) Affinitybased biochemical methods, and (d)
Physical methods.
Genetic methods
Classical genetics can be used to
investigate protein interactions
by combining different mutations
in the same cell or organism and
observing the resulting
phenotype.
Suppressor mutation: A
secondary mutation that can
correct the phenotype of a
primary mutation.
Suppressor mutation
Synthetic lethal effect
Bioinformatic methods
(A) The domain fusion method (or
Rosetta stone method):
The sequence of protein X (a singledomain protein from genome 1) is
used as a similarity search query on
genome 2. This identifies any singledomain proteins related to protein X
and also any multi-domain proteins,
which we can define as protein X-Y.
As part of the same protein, domain X
and Y are likely to be functionally
related.
The domain fusion method
(or Rosetta stone method)
The sequence of domain Y can then be used
to identify single-domain orthologs in
genome 1.
Thus, Gene Y, formerly an orphan with no
known function, becomes annotated due to
its association with Gene X. The two
proteins are also likely to interact.
The sequence of protein X-Y may also
identify further domain fusions, such as
protein Y-Z. This links three proteins into a
functional group and possibly identifies an
interacting complex.
The domain fusion method
(or Rosetta stone method)
Bioinformatic methods
(B) The phylogenetic profile:
It describes the pattern of presence or
absence of a particular protein across a
set of organisms whose genomes have
been sequenced. If two proteins have the
same phylogenetic profile (that is, the
same pattern of presence or absence) in
all surveyed genomes, it is inferred that
the two proteins have a functional link.
A protein’s phylogenetic profile is a nearly
unique characterization of its pattern of
distribution among genomes. Hence any
two proteins having identical or similar
phylogenetic profiles are likely to be
engaged in a common pathway or complex.
YPL207W clusters with
the ribosomal proteins
and can be assigned a
function in protein
synthesis.
When homology is present, the elements are shaped on a gradient
from light red (low level of identity) to dark red (high level of identity)
Affinity-based
biochemical methods
Affinity chromatography can be used
to trap interacting proteins. If protein
X is immobilized on Sepharose beads
(e.g., using specific antibodies), then
proteins (and other molecules)
interacting with protein X can be
captured from a cell lysate passed
through the column. After washing
away unbound proteins, the bound
proteins can be eluted, separated by
SDS-PAGE and analyzed by mass
spectrometry.
(A) Affinity chromatography
followed by SDS-PAGE & Mass
spectrometry
(B) Immunoprecipitation
The addition of antibodies specific for
protein X to a cell lysate will result in
the precipitation of the antibodyantigen complex.
The technique is usually carried out
with polyclonal antisera.
The precipitated complexes are
separated from the cell lysate by
centrifugation, washed and then
fractionated by SDS-PAGE, and the
bound proteins can be identified by
mass spectrometry.
Immunoprecipitation
(C) GST pulldown
The protein X is expressed as a fusion
to GST. After mixing the fusion
protein with a cell lysate and allowing
complexes to form, glutathionecoated beads are added to capture
the GST part of the fusion. The beads
are recovered by centrifugation,
washed and the recovered proteins
fractionated and identified by mass
spectrometry.
GST pulldown