Download protein-protein interactions

Research
Methodology of
Biotechnology:
Protein-Protein
Interactions
Yao-Te Huang
Aug 19, 2010
The website for downloading
lecture notes/lecture materials
http://mail.cmu.edu.tw/~
ythuang/teaching.summer
.2010.htm
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
(A) 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.
Affinity chromatography followed
by SDS-PAGE & Mass spectrometry
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
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
Crosslinking
Interacting proteins can be identified
by crosslinking. A labeled crosslinker
is added to protein X in vitro and the
cell lysate is added so that
interactions can occur. If the
crosslink is activated at this stage,
interacting proteins become
covalently attached to the bait. After
purification, the crosslink can be
cleaved and the interacting proteins
separated by 2D SDS-PAGE.
Crosslinking (contd.)
Physical methods
High-resolution methods: (e.g.,
X-ray crystallography & NMR)
providing data about the relative
spacing of atoms of interacting
molecules.
Low-resolution methods: e.g.,
electron crystallography and
electron tomography.
FRET (Fluorescence Resonance
Energy Transfer)
FRET
FRET is the energy
transfer that occurs
when two
fluorophores are
close together, and
one of fluorophores
(the donor) has
an emission
spectrum that
overlaps the
excitation spectrum
(absorption spectrum)
of the other
fluorophoe (the
acceptor).
Basic Theory of FRET:
kT(r) = (QD2)(1/Dr6)(9000 *In10)(1/1285NAn4)(∫FD()A() 4d )
= (1/D)(R0/r)6
where R0 is the Förster distance
r is the distance between the donor and the acceptor
J(), the so-called overlap integral= ∫FD()A() 4d 
E = 1/(1+(r/R0)6)
where E is the efficiency
of the energy transfer
FRET
FRET
R0: is the Förster distance
FRET: distancedependent
R0 is the Förster distance
r is the distance between
the donor and the acceptor
E is the efficiency of
the energy transfer
Note: when r=R0, E=0.5
FD: the fluorescence intensity of the
donor in the absence of the acceptor
FDA: the fluorescence intensity of the
donor in the presence of the acceptor
Library-based methods for the
global analysis of binary
interactions
Standard cDNA expression
libraries
Phage display method
The yeast two-hybrid
system
Standard cDNA
expression libraries
Expression libraries are usually
screened with labeled antibodies. In
place of antibodies, other proteins
can be used as probes. For example,
labeled calmodulin has been used to
screen for calmodulin-binding
proteins.
Low throughput
Does not provide the native
conditions for the folding of all
proteins, so a significant number of
interactions would not be detected.
Phage display method (1)
M13 (a filamentous phage containing ss-DNA encased in a protein
coat): contains five coat proteins, two of which are
gVIIIp (gene VIII protein) and gIIIp (gene III protein).
Phage display method (2)
Phage display method
(2): contd.
The phage display method
The yeast two-hybrid
system
Transcription factors generally comprise two
functionally independent domains, one for
DNA binding and one for transcriptional
activation. These do not have to be covalently
joined together, but can be assembled to form
a dimeric protein. This principle is exploited to
identify protein interactions. Bait proteins are
expressed in one yeast strain as a fusion with
a DNA-binding domain and candidate prey
proteins are expressed in another strain as
fusions with a transactivation domain. When
the two strains are mated, functional
transcription factors are assembled only if the
bait and prey interact. This can be detected by
including a reporter gene activated by the
hybrid transcription factor.
The yeast two-hybrid yeast
Limitations of the yeast
two-hybrid system
First, where independent groups have
carried out similar, large-scale
studies, the degree of overlap in the
reported interactions is very low (1015%). This suggest either that the
screens were not comprehensive or
that even minor differences in
experimental conditions could
influence the types of interactions
that are detected.
Limitations of the yeast
two-hybrid system
Secondly, a significant number of
well-characterized interactions are
not detected in the large-scale
screens, suggesting there is a high
level of false negatives.
Thirdly, a significant number of
interactions that are detected in
large-scale screens appear spurious
when investigated in more detail,
suggesting there is also high level of
false positives.
A variant of the yeast
two-hybrid system
Protein interaction maps
Node: proteins or protein complexes are
treated as nodes.
Edge (or link): interactions between them.
Some proteins serve as hubs for very large
numbers of interactions.
Binary interaction map including 1200 interacting proteins in yeast
Trends in Cell biology (2001), 11: 102-106
A simplified version in which yeast
proteins have been clustered
according to their function
Homework
Pick up your most favorite
method of protein-protein
interactions, and describe it in
detail.
Describe phage display method.