A DelicAte touch
The notion of a cell membrane serving to contain a large mixture of proteins randomly
moving about in a cytosol may once have been the grade school introduction to cellu-
lar biology. However, when one considers the nature and coordination of cellular ac-
tivities, it becomes evident that random collisions between proteins could not result
in the spatial and temporal reaction and information-carrying cascades now known
to occur in cells. Proteins must interact in very specific ways in order to coordinate
nearly all cellular processes including DNA replication and transcription, RNA splicing
and translation, protein modification and secretion, cell cycle control and apoptosis
as well as signal transduction and gene expression. Therefore, a disruption in the
interaction of proteins is likely to contribute to the onset of disease and is the reason
so much research effort is focused on understanding the nuances and implications of
such interactions. By Alfred Doig
Protein-protein interactions (PPIs) occurring on the exterior of the cell mem-brane provide signals as to the cell’s external environment. Within the cell membrane such signals are propagated by other specialized PPIs that serve to deliver the message to one or more of the compartmentalized cell struc-
tures, such as the nucleus or mitochondria, which might result in, for example, a
change in gene expression or ATP production.
PPIs are very diverse but all protein interactions occur in a highly specific manner
determined by structural and physiochemical properties of the interacting proteins.
At the molecular level PPIs can be characterized by their binding strength (permanent
or transient), specificity (specific or nonspecific), the location of interacting segments
(within one or more polypeptide chains), and the degree of similarity between inter-
acting protein subunits.
experimental Approaches
PPIs are affected by a number of variables including protein concentrations—as de-
termined by protein synthesis and degradation—and the location of the interacting
protein participants within the cell. In recent years, innovations in software, reagents,
and instrumentation as well as improvements to experimental protocols have given a
clearer understanding of the biological roles of many PPIs.
Methods available to study PPIs extend from very qualitative approaches to highly
quantitative measurements. The methods of choice are determined by the nature of
the experimental study and can range from the desire to discover new PPIs to de-
termining the dissociation kinetics between well-established interacting pairs. There
exist a number of in vivo and in vitro methods used to identify and characterize PPIs.
The techniques are based on a variety of biological, biophysical, or physiochemical
measurements and some lend themselves to high throughput format development.
Biological Methods
If a yes/no answer regarding the significance of a suspected two-protein interaction
is required, a synthetic lethality approach may be appropriate. This method involves
the construction of cells containing two mutations, one in each of a suspected PPI
pair. Neither of the mutations alone results in loss of cell viability, but if both should
occur in the same cell, death results. Another established method used in PPI re-
search is the two-hybrid approach, first demonstrated using yeast strains and subse-
quently adapted for use with mammalian and human cell lines. The yeast two-hybrid
methodology utilizes a nutrient-dependent yeast strain into which separate bait and
prey plasmids are introduced. One plasmid produces a known protein (the bait) with
a fused DNA-binding domain (BD) fragment while the other plasmid produces a pro-
tein product in which an activation domain (AD) fragment is fused onto
“In recent years, innovations
in software, reagents, and
instrumentation as well as
improvements to experimental
protocols have given a clearer
understanding of the biological
roles of many PPIs.”
Look for these upcoming Articles
Robotics/Automation — January 18
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continued »
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AAAS/Science Business Office FeatureProtein-Protein Interactions
the so-called prey protein. The prey protein can be either a single
known protein or a library of known or unknown proteins. If the bait
and prey proteins bind, transcription of a reporter gene takes place,
indicating the formation of a PPI.
This “bait and prey” approach has been incorporated into a vari-
ety of commercial reagent formats. BD Biosciences markets the BD
Matchmaker Two-Hybrid System 3, an enhanced GAL4 two-hybrid
system. The product uses yeast strain AH109, in which four reporter
genes are integrated in the host genome. A similar system, DUAL-
membrane from Dualsystems Biotech, is specifically designed to
detect interactions involving integral membrane proteins with other
such proteins, membrane-associated proteins, or soluble proteins.
A human cell-based “bait-prey” PPI assay system, known as GRIP, is
produced by Bioimage (part of Thermo Fisher Scientific). The GRIP
technology is based on the translocation of human cAMP phos-
phodiesterase PDE4A4 and provides a high throughput method to
screen for inhibitors of protein interactions.
Another in vivo method for visualizing PPIs involves use of the
fluorescence resonance energy transfer (FRET) approach. In this
technique, two different fluorescent molecules (fluorophores)—the
donor and acceptor—are genetically fused to the two proteins of in-
terest. Regular fluorescence occurs when the protein-bound fluoro-
phores emit energy at the emission frequency. When the two labeled
proteins interact and are stimulated by light energy at the excitation
frequency for the donor fluorophore, some of this energy transfers to
the acceptor, which then re-emits the light at its own emission wave-
length. The result is that the donor partner in the PPI emits less light
energy, while the acceptor emits more. FRET equipped microscopes
and fluorescence-based cell sorter systems are used in conjunction
with these FRET reagents to quantify the PPIs.
isolating Protein Pairs
In studies where the isolation of proteins involved in a PPI is
desired, “pull down” assays can be used. Pierce Biotechnology (now
a part of Thermo Fisher Scientific) offers its ProFound PPI pull down
kits for this application. The product is based on either GST- or His-
tagged fusion proteins. The tagged fusion protein is used as the “bait”
protein and the pull-down process is based on a bead-based, affinity
purification technique. The captured PPIs are eluted for analysis by
Western blot.
For recovering interacting proteins from mammalian cells,
Stratagene, an Agilent technologies company, produces its InterPlay
Mammalian TAP System. The method is based on expression of a
protein of interest fused to two affinity tags: a streptavidin binding
peptide (SBP) and a calmodulin binding peptide (CBP). “A two-step
tandem affinity purification protocol yields exceptionally pure and
intact interacting proteins through gentle elution and elimination of a
protease digestion step,” says Benjamin Pricer, the product manager
for functional biology at Stratagene. “The isolated proteins,”
he continues, “can be identified using Western blotting or mass
spectrometry.”
Array Screening
Protein arrays are another, more recent addition to the PPI toolbox.
As in the two-hybrid approach, a “bait and prey” strategy is used.
The methods involve incubating an array of surface anchored pro-
teins with cell supernatant, washing the assay surface, and analyz-
ing for PPIs. A variety of anchoring methods and formats is used
including glass slides, polymeric beads, and chromatographic me-
dia. hypromatrix supplies a general purpose PPI screening array
called the AntibodyArray, which comprises membrane tethered an-
tibodies against hundreds of well-studied proteins. The antigenic
protein binds to the antibody, thus capturing any PPIs between the
antigen protein and its interacting partners. Similarly, invitrogen’s
ProtoArray Human Protein Microarray contains approximately 8,000
unique human proteins, selected from multiple gene families and ar-
rayed in duplicate on a 1 inch x 3 inch nitrocellulose-coated glass
slide. To provide maximum flexibility, these microarrays are compat-
ible with fluorescent, chemiluminescent, radioisotopic, and other
detection methods.
Specialty arrays for PPI screening are also available and include
Sigma-Aldrich’s Panoram Human Cancer v1 Protein Array used to
screen for interactions with 130 fully functional cancer proteins.
Also, Panomics’ TF Protein Array is useful for determining how a
particular protein interacts with the 140 known transcription factor
proteins spotted on this array.
instrumentation for Bound Proteins
Over the past 15 years, specific instrumentation has evolved to mea-
sure the physiochemical properties of known interacting proteins.
These instruments are based on crystal resonance, surface plasmon
energy measurements, microcalorimetry, scanning tunneling micros-
copy, and total internal reflection fluorescence.
The principle of crystal resonance has been applied by Attana
to develop a quartz crystal microbalance (QCM) technology to ob-
tain label-free, real-time measurements of PPI kinetics, affinity, and
specificity. QCM uses a piezoelectric effect to oscillate a crystal at
its resonance frequency. This frequency shifts when molecules are
added to or removed from the surface of the crystal. By attaching a
specific bait protein to the biotin treated crystal surface,
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life science technologies AAAS/Science Business Office Feature
Protein-Protein Interactions
Agilent technologies
www.agilent.com
Attana
www.attana.com
BD Biosciences
www.bdbiosciences.com
Bioimage (part of thermo
Fisher Scientific)
www.bioimage.com
Bio-Rad
www.bio-rad.com
Dualsystems Biotech
www.dualsystems.com
Ge healthcare/Biacore
www.biacore.com
hypromatrix
www.hypromatrix.com
ingenuity Systems
www.ingenuity.com
Featured Participants
invitrogen
www.invitrogen.com
Microcal
www.microcal.com
Panomics
www.panomics.com
Pierce Biotechnology (part of
thermo Fisher Scientific)
www.piercenet.com
Sigma-Aldrich
www.sigmaaldrich.com
Stratagene
www.stratagene.com
tiRF technologies
www.tirftechnologies.com
Yale university
www.yale.edu
RNAi
life science technologiesAAAS/Science Business Office Feature
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Protein-Protein Interactions
an interaction with an introduced prey protein can be detected due
to the resulting change in protein mass.
Taking a slightly different tack, two companies, Ge healthcare/
Biacore Systems and Bio-Rad laboratories, market instrumentation
based on surface plasmon resonance (SPR). These instruments use
a gold foil detector surface and reflected light energy to generate
surface electron charge density waves (plasmons). PPIs forming be-
tween surface-tethered proteins and introduced proteins on the de-
tector surface interact with these plasmons resulting in measurable
refractive index changes that are proportional to changes in surface
mass. Stefan Löfås, chief scientist, Biacore Systems, GE Healthcare
Bio-Sciences AB, explains that “the combination of label-free SPR
detection, sophisticated microfluidics and a broad range of surface
chemistries allows characterization of almost any type of biomo-
lecular interaction at a very detailed level. The kinetic information
obtained greatly facilitates the characterization of molecular mecha-
nisms and biological processes.”
The introduction of higher throughput screening capacities
for SPR instruments is a focus for Biacore and Bio-Rad. As Cathy
Mainini, senior product manager, protein function division, Bio-Rad
Laboratories, points out, “We have combined surface plasmon reso-
nance technology with an innovative 6 x 6 microfluidic design that al-
lows one to measure an array of 36 biomolecular interactions simul-
taneously. This parallel approach can generate a complete kinetic
profile of a biomolecular interaction in a single experiment without
regeneration.”
tiRF technologies’ instruments are based on total internal reflec-
tion fluorescence (TIRF). The process uses light propagating within a
quartz crystal which, when it reaches an interface with a less dense
aqueous solution, although fully reflected, generates an evanescent
field that extends beyond the interface and into the aqueous solu-
tion. The fluorophores adsorbed, adhered, or bound to the surface
will fluoresce while fluorophores in bulk solution will not. The sensi-
tivity of the TIRF approach is reported to be 10,000 times higher than
SPR-based instruments.
Based on the scanning tunneling microscopy—the inventors of
which received half of the 1986 Nobel Prize in Physics—the atomic
force microscope (AFM) is an imaging and measurement tool that
provides a three-dimensional map of a sample’s surface. The AFM
instrument employs a sensitive probe, the deflection of which is a
measure of sample surface topography. With the capacity to observe
and manipulate biological surfaces under physiological conditions,
AFM can be used to explore biological structures at the single mole-
cule level and measure mechanical ligand-receptor interactions with
3D resolution and sensitivity down in the picoNewton (N-12) range.
W. Travis Johnson, senior scientist at Agilent Technologies, explains,
“We are combining AFM with surface chemistry and bioconjugation
chemistry in order to study individual, discrete ligand-receptor inter-
actions far from equilibrium. This is increasing our understanding of
how biological systems work at the single molecule level.”
instrumentation for Proteins in Solution
Microcal has found a way to adapt ultrasensitive calorimetry to the
study of PPIs. Utilizing isothermal titration calorimetry (ITC), the in-
strument measures the heat that is absorbed or generated when a
biomolecular interaction occurs. Uniquely, the method does not re-
quire that the target protein be labeled or bound to a surface, allow-
ing the proteins to be studied in solution in a native state. Ernesto
Freire, Henry Walters Professor, Biology and Biophysics, Johns Hop-
kins University, reports his laboratory uses “ITC in all our projects
involving protein-protein interactions. ITC not only provides the most
accurate determination of binding affinity, but also it is the only tech-
nique that reveals the nature and magnitude of the forces involved in
the binding process by being able to measure the binding enthalpy
and entropy in a single experiment.”
Bringing it All together
Understanding of the physiological and disease associated impor-
tance of protein-protein interactions continues to expand. For exam-
ple, researchers recently reported using a Biacore system to gener-
ate binding kinetics measurements to identify a site on the HIV-1 Env
protein that may be a target for vaccine development. PPI research is
moving forward aided by innovations in instrumentation, availability
of reagents, and the generation of robust data sets.
PPI research is also benefitting from advances in knowledge
software such as ingenuity Systems’ Pathways Analysis. Megan
Laurance, senior scientist at Ingenuity Systems, points out “our soft-
ware application contains a comprehensive network of protein-pro-
tein interactions and regulatory events [transcriptional effects, post-
translational modifications, epigenetic events] manually curated
from the scientific literature.” This enables researchers to put the
particular PPI under investigation into a broader context of normal
and abnormal cellular function. As Laurance puts it, this allows them
to “rapidly understand their experimental system as a whole, based
on what was detected at the protein interaction level.”
Emphasizing the importance of depending on multiple technolo-
gies, Michael Snyder of Yale university points out that in his labo-
ratory “we use a number of methods including two-hybrid, protein
arrays, affinity chromatography, and SPR to investigate the biologi-
cal significance of PPIs. All of these methods have their inherent
limitations; however, by integrating the resulting datasets we gain
confidence in our understanding of the function and importance
of specific PPIs.” These technologies are enabling exciting new re-
search on the characterization of multiprotein interactions involving
“scaffold” proteins responsible for bringing other proteins together
so they can interact.
Continued advancements in the ability to measure the various
parameters associated with PPIs coupled with knowledge-based
software will undoubtedly provide the understanding required for
systems biology modeling and serve to uncover new targets for ther-
apeutic developments.
Alfred Doig is a freelance bioscience writer and editor living in Natick, MA.
Doi: 10.1126/science.opms.p0700020
“The AFM instrument
employs a sensitive probe,
the deflection of which is a
measure of sample surface
topography.”
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Trypsin Removal
Mag-Trypsin provides a method for magnetically immobilizing tryp-
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