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Proteins
adorning the surfaces of human cells perform an array of essential
functions, including cell signaling, communication and the transport of
vital substances into and out of cells. They are critical targets for
drug delivery and many proteins are now being identified as disease
biomarkers—early warning beacons announcing the pre-symptomatic presence
of cancers and other diseases.
While
study of the binding properties of membrane proteins is essential,
detailed analysis of these complex entities is tricky. Now, Nongjian
(NJ) Tao, professor of electrical engineering, and director of the
Center for Bioelectronics and Biosensors at ASU’s Biodesign Institute,
has devised a new technique for examining the binding kinetics of
membrane proteins.
“This
is a very important but very difficult problem to solve,” Tao notes.
“We demonstrate a new method of approaching the issue, which provides a
quantitative analysis of protein interactions on the surface of a cell.”
The
technique—known as SPR microscopy—holds the potential to simplify the
study of membrane proteins, thereby streamlining the development of new
drugs, aiding the identification of diagnostic biomarkers and improving
the understanding of cell-pathogen interactions.
The group’s results appear in this week’s advanced online issue of the journal Nature Chemistry.
Typically,
proteins attached to or embedded in the cell membrane’s lipid bilayer
are either tagged with fluorescent markers or extracted from their
locations, purified and immobilized on a glass surface in protein
microarrays. These efforts may not accurately reflect native
configuration and function.
Membrane
proteins are complex structures whose subtle performance is often
related to alterations in conformation and the particular binding
kinetics at work. Existing techniques using florescent markers have been
applied to pinpoint binding events, but these only permit the
visualization of the protein before and after binding, omitting the
dynamic processes evolving over time. Further, the use of fluorescent
labels to tag protein molecules can interfere with the processes
researchers hope to observe.
Alternately,
proteins are extracted, purified and affixed to microarray slides—a
labor-intensive process that removes proteins from their native
environment, potentially affecting the shapes they naturally assume in
situ and/or altering protein function.
In
the current study, a label-free imaging technique is applied in situ to
membrane proteins, which are visualized using a property known as
surface plasmon resonance. This effect occurs when polarized light
strikes the surface of a glass slide coated with a thin metallic film of
gold. Under proper conditions of wavelength, polarization and incident
angle, free electrons in the metal film absorb incident photons,
converting them into plasmon waves, which propagate much like waves in
water.
When
nanoscale phenomena, including membrane proteins, interact and disrupt
plasmon waves, they cause a measurable change in light reflectivity,
which the new microscopy method converts into an image.
Surface
plasmon resonance had already been applied to extracted proteins to
study binding kinetics, though Tao explains that many steps are required
and proteins may lose their proper conformational characteristics. This
is particularly true for proteins normally embedded in a cell
membrane’s lipid matrix.
Another
important consideration for the study of membrane proteins is the fact
that that they arrange themselves heterogeneously across membrane
surfaces and modify their distribution during various cellular
activities. This behavior is particularly important during a process
known as chemotaxis, when cells direct their movements under the
influence of chemicals in the surrounding environment. For this reason, a
tool allowing for both spatial and temporal study of membrane protein
distribution in real time is highly desirable.
Tao’s
method uses surface plasmon resonance to provide high-resolution
spatial and temporal information, and also allows for simultaneous
optical and fluorescence observation of the sample, combining the
advantages of both label-based and label-free methods.
High
spatial resolution proved particularly useful for observing the ways
polarized membrane proteins (bearing hydrophobic and hydrophilic
regions) rearrange themselves, assisting cell migration directed by
surrounding chemicals. The phenomenon also plays an important role
during immune recognition. Using SPR microscopy, the spatial
distribution of membrane proteins in single cells during chemotaxis
could be mapped in detail for the first time, using a chemoattractant to
induce cell migration.
Cells
for study are cultured directly on a gold-coated slide, which can be
subjected to simultaneous bright-field, florescent and SPR imaging. A
liquid containing binding ligands is then applied over cells and the
binding events with cell surface proteins monitored with SPR.
The
technique permits millisecond resolution of temporal events and
sub-micron scale analysis of spatial distribution. (See Figure 1b). In
the current study, the method examined the binding of membrane
glycoproteins with lectin ligands, the spatial distribution of membrane
receptor molecules and membrane protein polarization and redistribution
events.
The
versatility of the new method, allowing for simultaneous imaging in
optical, fluorescent and SPR modes, promises to significantly expand the
study of membrane proteins in their native state, improving the
understanding of protein binding kinetics and speeding the development
of drugs targeting membrane proteins.
Tao
stresses that such techniques—by more closely approximating in vivo
conditions—provide a valuable window into biological processes relevant
to health and disease: “Cells are different from tissues which are
different from human beings, but at least now we can move from a system
on the surface of a glass slide to an actual cell surface.”
Source: Arizona State University
