4 research outputs found
Probing Structure and Function of Ion Channels Using Limited Proteolysis and Microfluidics
Even though gain, loss, or modulation of ion channel function is implicated in many diseases, both rare and common, the development of new pharmaceuticals targeting this class has been disappointing, where it has been a major problem to obtain correlated structural and functional information. Here, we present a microfluidic method in which the ion channel TRPV1, contained in proteoliposomes or in excised patches, was exposed to limited trypsin proteolysis. Cleaved-off peptides were identified by MS, and electrophysiological properties were recorded by patch clamp. Thus, the structure-function relationship was evaluated by correlating changes in function with removal of structural elements. Using this approach, we pinpointed regions of TRPV1 that affect channel properties upon their removal, causing changes in current amplitude, single-channel conductance, and EC50 value toward its agonist, capsaicin. We have provided a fast "shotgun" method for chemical truncation of a membrane protein, which allows for functional assessments of various peptide regions
Probing Structure and Function of Ion Channels Using Limited Proteolysis and Microfluidics
Even though gain, loss, or modulation
of ion channel function is
implicated in many diseases, both rare and common, the development
of new pharmaceuticals targeting this class has been disappointing,
where it has been a major problem to obtain correlated structural
and functional information. Here, we present a microÂfluidic
method in which the ion channel TRPV1, contained in proteoÂliposomes
or in excised patches, was exposed to limited trypsin proteolysis.
Cleaved-off peptides were identified by MS, and electroÂphysiological
properties were recorded by patch clamp. Thus, the structureâfunction
relationship was evaluated by correlating changes in function with
removal of structural elements. Using this approach, we pinpointed
regions of TRPV1 that affect channel properties upon their removal,
causing changes in current amplitude, single-channel conductance,
and EC<sub>50</sub> value toward its agonist, capsaicin. We have provided
a fast âshotgunâ method for chemical truncation of a
membrane protein, which allows for functional assessments of various
peptide regions
Microfluidic Flow Cell for Sequential Digestion of Immobilized Proteoliposomes
We have developed a microfluidic flow cell where stepwise
enzymatic
digestion is performed on immobilized proteoliposomes and the resulting
cleaved peptides are analyzed with liquid chromatographyâtandem
mass spectrometry (LCâMS/MS). The flow cell channels consist
of two parallel gold surfaces mounted face to face with a thin spacer
and feature an inlet and an outlet port. Proteoliposomes (50â150
nm in diameter) obtained from red blood cells (RBC), or Chinese hamster
ovary (CHO) cells, were immobilized on the inside of the flow cell
channel, thus forming a stationary phase of proteoliposomes. The rate
of proteoliposome immobilization was determined using a quartz crystal
microbalance with dissipation monitoring (QCM-D) which showed that
95% of the proteoliposomes bind within 5 min. The flow cell was found
to bind a maximum of 1 ÎŒg proteoliposomes/cm<sup>2</sup>, and
a minimum proteoliposome concentration required for saturation of
the flow cell was determined to be 500 ÎŒg/mL. Atomic force microscopy
(AFM) studies showed an even distribution of immobilized proteoliposomes
on the surface. The liquid encapsulated between the surfaces has a
large surface-to-volume ratio, providing rapid material transfer rates
between the liquid phase and the stationary phase. We characterized
the hydrodynamic properties of the flow cell, and the force acting
on the proteoliposomes during flow cell operation was estimated to
be in the range of 0.1â1 pN, too small to cause any proteoliposome
deformation or rupture. A sequential proteolytic protocol, repeatedly
exposing proteoliposomes to a digestive enzyme, trypsin, was developed
and compared with a single-digest protocol. The sequential protocol
was found to detect âŒ65% more unique membrane-associated protein
(<i>p</i> < 0.001, <i>n</i> = 6) based on peptide
analysis with LCâMS/MS, compared to a single-digest protocol.
Thus, the flow cell described herein is a suitable tool for shotgun
proteomics on proteoliposomes, enabling more detailed characterization
of complex protein samples