2 research outputs found
Identification of Destabilizing and Stabilizing Mutations of Ste2p, a G Protein-Coupled Receptor in <i>Saccharomyces cerevisiae</i>
The
isolation of mutations affecting the stabilities of transmembrane
proteins is useful for enhancing the suitability of proteins for structural
characterization and identification of determinants of membrane protein
stability. We have pursued a strategy for the identification of stabilized
variants of the yeast α-factor receptor Ste2p. Because it was
not possible to screen directly for mutations providing thermal stabilization,
we first isolated a battery of destabilized temperature-sensitive
variants, based on loss of signaling function and decreased levels
of binding of the fluorescent ligand, and then screened for intragenic
second-site suppressors of these phenotypes. The initial screens recovered
singly and multiply substituted mutations conferring temperature sensitivity
throughout the predicted transmembrane helices of the receptor. All
of the singly substituted variants exhibit decreases in cell-surface
expression. We then screened randomly mutagenized libraries of clones
expressing temperature-sensitive variants for second-site suppressors
that restore elevated levels of binding sites for fluorescent ligand.
To determine whether any of these were global suppressors, and thus
likely stabilizing mutations, they were combined with different temperature-sensitive
mutations. Eight of the suppressors exhibited the ability to reverse
the defect in ligand binding of multiple temperature-sensitive mutations.
Combining certain suppressors into a single allele resulted in levels
of suppression greater than that seen with either suppressor alone.
Solubilized receptors containing suppressor mutations in the absence
of temperature-sensitive mutations exhibit a reduced tendency to aggregate
during immobilization on an affinity matrix. Several of the suppressors
also exhibit allele-specific behavior indicative of specific intramolecular
interactions in the receptor
Nanochannel-Based Single Molecule Recycling
We present a method for measuring the fluorescence from
a single
molecule hundreds of times without surface immobilization. The approach
is based on the use of electroosmosis to repeatedly drive a single
target molecule in a fused silica nanochannel through a stationary
laser focus. Single molecule fluorescence detected during the transit
time through the laser focus is used to repeatedly reverse the electrical
potential controlling the flow direction. Our method does not rely
on continuous observation and therefore is less susceptible to fluorescence
blinking than existing fluorescence-based trapping schemes. The variation
in the turnaround times can be used to measure the diffusion coefficient
on a single molecule level. We demonstrate the ability to recycle
both proteins and DNA in nanochannels and show that the procedure
can be combined with single-pair Förster energy transfer. Nanochannel-based
single molecule recycling holds promise for studying conformational
dynamics on the same single molecule in solution and without surface
tethering