Givaudan-Roure Lecture - Association for Chemoreception Sciences

125 Slide [ ] Odorant Receptors & Transduction
DNA-BASED FLUORESCENT CHEMOSENSORS FOR DIRECT
DETECTION OF VOLATILE COMPOUNDS IN AN
ARTIFICIAL NOSE
White J.E. 1, Williams L.B. 1, Atkisson M.S. 2, Kauer J.S. 1 1Neuroscience,
Tufts University School of Medicine, Boston, MA; 2CogniScent, inc.,
Weston, MA
As reported previously (White et al., 2002, AChemS XXIV), we are
developing a portable artificial olfactory system to detect and
identify volatile compounds in the ambient environment. In a manner
based directly on animal behavior, the device actively samples air via
pulsatile sniffing. Air samples are drawn over an array of
broadly-responsive, optically-based chemical sensors, which are used
to detect and discriminate odorants in the sample. Device function
thus depends directly on the sensitivity and diversity of the sensors
in the array. Biopolymers, particularly nucleic acids, are attractive
for building sensors of great variety and wide utility. This is
because of the tremendous combinatorial complexity made possible
by constructing them from sequences of just a few building blocks. We
have found that dye-labeled DNA sensors, dried onto a substrate,
differentially respond to volatile chemical compounds. To our
knowledge, this is the first time that solid phase DNA-based sensors
have been used for directly detecting small, vapor phase molecules.
Furthermore, our experiments indicate that Cy3-labeled, single- strand
DNA sequences of similar length but different base sequence can
respond differently to the same odorant set. These observations
suggests that "sensor discovery" via high throughput screening of
large-scale DNA-based sensor libraries may be possible, thereby
providing a rapid means to easily identify sensors that are optimized
for defined odorant detection tasks. Supported by NIDCD and NSF.
32
126 Slide [ ] Odorant Receptors & Transduction
MAKING SENSE OF OLFACTION THROUGH MOLECULAR
MODELING
Floriano W.B. 1, Hall S. 1, Leonard O. 2, Hummel P.A. 1, Vaidehi N. 1,
Goddard W.A. 1 1Materials and Process Simulation Center, California
Institute of Technology, Pasadena, CA; 2University of California -
Berkeley, Berkeley, CA
We used the MembStruk first principles computational technique to
predict the three-dimensional (3D) structure of ten mouse olfactory
receptors (S6, S18, S19, S25, S46, S50, S1, MOR-EV, MOR-EG, and
MI7) for which experimental odorant recognition profiles are available.
We used the HierDock method to scan each predicted OR structure for
potential odorant binding site(s), and to calculate binding energies of
each odorant in these binding sites. The calculated binding affinity
profiles correctly identify the chemical classes recognized
experimentally by each OR, validating the predicted 3D structures and
binding sites. Correlation between calculated binding affinity and
elicited response is also found within each chemical class. However the
cutoff response/no-response is not always well defined.
For each of the ten ORs, the binding site is located between TMs 3
through 6, with contributions from EC loops 2 and 3. In particular, we
find six residue positions in TM3 and TM6 to be consistently involved
in odorant binding. These positions are consistent with mutation data on
ligand binding for other GPCRs and sequence hypervariability studies
for ORs.
Amino acid patterns associated with the recognition of chemical
classes were defined using the predicted binding modes. These
sequence fingerprints were used to probe the alignment of 869 OR
sequences from the mouse genome in order to identify other ORs
matching each fingerprint.
This research was initiated with support by an ARO-MURI grant
(Dr. Robert Campbell) and completed with finding from NIHBRGRO1-
GM625523, NIH-R29AI40567, and NIH-HD36385. The computational
facilities were provided by a SUR grant from IBM and a DURIP grant
from ARO.
127 Slide [ ] Odorant Receptors & Transduction
THE MULTIFACETED RECEPTORS OF THE HUMAN NOSE
Lancet D. 1, Alony R. 1, Atarot T. 1, Ben-Asher E. 1, Feldmesser E. 1, Gilad
Y. 1, Khen M. 1, Man O. 1, Menashe I. 1, Olender T. 1, Stern S. 1 1Molecular
Genetics, Weizmann Institute, Rehovot, Israel
Human olfactory receptor (OR) genes are highly multifaceted, as
manifested at several different levels. This is documented in the
updated Human Olfactory Receptor Data Exploratorium (HORDE
version 40, http://bip.weizmann.ac.il/HORDE/), which contains 853
entries. One dimension of diversity spans a continuum between intact
ORs and definite OR pseudogenes. To probe the undecided cases, we
have devised methods to identify subtle deviations from shared motifs.
Positional departures of initiation and stop codons may indicate inactive
ORs. Second generation rhodopsin-based homology models provide
new clues to sequence positions essential for functionally intact
structure, including helix-kinking residues. A new algorithm, based on
paralog-orthologs comparisons for three mammalian species (human,
mouse and dog), allowed us to identify potential odorant contact
residues. These harbor maximal intra-species variability, but also
constitute additional targets for inactivating mutations. Another
dimension of diversity is inter-individual variability, generated by
single nucleotide polymorphisms that occur at pseudogenizing sequence
positions. This results in a personal genetic “bar-code”, whereby every
human individual has a different combination of intact and inactive
ORs. These “segregating pseudogenes” are likely key determinants of
specific anosmia. Finally, OR gene diversity is probed at the 5´
untranslated exon sequences by computer-based and experimental
approaches. This may shed light on presumed roles of upstream
segments in OR messenger RNA stability and translation.