Olfaction: Topics in Chemical Biology
Charles S. Sell, Givaudan, Kent, England
Smell is an intriguing sense in many ways. Although it is not the dominant sense in humans, it has affects on emotion and memory which can be very profound. Odor has long baffled chemists who try to model it in mechanistic terms, and a great deal of research has been invested in trying to understand how various features of molecular structure provide different odor characters and intensities. Many models for prediction of odor properties from chemical structure exist, but none are accurate and precise consistently. The reasons for this are now becoming clearer. The greatest breakthroughs in our understanding of olfaction came with the discoveries of the gene family coding for the olfactory receptor proteins (1) and the combinatorial mode of operation of these receptors (2). Odorous molecules enter the nose either from the front, by inhalation, or from the back, by diffusion from the mouth and respiratory tract. In the nose, they interact with an array of receptors. Each receptor type responds to a range of odorants, and each odorant stimulates a range of receptors. The signals thus generated are coded onto centers, known as glomeruli, in the olfactory bulb. The olfactory signals pass on upward through the brain and are interpreted finally, in the cortex, as odor. During the neurotransmission process, many interactions occur between olfactory signals and cortical feedback mechanisms, which include effects from other senses. Therefore, it is not surprising that simple structure/odor correlations are so elusive.
Smell is the sense by which animals detect and discriminate volatile, odorous chemicals in the environment. Although it is not their dominant sense, smell affects humans in various ways. At a basic survival level, it is important to find food and to determine its quality. Smell also plays a role in triggering emotions and memories and, in addition, is important at the hedonic level. For instance, the smell of consumer goods, such as soaps, cosmetic creams, and laundry powders, is a key factor in determining consumer preference and so commercial as well as scientific reasons, exist for our desire to understand the phenomenon of odor. Performance and safety constraints lead to a continual need for new fragrance ingredients, and this, in turn, leads to a need to understand the relationship between molecular structure and odor. As with odor per se, both commercial and scientific motives drive the search to understand of these relationships, and much research has been devoted to the subject. Many structure/odor models have been developed, but they are essentially statistical in nature and recent developments in our understanding of olfaction suggest that they will remain so for the foreseeable future.
Biologic Aspects of Olfaction
The process of olfaction begins when odorant molecules enter the nasal cavity either by inhaled air or by diffusion from the mouth. In the nose, these molecules activate receptors in the olfactory epithelium. The signals from the olfactory receptor cells are picked up by the olfactory bulb where mapping of receptor types onto glomeruli occurs. The signal patterns leave the olfactory bulb, travel upward through the brain, and are interpreted eventually as odor in the cortex. Within tens of milliseconds of presentation of an odorant to the nose, olfactory signals are generated in the epithelium; cortical activity is detectable within 100 milliseconds. Figure 1 shows the location of some key olfactory organs in humans.
Figure 1. Organs involved in human olfaction (with permission from Givaudan).
What is odor?
Smell probably is the oldest of our senses. The ability to detect and to recognize specific chemicals gives even single-celled organisms useful information about their environment. For example, it is known (3) that sperm will swim toward certain odorous substances. The ability to analyze their environment by chemosensation allows organisms to identify opportunities and threats from chemical changes in that environment; therefore, chemosensation has an obvious survival value. Continual refinement of the mechanism throughout evolution has led to the highly developed sense that we humans know as smell. In this refined sense, usually odors are not derived from single chemical entities but from complex mixtures, and each mixture is perceived as a separate odor image (4, p. 63). Traditionally, the sense of smell is associated with the nose because that is where the signals are detected and research has tended to focus on the receptor events. However, it is now clear that the phenomenon of odor exists only in the higher brain and is a synthesis of inputs from the olfactory receptors, other senses, and various cortical feedback mechanisms. As stated by Wilson and Stevenson (4, p. 34), “With a relatively few exceptions, neither odor physico-chemical feature extraction at the receptor sheet, nor spatial maps of those features in the olfactory bulb, nor simple convergence of those features in cortical circuits are sufficient to account for the rich experience that is olfaction.”
We tend to talk about the nose as a single entity; yet a septum exists that divides it into two physically separate cavities. On the roof of each of these cavities, and extending down onto the septum, is a patch of tissue known as the olfactory epithelium. Odorants can reach the epithelium either from in front, by inhalation of air from the environment (referred to as the orthonasal route), or by diffusion from the mouth and respiratory and gustatory tracts (referred to as the retronasal route). The latter is vital in flavor detection as the tongue contains receptors only for sweet, sour, salt, bitter, and umami tastes; the rest of what is referred to normally as taste, actually, is retronasal smell. On the sides of the nasal cavities are bony plates called turbinates that cause turbulence in the air flow and ensure that odorants reach the epithelia. The air flow through each nostril is always different because one is faster than the other; the fast flow alternates continually from one nostril to the other (5). This alternation provides a mechanism for increased sensitivity and discrimination based on transport phenomena because the timing and pattern of signal spread across the epithelium will depend, in part, on the air flow. It has been shown that information on recognition features is exchanged at a higher level in the brain because a molecule whose odor has been learned using one nostril only will be recognized by the other nostril (6).
The olfactory epithelium and the olfactory receptors
The olfactory epithelium is a patch of greenish-yellow tissue several square centimeters in area and 100-200 micrometers thick. It contains receptor cells that run from the nasal cavity through the base of the skull (the cribriform of ethmoid) into the olfactory bulb. On the side of the nasal cavity, the receptor cells have hairs or cilia that are 20-200 micrometers long. These cilia are bathed in a mucus layer that is 35 micrometers thick and flows backward continually at a rate of 1-6 centimeters/minute. The receptor proteins are expressed in the cilia of the receptor cells. Individual receptor cells fire spontaneously at a rate of 3-60 impulses/second; this rate of firing is increased when the cells are stimulated by an odorant.
The mucus also contains cytochrome P450s, which are oxidative enzymes, and proteins known as odor-binding proteins. The odor-binding proteins belong to the family of lipocalins. The role in olfaction of these two classes of proteins is not certain. It is possible that they serve only to remove excess odorants and therefore contribute to provision of the time dimension of olfaction, which is an important feature in survival. However, other roles have also been postulated and will be discussed below.
The olfactory receptor proteins (ORs) belong to the family of 7-transmembrane G-protein coupled receptors (GPCRs). The gene family coding for the receptor proteins is the largest in the genome, which contains codes for over 1000 proteins (1). Most mammals express 800-900 of these proteins, but humans use a much reduced set, which contains only 350-400 proteins. Interestingly, the only other mammals (gorillas, chimpanzees, orangutans, and rhesus macaques) with such a restricted set of proteins are the only other mammals to have color vision. Variation between humans is such that statistically it is unlikely that any two humans use the same set of olfactory receptors (7). Events that follow receptor activation involve the normal train of G-protein (G-olf, in the case of olfaction), second messenger (cAMP or IP3, in the case of olfaction), and ion channel chemistry, which leads to polarization of the cell and hence generation of a discharge at the synapse that leads to the olfactory bulb (8). Intriguingly, it has been shown that the signal pattern elicited by a mixture of two substances is not necessarily a simple additive of the signals elicited by the two components individually, but receptors that are not activated by either component can be activated by the mixture (9).
The olfactory bulb
The olfactory bulb sits at the base of the brain on the cribriform plate. Humans have two olfactory bulbs, the right bulb that receives signals from the right olfactory epithelium and the left bulb that receives signals from the left olfactory epithelium. In the bulb, centers called glomeruli exist, each of which receives signals from only one type of receptor cell, irrespective of where those receptors are located on the epithelium. Olfactory receptor cells have short lifetimes (about 2 weeks), and new receptor cells develop in such a way that each makes its connection with the correct glomerulus. Receptor types with similar substrate selectivities tend to be associated with glomeruli that are found close to each other in the olfactory bulb.
Olfactory signals that leave the olfactory bulbs travel by several routes to the higher centers of the brain where the phenomenon of odor develops eventually. Remarkably, the architecture of the olfactory parts of the brain is consistent across all mammalian species; research on other animals throws considerable light on the function in humans. The entire process is well reviewed by Wilson and Stevenson (4) and by Delano and Sobel (10).
The signals travel initially to the piriform cortex and the amygdala; hence, both the thalamus and the limbic system are involved. It is postulated that the direct link to the limbic system accounts for the influence of odor on memory and emotion. From the piriform cortex, signals go to the amygdala, then to the thalamus, and directly to the orbitofrontal cortex. Processed signals from the thalamus also go to the orbitofrontal cortex. This structure also receives signals directly from the amygdala. Many of these signal pathways are two-way in nature with signals that come down from higher centers that affect the ascending signals. This accounts for the well-known effects of experience, expectancy, and context in distorting odor perception. A classic example is the inability of experts to describe the aroma of a white wine correctly to which a tasteless red dye has been added (11). Interference from other senses is also important to determine the ultimate odor percept. For instance, 70% of odorants also stimulate the trigeminal system in the nasal cavity and visual input has been shown to affect olfactory signal processing (12). Essentially, recognition of “odor” is a process of matching pattern of this final signal combination against reference patterns stored in the brain. For example, in perfumery, it is well known that development of an odor language is essential to train perfumers, and their powers of discrimination improve as their language improves.
Anosmia, the inability to smell, can be divided into two classes. General anosmia, the inability to smell any odors at all, usually is the result of disease or accident. More common is specific anosmia, in which an individual either cannot detect a specific chemical substance that most people can detect or displays a threshold of detection for it which is significantly above the normal range. At one time, specific anosmias were linked to the concept of primary odors (13), but confirmation of the combinatorial mechanism of olfaction has put paid to this concept. Interestingly, it has been demonstrated that exposure to the substance can affect anosmia and individuals can begin to smell materials to which they were previously anosmic. This effect has been demonstrated for androstenone, amyl acetate, geranyl nitrile, and isoborneol (14-18).
Chemical Aspects of Olfaction
For various reasons, it is very difficult to develop an understanding of the chemistry involved in olfaction. The olfactory receptors are found in the membrane of the receptor cells; therefore, their active states are not amenable to structural determination by X-ray diffraction or other physical tools. Odor is a mental image rather than a physical property that can be measured and quantified. Odor perception is a multistep process. Olfaction is combinatorial in nature. All of these facts indicate that building of either substrate or receptor models are fraught with significant difficulties.
Odor, however, is a very obvious property of any chemical compound, and thus, speculation about the mechanism of perception is very tempting. Experienced fragrance chemists can predict odor type with much better than random accuracy, and a commercial driver exists in terms of design of novel materials for the fragrance industry. Therefore, it is not surprising that many structure/odor correlations and olfaction models have been reported and debated, often very hotly, in the literature.
Most olfaction models in the literature are far too simplistic and too mechanical in nature, and none of them have succeeded in accounting for all of the observations about olfaction. As described, recent advances in our understanding have confirmed that odor perception, as predicted by Polak (19), starts with a combinatorial mechanism at the receptor level (1) and involves pattern recognition in the higher brain (4). No single odorant-receptor interaction will be the sole determinant of odor percept, and even knowledge of the pattern elicited at the olfactory bulb is insufficient to enable prediction of the cortical image of odor. Therefore, structure/odor models are and, for the foreseeable future, will remain statistical tools rather than mechanistic indicators.
To understand olfaction at a chemical level, it is necessary to have good data that link chemical structure to odor properties. This task is much more difficult than it would seem, for instance, to a chemist who sniffs a sample that he has just synthesized in the laboratory and applies an odor descriptor to the molecular structure of his synthetic target. These difficulties stem from both chemical and sensory issues. Techniques for odor measurement and the difficulties involved have been reviewed by Neuner-Jehle and Etzweiler (20).
Chemical purity and organoleptic purity are not synonymous. For example, the aldehyde (Structure 1) was discovered when a sample of the alcohol (Structure 2) was found to have the expected muguet (lily of the valley) odor (21). The alcohol was prepared from 4-t-amylcyclohexanone (Structure 3) by the scheme shown in Fig. 2. The reduction product contained mostly the desired alcohol (Structure 2) but with some of the isomeric material (Structure 4) in which the double bond had moved into the ring. However, gc-sniffing revealed that neither of these was responsible for the muguet odor of the sample, but rather that it was caused entirely by a tiny trace of aldehyde (Structure 1). Unless organoleptic purity is verified by such techniques, a risk of mistaken attribution of an odor to a structure always exists, and doubtless, many instances of incorrect odor descriptions exist in the literature as a result.
Figure 2. Synthesis of 2-(4-tert.amylcyclohex-1-yl)acetaldehyde.
Odor is subjective even at the most basic level. It is unlikely that any two humans (except identical twins) use the same set of receptors in their epithelial array (7). Differences between individuals in subsequent neuroprocessing, because of physiologic and experiential factors, increase interindividual differences in odor perception. Therefore, information going into structure/odor models is either relevant for one individual or is an average figure. Comparison of data from one individual to another or from one average to another is always suspect and can be totally irrelevant.
A good example of subjectivity in odor character measurement is provided by Ohloff et al. (22). When 27 panelists were asked to allocate the odor of the cyclic ether (Structure 5) to one of various odor categories, 14 participants described it as minty/camphoraceous; 6 participants described it as fruity; 3 participants described it as balsamic, and 4 participants described it as musky/woody. Therefore, classification as minty (based on the largest subject group) would only be correct for 50% of the panel. Similarly, it is easy to demonstrate that Bangalol (Givaudan, Vernier, Geneva, Switzerland) (Structure 6) is perceived by some subjects as sandalwood in character but by others as musk (23). Odor intensity is also subjective. For example, the average odor threshold for (-)-geosmin (Structure 7) was found to be one tenth that for the (+)-enantiomer (Structure 8). However, some individuals were 40 times more sensitive to one enantiomer than to the other, some experienced similar thresholds for both enantiomers, and some were more sensitive to the (+)-enantiomer (24). As with all sensory magnitude estimation, odor intensity measurement must take into account the fact that humans adjust mental scales unconsciously to suit the task in hand.
Odor classification is particularly difficult. For the senses of touch or sight, it is easy to pick physical reference points (hardness of standard substances, wavelength of light, etc.) and then to classify sensory properties in relation to these points. No such classification exists in odor. No primary odors and no physical reference points exist. Consequently, all odor classification is by comparison with other odors. For example, it might make sense to see apples and pears as subclasses of fruit in botanical terms, but in terms of their odors, putting apple and pear under the general heading of fruity odors leads to difficulties in structure/odor correlation (25). Indeed, the brain sees each new odor as a new percept rather than as a combination of existing percepts (4). Therefore, odor classification, although a useful tool in perfumery, is essentially meaningless scientifically. For example, mixing together in suitable proportions, hexylcinnamic aldehyde (Structure 9) (fatty odor), benzyl acetate (Structure 10) (fruity odor), and indole (Structure 11) (fecal odor) will give a perfume that is recognizable as jasmine in character. However, a sample of pure cis-jasmone (Structure 12) would also attract the descriptor of jasmine. So, of what value is the term “jasmine?”
Transport to the receptors
It is self-evident that transport properties must be of importance in olfaction because if odorants cannot reach the olfactory epithelium, they will not be detected by the olfactory receptors and no odor will result. Volatility is the most obvious requirement, and for organic compounds, this requirement results in a cutoff point at about 18-20 carbon atoms in the molecule equivalent to a molecular weight of about 300 Daltons. Larger molecules are simply not volatile enough to reach the olfactory epithelium in sufficient concentration to be perceived. Solubility is also important, partially because water solubility implies a polar molecular structure, and this, in turn, implies a low vapor pressure relative to the molecular weight because of intermolecular hydrogen bonding. However, solubility properties per se also seem to be important, which is perhaps related to the ability of molecules to cross the aqueous mucus layer to reach the receptor proteins. Fragrance molecules generally have logPoct/water in the region of 2-5.
Proteins that belong to the lipocalin family and are present in the olfactory mucus were first identified as involved in binding of pyrazines and so were first named pyrazine-binding proteins (26). However, it soon became clear that they are capable of binding a very wide range of odorant molecules, and the name odorant-binding proteins (OBPs) was coined (27-30). Possible roles of odor-binding proteins include transport of odorants across the mucus to the receptor, signal attenuation, or removal of excess odorant, or it is also possible that the receptor proteins distinguish between free and liganded lipocalins rather than detecting free odorants. However, because no lipocalins are present in the experiments of Spehr et al. (1), it is clear that odorants can be detected by receptor proteins in the absence of OBPs. OBPs are more important to insects than to mammals. For instance, Drosophila melanogaster has about 50 different types of OBPs and 70 different types of ORs, whereas mammals have only about five types of OBPs and a potential pool of about 1000 different ORs (31). No kinetic studies have been carried out on OBP solubilization of odorants, but if simple model systems are relevant, then the work of Rebek on self-assembling clathrates (32-35) would suggest, by analogy, that the time required for trapping and release of odorants by OBPs might be rather long in terms of the total time involved in olfaction. Overall, insufficient information exists to enable a clear picture of the role of OBPs to be drawn.
The most obvious role for oxidative enzymes in the olfactory mucosa would be removal of excess or spent signal material. However, some evidence suggests that products of cytochrome oxidation are detected by the olfactory receptors and thus constitute a part of odor perception (36).
The receptor event
Based on homology models in humans and in mice, Lancet and coworkers have suggested that odorant binding to olfactory receptor proteins occurs in the transmembrane part of the receptor protein, which bridges between amino acid residues of helices 2 to 7 (37, 38). Interestingly, this region is the same region in which 11-cis-retinal binds in rhodopsin and also corresponds with the binding sites of other GPCRs (39). Furthermore, it is also in broad agreement with binding sites proposed in several reported model studies on specific odorant/olfactory receptor binding (vide infra).
The olfactory receptors are tuned broadly because each receptor type responds to a range of odorants, and each odorant fires a range of receptor types (16). For some receptor protein/odorant combinations, the binding affinity is concentration dependent (40, 41), and this can correlate with observed changes in character as concentration varies (2, pp. 71-74).
The relationship between molecular structure and odor has been the subject of much research during the last 150 years. The motivation has come partly from commercial interest to learn how to design improved ingredients for fragrances and partly from the scientific interest to understand the sense of smell. Attempts to translate structure/odor models into mechanism and vice versa have led to much confusion and to very heated debates. It is now clear that structure/odor models are useful tools to aid the design of novel odorants but that understanding of the mechanism of olfaction will come from biochemistry, molecular biology, and neuroscience rather than from these models. Conversely, developments in our understanding of receptor events in olfaction will not necessarily improve the structure/odor models.
The structure/activity relationship (SAR) tools employed in odor research are essentially the same standard tools used in all applications, and the models developed fall into the categories of substrate and receptor models. The pharmaceutical industry is the leader in SAR techniques, and the fragrance industry tends to follow its lead. Early models were substrate based, but the discovery of the genes that code for the olfactory receptor proteins has also allowed receptor models to be constructed.
For substrate models, classic chemical methods such as Hansch analysis are used, as are statistical techniques such as principal components analysis (PCA). Hansch uses regression analysis to correlate electronic, steric, and hydrophobic properties with the biologic activity in question, whereas PCA is a statistical technique that reduces multidimensional input (physical properties of molecules) to two or three dimensions that aids in correlation with the biologic activity. Molecular modeling tools such as COMFA (comparative molecular field analysis) and the olfactophore approach, the odor equivalent of pharmacophores, have also been used successfully. In these techniques, the stereoelectronic properties of a test set of molecules are used to build a model of an idealized substrate with which putative novel materials can then be compared. A comparison of Hansch analysis, PCA and COMFA in the correlation of structure with fruity odors provides a useful introduction to the three techniques and shows that, in this instance at least, they provide similar results (42). A good illustration of the use of the olfactophore approach is Kraft and Eichenberger’s design of a novel marine odorant using the technique (43). The approaches used in structure/odor modeling have been comprehensively reviewed by Rossiter (44), Frater et al. (45), and Kraft et al. (46).
The commercial driver for substrate models has been the search for novel fragrance ingredients, and odor has tended be the defined biologic activity. However, the combinatorial mechanism of olfaction presents a serious obstacle for such substrate modeling and renders it almost meaningless in terms of understanding the mechanism of olfaction. Medicinal chemists who work on drug SARs usually are targeting a specific active site in a single protein. Fragrance chemists must target interactions of odorants simultaneously with the active sites of an unknown number of proteins because no single protein-single percept relationship exists. This lack of relationship can be the case even with animals simpler than humans. For example, it has been shown that the fruit fly Drosophila requires simultaneous activation of two different receptors to detect carbon dioxide (47).
Frustrating factors abound in structure/odor correlation. Sometimes, a small structural change in a molecule produces a large odor change whereas in other cases, gross structural changes produce little change in odor (44). Sometimes the chemical functional group in a molecule is important, as in the ester group and the fruity odor (25), whereas as other times the shape of the molecule is more important, as for the camphor odor (48). Absolute stereochemistry of a molecule sometimes affects its odor and sometimes it does not (49). Similarly, the effects on odor of a given change in molecular structure are unpredictable (50). In general, SARs suffer from the limitations of being interpolative and their reliability is proportional inversely to the number of steps in the process being modeled. In view of all of this and of all that has been said above about the process of olfaction, its combinatorial nature, its subjectivity, the difficulty in measuring odor and the fact that odor is a mental construction rather than a physical reality, it might be expected that structure/odor models would be impossible to find. However, many useful models do exist. The following accounts provide a few examples of useful models. For a more comprehensive list, the reader is referred to the reviews cited above.
The most consistently accurate structure/odor model is Amoore’s camphor model (48). As shown in Fig. 3, the model indicates that hydrophobic molecules with an ellipsoidal shape that have a long axis of 9.5 A and a short axis of 7.5 A will possess a camphoraceous odor. Another example of a simple but effective model is Boelens’s model for jasmine odorants (51). Shown in Fig. 4, this model proposes that, to possess a jasmine odor, a molecule should contain a central carbon atom surrounded by a strongly polar group, a weakly polar group, and an alkyl chain.
Figure 3. Amoore's model for camphoraceous odorants.
Figure 4. Boelens's model for jasmine odorants.
The first published model for the ambergris odor is that of Ohloff’s triaxial rule (52). This model proposes that, to possess an ambergris odor, a molecule should have a trans-decalin structure with three axial substituents in a 1,2,4-relationship, as shown in Fig. 5, and that one of these should be an oxygen function. A more sophisticated model is that of Bersuker et al., which is based on the electron topological theory of odor (53). They propose that ambergris odorants have two hydrogen atoms and one atom located at the corners of a triangle, the dimensions of which are shown in Fig. 5. They also require that all three atoms make a significant contribution to the LUMO of the molecule, that the coefficients of both of these hydrogen atoms should coincide, that the negative charge on the oxygen atom should be between 0.24 and 0.31 of that of an electron, that the charge on Hi should be negative, and that the charge density over the triangle should be -0.1e/A2. More recently, Bajgrowicz and Broger constructed an olfactophore model for the ambergris odor (54). Ohloff’s model is reasonably representative for decalin systems similar to the natural ambergris chemicals. However, many ambergris odorants are known now for which this model in inapplicable. Therefore, the more recent models are of more use when studying the ambergris odor.
Figure 5. Ambergris models.
Even a cursory inspection of the known sandalwood odorants suggests a model that involves an alcohol function with a center of hydrophobic bulk at a set distance from it. This basic model has been refined by many workers that use different SAR techniques to define more closely the exact requirements of the hydrophobe and the distance between it and the alcohol function (55-60). Bajgrowicz et al. built an olfactophore model around this basic concept and used it to design a potent new sandalwood odorant successfully (61).
The ability to insert active olfactory receptors into functioning cells means that it is now possible to profile the activity of receptor proteins and move from the realm of guesswork into experimental reality. For example, Sanz et al. have profiled and compared the selectivity of two human olfactory receptors that belong to different phylogenetic classes (62). They found that the class I receptor, OR52D1 has a relatively narrow range, which accepts alcohols, esters, ketones, and acids with a molecular length that corresponds to a chain of 8 or 9 carbon atoms; whereas the class II receptor, OR1G1, is much more broadly tuned and responds to a wide range of functional groups and with a preference for slightly longer carbon chains of 8 or 9 atoms. They also found that some odorants are capable of acting as antagonists and of blocking receptor activity from other molecules that would function normally as agonists. This finding is similar to that of Spehr et al. (1) who observed the same phenomenon with hOR17-4. Therefore, it seems likely that such antagonism is widespread and this will have implications for the perception of odor in mixtures. Because almost everything we smell is a mixture, such interactions are of considerable importance.
Models of the receptor sites are based on analogy with those of other GCPRs and of rhodopsin in particular. Using homology modeling, it would seem that the odorants are most likely to be bound in the regions between the 3-, 4-, 5-, and 6-transmembrane sections, and the region of rhodopsin is where 11-cis-retinal is bound. A good example of this approach is that of Pilpel and Lancet (63).
Several assumptions are made when building such models. For example, it is assumed that the tertiary structure adopted by rhodopsin in crystalline form is similar to that which it adopts when in the cell membrane; that olfactory receptor proteins adopt a similar tertiary structure to that of rhodopsin, and that ligand docking in olfactory receptors is similar to cofactor docking in rhodopsin. However, Vaidehi et al. (64) have used MembStruk and HierDock software (which can be obtained from William A Goddard III at Caltech, Pasadena, CA) to show that consistency exists between GPCRs of different types (mouse and rat I7 olfactory receptors, the human sweet receptor, endothelial differential gene 6, and the β-adrenergic receptor) (64). They also showed that these modeling techniques work across this range of receptors and they used these models to predict both the tertiary structure and binding site of rhodopsin with a reasonable degree of accuracy. Moreover, it is possible to test receptor models experimentally by comparing the predictions of the model with the measured selectivity of the receptor in a live cell. Research in this area over the last decade has yielded significant results and we can be confident that we now have a reasonable concept of how olfactory receptors recognize their agonists.
One of the first exercises of this type was the work of Singer and Shepherd on the rat receptor OR5 which is known to respond to Lyral (International Flavors and Fragrances of Union Beach, New Jersey) (65). They proposed a model for the binding site of Lyral that involved transmembrane helices III to VII. However, a later model identified a somewhat different site using only helices III, IV, and V (66).
Floriano et al. investigated the mouse receptor ORS25 (67). Using HierDock software, they identified binding sites and calculated binding energies for 24 potential agonists. The binding site was found to involve 10 amino acids from transmembrane helices III-VI and the energies indicated that hexanol and heptanol should bind most strongly. Experiments with the receptor in cells showed that, indeed, of the 24 test materials, only these two alcohols elicited a response. They then went on to screen an additional six mouse receptors (S6, S18, S19, S25, S46, and S50) against the same 24 odorants (C4-C9 alcohols, acids, bromoacids, and dicarboxylic acids) (68). As before, they found good agreement between the HierDock calculated binding energies and experimental receptor activation. They confirmed that the crucial transmembrane helices are III-VI and that extra-cellular loops II and III also contribute to binding. Six of the amino acid positions are key to binding and, in the examples studied, the selectivity of the receptors were determined largely by two of these positions. This latter result is also supported by mutation data. Extrapolating these results to all 869 olfactory receptors in the murine genome, they suggest that 34 receptors are involved in perception of acids and 36 receptors are involved in the perception of alcohols. The original six receptors under study were also found to respond to aldehydes and esters. They have also screened 56 odorants against mouse and rat OR I7 and again found good agreement between the predictions from their model and in vivo results (69).
Araneda et al. tested 90 odorants for activity with the rat receptor I7 (70). They used an adenovirus to increase the level of I7 in the epithelium and to introduce green fluorescent protein (GFP) simultaneously as a way to detect activation. I7 was found to be very selective toward aldehydes and with a preference for eight carbon atoms in the chain. Both saturated and olefinic aldehydes were found to be active and some tolerance exists for substituents, mostly methyl groups, on the chain. Using models, they could define more precisely the steric constraints on the binding site.
Katada et al. studied the responsiveness of the eugenol sensitive mouse receptor mOR-EG using 22 shikimate derivatives related to eugenol or vanillin (71). This receptor also has a broad but selective range. The oxygen of the hydroxy group of eugenol (and the corresponding oxygen atom of the other agonists) is hydrogen bonded to serine 113 and eight other amino acid residues form the general shape of the binding pocket. They showed that recognition occurs through electrostatic (hydrogen bonding), van der Waals, and hydrophobic interactions and their results from modeling were confirmed by in vivo testing of the receptors.
A new dimension in modeling was introduced by Lai et al. who built a dynamic model of rat receptor ORI7 (72). They incorporated 10 potential aldehydic ligands into the binding site and then set the whole assembly into normal motion. Some test materials remained in the binding site whereas others migrated out. Correlation with in vivo results was 100%. Those molecules that the model predicted would remain in the binding site were found to be agonists whereas those that migrated out failed to activate the receptor in vivo. Moreover, the model elucidated the route into the binding site from the extracellular side of the protein. Similarly to the results of Katada et al. (71) on mOR-EG, they found that the oxygen atom of the agonists was tethered by electrostatic forces, in this instance to lysine 164, and the steric fit between the residues of the binding pocket and the agonist determined its stability in the binding site.
A consistent feature exists in all of these models, in that, for a good odorant/receptor fit, each model requires a polar group in the odorant that can form a hydrogen bond or similar interaction with a donor site in the receptor and the rest of the fit is determined by a spatial match with the shape of the (largely hydrophobic) binding pocket. Saturated hydrocarbons presumably lack the polar interaction and, in some cases at least, it would seem that weaker nonbonded interactions, such as π-stacking exist in the hydrophobic pocket.
Dyson proposed an alternative theory of odor in 1938, in which odorant molecules are recognized by their vibrational frequencies (73). He had no suggestions for a biologic mechanism for such recognition but one was proposed by Turin in 1996 (74). The vast weight of experimental evidence (which includes that summarized above) is in favor of stereoelectronic rather than vibrational recognition and this theory is supported by a recent study in which sila-analogs of the odorants Bourgeonal and Lilial were synthesized and a good correlation was found between their modeled stereoelectronic fit with hOR17-4 and both the response of the receptor to the molecules and their perceived odor thresholds (75).
Olfaction research is at a very interesting stage currently. The advances in molecular biology have enabled us to isolate receptor proteins and to understand their mode of operation. However, what we have learned has shown that the correlation between molecular structure and odor is much more complex than early lock and key concepts envisaged and the complexity of signal interpretation shows that olfaction still holds much for us to discover. Recent advances in understanding may not have helped a great deal in the design of novel odorants, but we are left with the intriguing puzzle that structure/odor correlations do work to some degree when the combinatorial mechanism suggests that they should not. This and the enormous complexity of olfactory signal processing indicate that generations of future researchers will have plenty to stimulate their curiosity, to stretch their minds, and to fascinate them.
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