Investigation of Human Olfactory Receptor 17-40 Interaction with Odorant Molecules by Means of Surface Plasmon Resonance

Registration of Odorant Molecules by Artificially Created Sensitive Structures (Bioelectronic Nose)

Recent advances in the pharmacology of olfactory receptors (ORs) result mainly from the development of drugs that can interact with ORs and initiate the physiological characteristics of the response of the receptor, triggering a chain of intracellular biochemical processes in the body [28]. There is a growing interest in the elaboration of biosensors based on ORs, employed in the membrane fraction or in the whole cell coupled to a solid transducer (bioelectronic nose) [29]. Such biosensor platform can be based on the direct monitoring either of developed drug binding to receptor or on an external monitoring of an organism's response caused by drug after binding to the receptor [30].

The pharmacological data available on mammalian ORs include various dose-response profiles. A typical adsorption curve of odorant octanal with a remarkably broad linear part (10_11-10~3 M) was obtained by means of the QCM technique for rat OR 17 [31]. Signal profile of OR 17 from isolated olfactory neurons was sigmoid within the concentration range 1СГ7-10~5 M of octanal [32]. Other data obtained from intracellular calcium and bioluminescence assays revealed the response pattern of OR 17 and OR 17-40 to be bell-shaped within the concentration range 10"14-10“3 M of odorant helional [33].

This section covers the study of the registration of odorant molecules by artificially created sensitive structures using SPR and complementary methods [34]. The goal of this study was to investigate a pattern of G protein-coupled OR 17-40 response accompanying its biospecific interaction with odorant helional molecule using artificially created structures.

Investigation of the Interaction of Receptor OR 17-40 with Odorant Molecules Using SPR and Complementary Methods

Two different biofilm architectures were studied: Au + (16-mercaptohexadecanoic acid (MHDA) + 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-biotinyl sodium salt (biotinyl- PEA)) + neutravidin + biotinylated anti-cmyc monoclonal antibody (Ab) + OR 17-40 ("Al” biofilm), and Au + biotinylated Ab + OR 17-40 C"A2” biofilm).

To obtain self-organized heterogeneous layer onto gold in the case of Al biofilm, 1 mM MHDA and 0.1 mM biotinyl-PEA were dissolved in ethanol and incubated with freshly cleaned SPR chip for 21 h at room temperature. MHDA was fixed onto Au via chemisorption, whereas biotinyl-PEA was inserted between long- chain thiols via hydrophobic interactions [35]. Such self-assembled monolayer (SAM) provided a good basis for the further anchoring of biomolecules to the surface. To elute unfixed molecules, the chip was rinsed with ethanol and dried under nitrogen flow. Neutravidin (0.5 pM in PBS) was bound to biotin through biospecific interaction; the rest of its specific sites served to attach biotinylated Ab also through biospecific interaction. In the process, due to the spatial structure of neutravidin, the Ab adopted a relative orientation toward the surface of gold, that is, the structure of the layer became more compact. In a comparative A2 biofilm, Abs were attached to the gold surface by random adsorption. Before the formation of any upper molecular layer, the previous one was rinsed with PBS for 5-15 min. In order to saturate all non-specific adsorption sites on modified surface, Ab layer was blocked by BSA (0.5 mg/ml in PBS). A non-specific to OR 17-40 odorant heptanal was used to control the selectivity of the biosensor structure.

Stock suspension of OR 17-40 in membrane fraction was diluted in PBS on ice down to the protein concentration 70 pg/ml, and 0.3 ml of this suspension was treated in the ultrasonic bath in ice-cold water for 20 min in order to obtain a homogeneous suspension of membrane vesicles called nanosomes due to their size [36]. Afterward, the suspension was immediately deposited onto the modified surface. One nanosome of 50 nm diameter could bear up to 10 ORs [37].

(a) Sensorgram of layer-by-layer assembly of A1 biofilm,

Figure 7.6 (a) Sensorgram of layer-by-layer assembly of A1 biofilm, (b) Scheme of A1 biofilm. Possible orientations (1, 2, 3) of Gao!f coupled receptors in a nanosome are described in Table 7.2. Adapted by permission from Springer Customer Service Centre GmbH: Springer European Biophysics Journal, Ref. [34], Copyright 2008.

Stock 0.1 M solutions of odorants were prepared freshly on the day of experiment in dimethyl sulfoxide; further dilutions (from 10'4 to 10'12 M) were obtained by successive 1:10 dilutions in PBS. The blank probes at the various dilutions were prepared replacing the odorant by PBS. Additionally, each odorant and blank probe contained 10 pM of guanosine 5'-0-[gamma-thio]triphosphate (GTP-y-S) prepared on ice from the 1 mM solution. Measurement of the presence of odorant as an analyte was performed in the presence of GTP-y-S, which acted as an activator of the OR 17-40 activity [36].

Receptors carried by nanosomes were immobilized via interactions of cmyc sequence with anti-cmyc monoclonal Ab attached to the gold in orientated or random way. As it was already mentioned, in the first case, Abs were uniformly attached to the neutravidin layer (Fig. 7.6). In the second case, random immobilization involved Abs' adsorption on the freshly cleaned gold (Fig. 7.7). SPR spectrum minimum shift for specific anchoring of biotinylated Ab to the neutravidin was about two times lower than a response to its direct adsorption on gold probably due to the limited quantity of biotinyl-PEA affinity sites at the surface. To estimate the thickness of each molecular layer, the experimental SPR spectra were fitted to the theoretical curves [38]. As a basis, an effective refractive index of n = 1.36 was used for protein layers [38] and of n = 1.46 for a membrane vesicle [39]. The calculated values of thickness are presented in Table 7.1.

(a) Sensorgram of layer-by-layer assembly of A2 biofilm, (b)

Figure 7.7 (a) Sensorgram of layer-by-layer assembly of A2 biofilm, (b)

Scheme of A2 biofilm. Possible orientations (1, 2, 3) of Gao!f coupled receptors in a nanosome are described in Table 7.2. Adapted by permission from Springer Customer Service Centre GmbH: Springer European Biophysics Journal, Ref. [34], Copyright 2008.

Table 7.1 Calculated thickness of each molecular layer in A1 and A2 biofilms

Layer

Al, effective thickness, nm

A2, effective thickness, nm

MHDA

1.9 [40]

Neutravidin

15 ± 1

Biotinylated antibodies

12.5 ±0.75

18.5 ± 1

BSA

0.5 ± 0.1

0.9 ± 0.2

OR 17-40-bearing nanosomes

11 ± 0.75 (V= 1)

11 ±0.75 (V = 0.25)

Total thickness of multilayer, nm

40.9

30.4

Кis a coefficient of surface coverage with lipidic biomaterial estimated from the AFM data shown in Fig. 7.9.

Source: Adapted by permission from Springer Customer Service Centre GmbH: Springer European Biophysics Journal, Ref. [34], Copyright 2008.

As it was mentioned in Section 4.3, the spatial orientation of immobilized Ab is crucial for analyte (here OR) capture since the latter is based on the highly specific interaction via cmyc tag. While the ratio Absmanosomes in the case of biofilm A1 could be close to 1 due to the oriented Ab layer, it should be much lower for the biofilm A2 suggesting that probably only a part of immobilized antibodies is properly oriented. Therefore, a random orientation of Ab layer resulted in a nanosome layer of comparatively low density (Table 7.1). At it was revealed by cyclic voltammetry (CV) measurements, A1 biofilm with an oriented architecture was highly insulating (Fig. 7.8). At the same time, electron transfer through the A2 biofilm was approximately 50% weaker in comparison with redox kinetics on bare Au (Fig. 7.8, inset).

In order to clarify this phenomenon, a biofilm similar to A2 consisting only of randomly adsorbed Ab was probed by means of CV under the same conditions. This layer of antibodies demonstrated an increased penetrability to redox couple after 12 h of contact with PBS (data not shown). Therefore, an increase in the insulating properties of A2 biofilm can be attributed to the nanosomes' fusion on the top of sensor surface. Since redox peaks did not completely disappear, one might conclude that the membrane vesicles did not merge into a continuous layer; therefore, the fusion of nanosomes on the electrode surface could be only partial, in agreement with reported AFM-based data [37].

Cyclic voltammograms of biofilms A1 and A2. Inset

Figure 7.8 Cyclic voltammograms of biofilms A1 and A2. Inset: cyclic voltammogram of bare gold substrate. Adapted by permission from Springer Customer Service Centre GmbH: Springer European Biophysics Journal, Ref. [34], Copyright 2008.

Atomic force microscopy images with cross-sectional profiles corresponding to working "spots" of A1

Figure 7.9 Atomic force microscopy images with cross-sectional profiles corresponding to working "spots" of A1 (a) and A2 (b) biofilms. Images were taken in tapping mode after the second day of using A1 and A2 biofilms for detection of odorants at room temperature. Reprinted by permission from Springer Customer Service Centre GmbH: Springer European Biophysics Journal, Ref. [34], Copyright 2008.

AFM images of biofilms were taken after the second day of odorant screening. Clear difference in relief porosity was observed between A1 (Fig. 7.9a) and A2 (Fig. 7.9b) structures. The surface profile of A1 biofilm was rather smooth due to the proper orientation of multilayer or to the collapsing of immobilized nanosomes, whether initial or after work with the surface. At the same time, the AFM image of biofilm A2 implies the shrinkage and clustering of the nanosome layer. In the calculations of thickness (Table 7.1), the coefficient of coverage of A1 biofilm with membrane biomaterial was taken as 1. The A2 biofilm surface coverage was estimated as 0.25 from the level of porosity observed.

Surface-grafted membrane fragments bearing Gaolf and ORs present a complex biorecognition unit where the above-described conformational changes of receptor and Gaolf are thought to occur upon OR stimulation with odorant. An olfactory signal is transmitted into sensory neurons via an interaction of OR with heterotrimeric G protein located on the cytoplasmic face of neuron ciliae membrane. Activated OR promotes the liberation of GTP-bound Got subunit from Gpy dimer [41]. Possible orientations of Gaolf-coupled OR in nanosome are shown in Figs. 7.6b and 7.7b, and a potential biorecognition efficiency of each configuration is schematized in Table 7.2.

Table 7.2 Biorecognition efficiency of various configurations (1, 2, B) of G protein-coupled olfactory receptor in a nanosome (see Figs. 7.6b, 7.7b)

Configuration

1

2

3

Flexibility of N-ends in aromatic compounds, crucial for odorant binding

-

+

+

Accessibility of Gaolf to GTP-y-S

-

-

+

Biorecognition efficiency

Low

Middle

High

Source: Adapted by permission from Springer Customer Service Centre GmbH: Springer European Biophysics Journal Ref. [34], Copyright 2008.

Two odorants, helional and heptanal, were tested on A1 and A2 biofilms in the concentration range from 10'12 to 10'5 M. Helional is documented as a cognate odorant for OR 17-40 [42]. SPR measurements were carried out in the differential mode, and

Dependences of SPR response for A1 and A2 biofilms on the helional concentration. The error values mentioned represent intersensor standard deviation (n = 2-3). Inset

Figure 7.10 Dependences of SPR response for A1 and A2 biofilms on the helional concentration. The error values mentioned represent intersensor standard deviation (n = 2-3). Inset: typical kinetics of responses to helional obtained from the A2 biofilm in differential mode. Adapted by permission from Springer Customer Service Centre GmbH: Springer European Biophysics Journal, Ref. [34], Copyright 2008.

Profile of the A2 biofilm responses to helional during 2 days of work

Figure 7.11 Profile of the A2 biofilm responses to helional during 2 days of work. Heptanal was used as a negative control. Data set was collected from the same sensor chip. Adapted by permission from Springer Customer Service Centre GmbH: Springer European Biophysics Journal, Ref. [34], Copyright 2008.

the registered kinetics of the SPR angular position change had a negative direction (toward the smaller angles of incidence of light) (Fig. 7.10, inset). Olfactory sensitivity of thicker A1 biofilm with comparatively dense nanosomes layer was weaker than that of the A2 (Fig. 7.10); therefore, further measurements of the response profile through the whole range of odorant concentrations were performed with A2 biofilm only. Sensitivity of receptors to helional after the overnight storage of SPR substrate coated with biofilm A1 decreased essentially, while A2 demonstrated only a slight relative decrease in response (Fig. 7.10). During 2 days of work with the same A2 biofilm, the same pattern of response to helional was observed (Fig. 7.11). Meanwhile, A2 sensitivity to the unrelated odorant heptanal remained insignificant somewhat increasing at 10'9 M after the overnight storage (Fig. 7.11).

Comparative Sensitivity Analysis for Different Types of Biofilm Architecture

As it can be seen from Table 7.1, the biofilm A2 based on the randomly oriented Abs was at least 27% thinner than the A1 biofilm. Low thickness of A2 biofilm and its high porosity (Fig. 7.9b) resulted in better accessibility of Gaolf protein to GTP-y-S, which could explain a surprisingly better sensitivity of A2 to helional. The presence of pores in A2 initially originates from the random orientation of Abs and relates to their inability to bind a large amount of nanosomes. From this point of view, the thickness of both A1 and A2 nanosome layer is similar, whereas the filling ratio of the latter is smaller. The thickness of nanosome layer in both cases, close to twice a lipid bilayer thickness, demonstrates a flattening of the nanosomes down to partial collapsing.

The possible reasons of signal decrease after 12 h storage of SPR chip at room temperature are: (1) loss in receptor and/or Gaolf protein activity at room temperature, and (2) depletion of the available Gaolf protein pool. The a subunit of G protein has a molecular mass of ~40-50 kDa [43]; therefore, its desorption is reliably detected by the SPR technique. However, quite low amplitude of signals measured can be ascribed to the fewer number of receptors oriented in the direction allowing full access of Gaolf to GTP-y-S. Indeed, the latter cannot penetrate the lipid bilayer to access Gaolf located inside the nanosome, whereas the hydrophobic odorants may penetrate the bilayer [44] to reach the receptor ligand binding pocket and activate the receptor. Another phenomenon that could contribute to the negative SPR angular shift was the intrinsic conformational change in activated OR. The latter is composed of a bundle of seven transmembrane a-helices connected by loops [45]. The odorant stimulation of OR seems to induce a rearrangement of helices leading to separation of transmembrane domains in the helix bundle [41]. Recently, it has been suggested that the signal changes observed by SPR can be also ascribed to the protein secondary structure changes.

Thus, May and Russell [46] have correlated a decrease in SPR signal to the formation of (3-sheets, turn or unordered protein secondary structures; compact helical structure was thought to possess higher refractive index and thus to increase an SPR signal. In this way, the OR conformational changes on SPR signals may also be taken into consideration.

Two maxima bell-shaped curves such as that found previously [36] can be superimposed to the experimental points (Fig. 7.11) to account for the functional response observed at the A2 biofilm stimulation by the odorant specific for OR 17-40. The exceptions are data for measurements at a concentration of 10'9 M with a rather low signal level, which require additional research. The presence of two maxima in the response profile can be explained by the presence of a significant number of specific binding sites with different receptor affinity in a receptor-odorant pair [36]. It should be noted that the detailed molecular mechanism of functional interactions between the odorant and the receptor is still the subject of research.

 
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