Laser-Based Mid-Infrared Spectroscopy for Monitoring Temperature-Induced Denaturation of Bovine Serum Albumin and De-/Stabilization Effects of Sugars.

Stability of high-concentration protein formulations is considered a major challenge in current biopharmaceutical development. In this work, we introduce laser-based mid-infrared (IR) spectroscopy as a versatile technique to study the effect of protein concentration and presence of sugars on the thermal denaturation of the model protein bovine serum albumin (BSA). Many analytical techniques struggle to characterize the complex structural transition that occurs during protein denaturation. To this end, a commercially available laser-based mid-IR spectrometer equipped with a customized flow cell was employed to record IR spectra of BSA in the temperature range of 25-85 °C. The temperature perturbation induces a conformational change from a native α-helical to an intermolecular ß-sheet secondary structure in BSA. Systematic investigation of the concentration dependence of the α-ß transition temperature between 30 and 90 mg mL-1 shows a trend of decreasing denaturation temperatures at higher BSA concentrations. In-depth chemometric analysis by a multivariate curve resolution-alternating least squares (MCR-ALS) analysis of the spectra, suggested the formation of not one but two intermediates in the denaturation of BSA. Subsequently, the impact of sugars on denaturation temperatures was investigated, revealing both stabilizing (trehalose, sucrose, and mannose) and destabilizing (sucralose) effects, illustrating the applicability of this method as an investigative tool for stabilizers. These results highlight the potential and versatility of laser-based IR spectroscopy for analysis of protein stability at high concentrations and varying conditions.

QCL-IR Spectroscopy for In-Line Monitoring of Proteins from Preparative Ion-Exchange Chromatography.

In this study, an external cavity-quantum cascade laser-based mid-infrared (IR) spectrometer was applied for in-line monitoring of proteins from preparative ion-exchange chromatography. The large optical path length of 25 µm allowed for robust spectra acquisition in the broad tuning range between 1350 and 1750 cm-1, covering the most important spectral region for protein secondary structure determination. A significant challenge was caused by the overlapping mid-IR bands of proteins and changes in the background absorption of water due to the NaCl gradient. Implementation of advanced background compensation strategies resulted in high-quality protein spectra in three different model case studies. In Case I, a reference blank run was directly subtracted from a sample run with the same NaCl gradient. Case II and III included sample runs with different gradient profiles than the one from the reference run. Here, a novel compensation approach based on a reference spectra matrix was introduced, where the signal from the conductivity detector was employed for correlating suitable reference spectra for correction of the sample run spectra. With this method, a single blank run was sufficient to correct various gradient profiles. The obtained IR spectra of hemoglobin and ß-lactoglobulin were compared to off-line reference measurements, showing excellent agreement for all case studies. Moreover, the concentration values obtained from the mid-IR spectrometer agreed well with conventional UV detectors and high-performance liquid chromatography off-line measurements. LC-QCL-IR coupling thus holds high potential for replacing laborious and time-consuming off-line methods for protein monitoring in complex downstream processes.

Application of Quantum Cascade Laser-Infrared Spectroscopy and Chemometrics for In-Line Discrimination of Coeluting Proteins from Preparative Size Exclusion Chromatography.

An external-cavity quantum cascade laser (EC-QCL)-based flow-through mid-infrared (IR) spectrometer was placed in line with a preparative size exclusion chromatography system to demonstrate real-time analysis of protein elutions with strongly overlapping chromatographic peaks. Two different case studies involving three and four model proteins were performed under typical lab-scale purification conditions. The large optical path length (25 µm), high signal-to-noise ratios, and wide spectral coverage (1350 to 1750 cm-1) of the QCL-IR spectrometer allow for robust spectra acquisition across both the amide I and II bands. Chemometric analysis by self-modeling mixture analysis and multivariate curve resolution enabled accurate quantitation and structural fingerprinting across the protein elution transient. The acquired concentration profiles were found to be in excellent agreement with the off-line high-performance liquid chromatography reference analytics performed on the collected effluent fractions. These results demonstrate that QCL-IR detectors can be used effectively for in-line, real-time analysis of protein elutions, providing critical quality attribute data that are typically only accessible through time-consuming and resource-intensive off-line methods.

The Next Generation of IR Spectroscopy: EC-QCL-Based Mid-IR Transmission Spectroscopy of Proteins with Balanced Detection.

We report a mid-IR transmission setup for the analysis of the protein amide I and amide II band in aqueous solutions that achieves a limit of detection as low as 0.0025 mg mL-1 (outperforming our previous results and other state-of-the-art mid-IR-based techniques by almost an order of magnitude). This large improvement is made possible by combining the latest-generation external cavity-quantum cascade laser (EC-QCL) operated at room temperature with an optimized double-beam optical setup that adjusts the path length (26 µm) to ensure robust sample handling. For minimizing the noise introduced by the high-intensity laser light source, a thermoelectrically cooled mercury cadmium telluride balanced detection module was employed. In this way, noise levels better by a factor of up to 20 were achieved compared with single-channel measurements. Characteristic spectral features of proteins with different secondary structures were successfully identified at concentrations as low as 0.1 mg mL-1. Furthermore, a highly linear response was demonstrated for concentrations between 0.05 and 10 mg mL-1. The total acquisition time of the setup can be adapted to fulfill the required sensitivity of the protein measurements and to ensure maximum flexibility for future applications. The presented setup combines high sensitivity, large optical path lengths, and short measurement times and thus outperforms previous research type EC-QCL setups as well as commercially available instruments. This opens a wide range of future applications including protein-ligand interaction studies as well as qualitative and quantitative analyses of proteins in complex matrices such as those found in up- and downstream bioprocess monitoring and similar challenging applications which can not be readily met by conventional FT-IR spectroscopy.

Mid-IR refractive index sensor for detecting proteins employing an external cavity quantum cascade laser-based Mach-Zehnder interferometer.

Novel laser light sources in the mid-infrared region enable new spectroscopy schemes beyond classical absorption spectroscopy. Herein, we introduce a refractive index sensor based on a Mach-Zehnder interferometer and an external-cavity quantum cascade laser that allows rapid acquisition of high-resolution spectra of liquid-phase samples, sensitive to relative refractive index changes down to 10-7. Dispersion spectra of three model proteins in deuterated solution were recorded at concentrations as low as 0.25 mg mL-1. Comparison with Kramers-Kronig-transformed Fourier transform infrared absorbance spectra revealed high conformance, and obtained figures of merit compare well with conventional high-end FTIR spectroscopy. Finally, we performed partial least squares-based multivariate analysis of a complex ternary protein mixture to showcase the potential of dispersion spectroscopy utilizing the developed sensor to tackle complex analytical problems. The results indicate that laser-based dispersion sensing can be successfully used for qualitative and quantitative analysis of proteins.

Beyond Beer's Law: Why the Index of Refraction Depends (Almost) Linearly on Concentration.

Beer's empiric law states that absorbance is linearly proportional to the concentration. Based on electromagnetic theory, an approximately linear dependence can only be confirmed for comparably weak oscillators. For stronger oscillators the proportionality constant, the molar attenuation coefficient, is modulated by the inverse index of refraction, which is itself a function of concentration. For comparably weak oscillators, the index of refraction function depends, like absorbance, linearly on concentration. For stronger oscillators, this linearity is lost, except at wavenumbers considerably lower than the oscillator position. In these transparency regions, linearity between the change of the index of refraction and concentration is preserved to a high degree. This can be shown with help of the Kramers-Kronig relations which connect the integrated absorbance to the index of refraction change at lower wavenumbers than the corresponding band. This finding builds the foundation not only for refractive index sensing, but also for new interferometric approaches in IR spectroscopy, which allow measuring the complex index of refraction function.

Production of Active Recombinant Hyaluronidase Inclusion Bodies from Apis mellifera in E. coli Bl21(DE3) and characterization by FT-IR Spectroscopy.

The bacterium E. coli is one of the most important hosts for recombinant protein production. The benefits are high growth rates, inexpensive media, and high protein titers. However, complex proteins with high molecular weight and many disulfide bonds are expressed as inclusion bodies (IBs). In the last decade, the overall perception of these IBs being not functional proteins changed, as enzyme activity was found within IBs. Several applications for direct use of IBs are already reported in literature. While fluorescent proteins or protein tags are used for determination of IB activity to date, direct measurements of IB protein activity are scacre. The expression of recombinant hyaluronidase from Apis mellifera in E. coli BL21(DE3) was analyzed using a face centered design of experiment approach. Hyaluronidase is a hard to express protein and imposes a high metabolic burden to the host. Conditions giving a high specific IB titer were found at 25 °C at low specific substrate uptake rates and induction times of 2 to 4 h. The protein activity of hyaluronidase IBs was verified using (Fourier transform) FT-IR spectroscopy. Degradation of the substrate hyaluronan occurred at increased rates with higher IB concentrations. Active recombinant hyaluronidase IBs can be immediately used for direct degradation of hyaluronan without further down streaming steps. FT-IR spectroscopy was introduced as a method for tracking IB activity and showed differences in degradation behavior of hyaluronan dependent on the applied active IB concentration.

Native Nano-electrospray Differential Mobility Analyzer (nES GEMMA) Enables Size Selection of Liposomal Nanocarriers Combined with Subsequent Direct Spectroscopic Analysis.

Gas-phase electrophoresis employing a nano-electrospray differential mobility analyzer (nES DMA), aka gas-phase electrophoretic mobility molecular analyzer (nES GEMMA), enables nanoparticle separation in the gas-phase according to their surface-dry diameter with number-based concentration detection. Moreover, particles in the nanometer size range can be collected after size selection on supporting materials. It has been shown by subsequent analyses employing orthogonal methods, for instance, microscopic or antibody-based techniques, that the surface integrity of collected analytes remains intact. Additionally, native nES GEMMA demonstrated its applicability for liposome characterization. Liposomes are nanometer-sized, biodegradable, and rather labile carriers (nanoobjects) consisting of a lipid bilayer encapsulating an aqueous lumen. In nutritional and pharmaceutical applications, these vesicles allow shielded, targeted transport and sustained release of bioactive cargo material. To date, cargo quantification is based on bulk measurements after bilayer rupture. In this context, we now compare capillary electrophoresis and spectroscopic characterization of vesicles in solution (bulk measurements) to the possibility of spectroscopic investigation of individual, size-separated/collected liposomes after nES GEMMA. Surface-dried, size-selected vesicles were collected intact on calcium fluoride (CaF2) substrates and zinc selenide (ZnSe) prisms, respectively, for subsequent spectroscopic investigation. Our proof-of-principle study demonstrates that the off-line hyphenation of gas-phase electrophoresis and confocal Raman spectroscopy allows detection of isolated, nanometer-sized soft material/objects. Additionally, atomic force microscopy-infrared spectroscopy (AFM-IR) as an advanced spectroscopic system was employed to access molecule-specific information with nanoscale lateral resolution. The off-line hyphenation of nES GEMMA and AFM-IR is introduced to enable chemical imaging of single, i.e., individual, liposome particles.

An Acoustic Trap for Bead Injection Attenuated Total Reflection Infrared Spectroscopy.

In this work, we introduce a system combining an acoustic trap for bead injection with attenuated total reflection (ATR) infrared (IR) spectroscopy. By mounting an acoustofluidic cell hosting an ultrasound source on top of a custom-built ATR fixture we were able to trap beads labeled with the enzyme alkaline phosphatase without requiring any mechanical retention elements. Sequential injection analysis was employed for reproducible sample handling and bead injection into the acoustic trap. To showcase potential applications of the presented setup for kinetic studies, we monitored the conversion of p-nitrophenylphosphate into p-nitrophenol and phosphate via beads carrying the immobilized enzyme using ATR-IR spectroscopy. Retaining the labeled beads via ultrasound particle manipulation resulted in excellent experimental reproducibility (relative standard deviation, 3.91%). It was demonstrated that trapped beads remained stably restrained with up to eight cell volumes of liquid passing through the acoustofluidic cell. Beads could be discarded in a straightforward manner by switching off the ultrasound, in contrast to systems containing mechanical retention elements, which require backflushing. Multiple experiments were performed by employing different substrate concentrations with the same batch of trapped beads as well as varying the amount of enzyme present in the cell, enabling enzyme kinetic studies and emphasizing the application of the proposed setup in studies where enzymatic reuse is desired. This proves the potential of the acoustic trap combined with ATR-IR spectroscopy to monitor the activity of immobilized enzymes and its ability to perform complex bead-based assays.

High-throughput quantitation of bovine milk proteins and discrimination of commercial milk types by external cavity-quantum cascade laser spectroscopy and chemometrics.

Analysis of bovine milk proteins is crucial in many food and non-food industrial applications, nevertheless labour-intensive wet-chemical, low-throughput methods are still routinely used. In this work, external cavity-quantum cascade laser (EC-QCL) mid-infrared spectroscopy is employed as a rapid method for protein analysis of commercial bovine milk. Combined analysis of the amide I and II bands enabled quantitation of individual proteins (casein, ß-lactoglobulin, α-lactalbumin) and total protein content. IR spectra of spiked and diluted milk samples were employed for calibration of the target analytes in the presence of a complex matrix by partial least squares (PLS) regression modelling. A sample set of different milk types (pasteurized; differently processed extended shelf life, ESL; ultra-high temperature, UHT) was analysed, and results agreed well with reference methods. Quantitation of temperature sensitive proteins enables detailed distinction between milk types experiencing different heat loads during processing, and discrimination between diverse bovine milk types is successfully demonstrated.

Beyond Fourier Transform Infrared Spectroscopy: External Cavity Quantum Cascade Laser-Based Mid-infrared Transmission Spectroscopy of Proteins in the Amide I and Amide II Region.

In this work, we present a setup for mid-IR measurements of the protein amide I and amide II bands in aqueous solution. Employing a latest generation external cavity-quantum cascade laser (EC-QCL) at room temperature in pulsed operation mode allowed implementing a high optical path length of 31 µm that ensures robust sample handling. By application of a data processing routine, which removes occasionally deviating EC-QCL scans, the noise level could be lowered by a factor of 4. The thereby accomplished signal-to-noise ratio is better by a factor of approximately 2 compared to research-grade Fourier transform infrared (FT-IR) spectrometers at equal acquisition times. Employing this setup, characteristic spectral features of three representative proteins with different secondary structures could be measured at concentrations as low as 1 mg mL-1. Mathematical evaluation of the spectral overlap confirms excellent agreement of the quantum cascade laser infrared spectroscropy (QCL-IR) transmission measurements with protein spectra acquired by FT-IR spectroscopy. The presented setup combines performance surpassing FT-IR spectroscopy with large applicable optical paths and coverage of the relevant spectral range for protein analysis. This holds high potential for future EC-QCL-based protein studies, including the investigation of dynamic secondary structure changes and chemometrics-based protein quantification in complex matrices.

Custom made inclusion bodies: impact of classical process parameters and physiological parameters on inclusion body quality attributes.

BACKGROUND: The bacterium E. coli is a major host for recombinant protein production of non-glycosylated products. Depending on the expression strategy, the recombinant protein can be located intracellularly. In many cases the formation of inclusion bodies (IBs), protein aggregates inside of the cytoplasm of the cell, is favored in order to achieve high productivities and to cope with toxic products. However, subsequent downstream processing, including homogenization of the cells, centrifugation or solubilization of the IBs, is prone to variable process performance or can be characterized by low extraction yields as published elsewhere. It is hypothesized that variations in IB quality attributes (QA) are responsible for those effects and that such attributes can be controlled by upstream process conditions. This contribution is aimed at analyzing how standard process parameters, such as pH and temperature (T) as well as different controlled levels of physiological parameters, such as specific substrate uptake rates, can vary IB quality attributes. RESULTS: Classical process parameters like pH and T influence the expression of analyzed IB. The effect on the three QAs titer, size and purity could be successfully revealed. The developed data driven model showed that low temperatures and low pH are favorable for the expression of the two tested industrially relevant proteins. Based on this knowledge, physiological control using specific substrate feeding rate (of glucose) qs,Glu is altered and the impact is tested for one protein. CONCLUSIONS: Time dependent monitoring of IB QA-titer, purity, IB bead size-showed a dependence on classical process parameters pH and temperature. These findings are confirmed using a second industrially relevant strain. Optimized process conditions for pH and temperature were used to determine dependence on the physiological parameters, the specific substrate uptake rate (qs,Glu). Higher qs,Glu were shown to have a strong influence on the analyzed IB QAs and drastically increase the titer and purity in early time stages. We therefore present a novel approach to modulate-time dependently-quality attributes in upstream processing to enable robust downstream processing.

Teaching an old pET new tricks: tuning of inclusion body formation and properties by a mixed feed system in E. coli.

Against the outdated belief that inclusion bodies (IBs) in Escherichia coli are only inactive aggregates of misfolded protein, and thus should be avoided during recombinant protein production, numerous biopharmaceutically important proteins are currently produced as IBs. To obtain correctly folded, soluble product, IBs have to be processed, namely, harvested, solubilized, and refolded. Several years ago, it was discovered that, depending on cultivation conditions and protein properties, IBs contain partially correctly folded protein structures, which makes IB processing more efficient. Here, we present a method of tailored induction of recombinant protein production in E. coli by a mixed feed system using glucose and lactose and its impact on IB formation. Our method allows tuning of IB amount, IB size, size distribution, and purity, which does not only facilitate IB processing, but is also crucial for potential direct applications of IBs as nanomaterials and biomaterials in regenerative medicine.

Quantum cascade lasers (QCLs) in biomedical spectroscopy.

Quantum cascade lasers (QCL) are the first room temperature semiconductor laser source for the mid-IR spectral region, triggering substantial development for the advancement of mid-IR spectroscopy. Mid-IR spectroscopy in general provides rapid, label-free and objective analysis, particularly important in the field of biomedical analysis. Due to their unique properties, QCLs offer new possibilities for development of analytical methods to enable quantification of clinically relevant concentration levels and to support medical diagnostics. Compared to FTIR spectroscopy, novel and elaborated measurement techniques can be implemented that allow miniaturized and portable instrumentation. This review illustrates the characteristics of QCLs with a particular focus on their benefits for biomedical analysis. Recent applications of QCL-based spectroscopy for analysis of a variety of clinically relevant samples including breath, urine, blood, interstitial fluid, and biopsy samples are summarized. Further potential for technical advancements is discussed in combination with future prospects for employment of QCL-based devices in routine and point-of-care diagnostics.

EC-QCL mid-IR transmission spectroscopy for monitoring dynamic changes of protein secondary structure in aqueous solution on the example of ß-aggregation in alcohol-denaturated α-chymotrypsin.

In this work, a novel EC-QCL-based setup for mid-IR transmission measurements in the amide I region is introduced for monitoring dynamic changes in secondary structure of proteins. For this purpose, α-chymotrypsin (aCT) acts as a model protein, which gradually forms intermolecular ß-sheet aggregates after adopting a non-native α-helical structure induced by exposure to 50 % TFE. In order to showcase the versatility of the presented setup, the effects of varying pH values and protein concentration on the rate of ß-aggregation were studied. The influence of the pH value on the initial reaction rate was studied in the range of pH 5.8-8.2. Results indicate an increased aggregation rate at elevated pH values. Furthermore, the widely accessible concentration range of the laser-based IR transmission setup was utilized to investigate ß-aggregation across a concentration range of 5-60 mg mL(-1). For concentrations lower than 20 mg mL(-1), the aggregation rate appears to be independent of concentration. At higher values, the reaction rate increases linearly with protein concentration. Extended MCR-ALS was employed to obtain pure spectral and concentration profiles of the temporal transition between α-helices and intermolecular ß-sheets. Comparison of the global solutions obtained by the modelled data with results acquired by the laser-based IR transmission setup at different conditions shows excellent agreement. This demonstrates the potential and versatility of the EC-QCL-based IR transmission setup to monitor dynamic changes of protein secondary structure in aqueous solution at varying conditions and across a wide concentration range. Graphical abstract EC-QCL IR spectroscopy for monitoring protein conformation change.

Method for time-resolved monitoring of a solid state biological film using photothermal infrared nanoscopy on the example of poly-L-lysine.

We report time-resolved photothermal infrared nanoscopy measurements across a spectral range of more than 100 cm(-1) (1565 cm(-1) to 1729 cm(-1)) at nanoscale spatial resolution. This is achieved through a custom-built system using broadly tunable external cavity quantum cascade lasers in combination with a commercially available atomic force microscope. The new system is applied to the analysis of conformational changes of a polypeptide (poly-l-lysine) film upon temperature-induced changes of the humidity in the film. Changes of the secondary structure from ß-sheet to α-helix could be monitored at a time resolution of 15 s per spectrum. The time-resolved spectra are well comparable to reference measurements acquired with conventional Fourier transform infrared microscopy.

External-Cavity Quantum Cascade Laser Spectroscopy for Mid-IR Transmission Measurements of Proteins in Aqueous Solution.

In this work, we report mid-IR transmission measurements of the protein amide I band in aqueous solution at large optical paths. A tunable external-cavity quantum cascade laser (EC-QCL) operated in pulsed mode at room temperature allowed one to apply a path length of up to 38 µm, which is four times larger than that applicable with conventional FT-IR spectrometers. To minimize temperature-induced variations caused by background absorption of the ν2-vibration of water (HOH-bending) overlapping with the amide I region, a highly stable temperature control unit with relative temperature stability within 0.005 °C was developed. An advanced data processing protocol was established to overcome fluctuations in the fine structure of the emission curve that are inherent to the employed EC-QCL due to its mechanical instabilities. To allow for wavenumber accuracy, a spectral calibration method has been elaborated to reference the acquired IR spectra to the absolute positions of the water vapor absorption bands. Employing this setup, characteristic spectral features of five well-studied proteins exhibiting different secondary structures could be measured at concentrations as low as 2.5 mg mL(-1). This concentration range could previously only be accessed by IR measurements in D2O. Mathematical evaluation of the spectral overlap and comparison of second derivative spectra confirm excellent agreement of the QCL transmission measurements with protein spectra acquired by FT-IR spectroscopy. This proves the potential of the applied setup to monitor secondary structure changes of proteins in aqueous solution at extended optical path lengths, which allow experiments in flow through configuration.

Insights into structural features determining odorant affinities to honey bee odorant binding protein 14.

Molecular interactions between odorants and odorant binding proteins (OBPs) are of major importance for understanding the principles of selectivity of OBPs towards the wide range of semiochemicals. It is largely unknown on a structural basis, how an OBP binds and discriminates between odorant molecules. Here we examine this aspect in greater detail by comparing the C-minus OBP14 of the honey bee (Apis mellifera L.) to a mutant form of the protein that comprises the third disulfide bond lacking in C-minus OBPs. Affinities of structurally analogous odorants featuring an aromatic phenol group with different side chains were assessed based on changes of the thermal stability of the protein upon odorant binding monitored by circular dichroism spectroscopy. Our results indicate a tendency that odorants show higher affinity to the wild-type OBP suggesting that the introduced rigidity in the mutant protein has a negative effect on odorant binding. Furthermore, we show that OBP14 stability is very sensitive to the position and type of functional groups in the odorant.

Structural proteins from whelk egg capsule with long range elasticity associated with a solid-state phase transition.

The robust, proteinaceous egg capsules of marine prosobranch gastropods (genus Busycotypus ) exhibit unique biomechanical properties such as high elastic strain recovery and elastic energy dissipation capability. Capsule material possesses long-range extensibility that is fully recoverable and is the result of a secondary structure phase transition from α-helical coiled-coil to extended ß-sheet rather than of entropic (rubber) elasticity. We report here the characterization of the precursor proteins that make up this material. Three different proteins have been purified and analyzed, and complete protein sequences deduced from messenger ribonucleic acid (mRNA) transcripts. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy indicate that the proteins are strongly α-helical in solution and primary sequence analysis suggests that these proteins have a propensity to form coiled-coils. This is in agreement with previous wide-angle X-ray scattering (WAXS) and solid-state Raman spectroscopic analysis of mature egg capsules.

Honey bee odorant-binding protein 14: effects on thermal stability upon odorant binding revealed by FT-IR spectroscopy and CD measurements.

In the present work, we study the effect of odorant binding on the thermal stability of honey bee (Apis mellifera L.) odorant-binding protein 14. Thermal denaturation of the protein in the absence and presence of different odorant molecules was monitored by Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). FT-IR spectra show characteristic bands for intermolecular aggregation through the formation of intermolecular ß-sheets during the heating process. Transition temperatures in the FT-IR spectra were evaluated using moving-window 2D correlation maps and confirmed by CD measurements. The obtained results reveal an increase of the denaturation temperature of the protein when bound to an odorant molecule. We could also discriminate between high- and low-affinity odorants by determining transition temperatures, as demonstrated independently by the two applied methodologies. The increased thermal stability in the presence of ligands is attributed to a stabilizing effect of non-covalent interactions between odorant-binding protein 14 and the odorant molecule.