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SCIENTIFIC REPORTS Anesthetic Alterations of Collective Terahertz Oscillations in Tubulin Correlate with Clinical Potency: Received: 8 May 2017 Accepted: I August 2017 Implications for Anesthetic Action Published online: 29 August 2017 and Post-Operative Cognitive Dysfunction Travis J. A. Craddock. 1, Philip Kurian2, Jordane Preto3, Kamlesh Sahitts, Stuart R. Hameroffs, Mariusz Klobukowski2 & Jack A. Tuszynski3,4 Anesthesia blocks consciousness and memory while sparing non•conscious brain activities.While the exact mechanisms of anesthetic action are unknown, the Meyer•Overton correlation provides a link between anesthetic potency and solubility in a lipid -like, non•polar medium. Anesthetic action is also related to an anesthetic's hydrophobicity, permanent dipole, and polarizability, and is accepted to occur in lipid-like, non-polar regions within brain proteins. Generally the protein target for anesthetics is assumed to be neuronal membrane receptors and ion channels, however new evidence points to critical effects on intra•neuronal microtubules, a target of interest due to their potential role in post-operative cognitive dysfunction (POCD). Here we use binding site predictions on tubulin, the protein subunit of microtubules, with molecular docking simulations, quantum chemistry calculations, and theoretical modeling of collective dipole interactions in tubulin to investigate the effect of a group of gases including anesthetics, non•anesthetics, and anesthetic/convulsants on tubulin dynamics. We found that these gases alter collective terahertz dipole oscillations in a manner that is correlated with their anesthetic potency. Understanding anesthetic action may help reveal brain mechanisms underlying consciousness, and minimize POCD in the choice and development of anesthetics used during surgeries for patients suffering from neurodegenerative conditions with compromised cytoskeletal microtubules. Anesthesia is one of the world's greatest serendipitous pharmacological discoveries, selectively and reversi- bly blocking consciousness while sparing non-conscious brain activities, enabling modern surgery. Yet, the mechanism by which anesthesia acts, and how the brain produces conscious experience remain unknown. Understanding anesthesia may help explain consciousness, and vice versa. On a more practical level, discov- ering sites and mechanisms of anesthetic action can help in clinical decisions (e.g. the choice of anesthetic in patients with cancer, neurodegenerative, and other disorders), lead to new anesthetics, and reduce the risk of anesthesia-related post-operative cognitive dysfunction (POCD). 'Departments of Psychology & Neuroscience, Computer Science, and Clinical Immunology, and the Clinical Systems Biology Group, Institute for Neuro-Immune Medicine, Nova Southeastern University, Fort Lauderdale, Florida, USA. 'National Human Genome Center and Department of Medicine, Howard University College of Medicine, and Computational Physics Laboratory, Howard University, Washington, DC, USA. 'Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada. `Department of Physics, University of Alberta, Edmonton, Alberta, Canada. 'Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada. 'Departments of Anesthesiology and Psychology, Center for Consciousness Studies, The University of Arizona Health Sciences Center, Tucson, Arizona, USA. 'Department of Chemistry, University of Alberta. Edmonton. Alberta. Canada. Correspondence and requests for materials should be addressed to T.J.A.C. SCIENTIFIC REPORTS17: 9877 IDO1:10.1038N41598-017-09992-7 EFTA00606068  Desfhwase tellurene Halothane Nitrou• Oxide Figure 1. Chemical structure of investigated agents. Blue - anesthetics; Red - non-anesthetics; Green - anesthetic/convulsant. The link between anesthesia, POCD, and the exacerbation of neurodegenerative disorders is a major con- cern'''. After surgery at least 30% of adults show cognitive dysfunction two days following surgery, and lasting up to at least 3-months in 12% of the elderly'. This is clearly problematic for patients with neurodegenerative condi- tions, with anesthesia having a significant impact on disease progression and cognitive decline". As Alzheimer's disease (AD) and other dementias currently cost the American taxpayer $236 billion annually's and with increas- ing anesthesia required for age-related surgery, this cost is only expected to rise as the population ages. The mystery of anesthesia stems from the baffling structure-activity relationship of general anesthetics, as effective agents can span a 35-fold range in molecular volume from a single atom (xenon) to 56-atom steroids, with numerous types of chemical structures, including ethers and halogenated hydrocarbons, in between" (Fig. l). Examining the anesthetic action of gases with such disparate structures, Hans Meyer" and Charles Overton" discovered that anesthetic potency (e.g. the inverse of the minimum alveolar concentration (MAC) at which half of animals tested would lose purposeful behavior) correlates highly with their solubility in a par- ticular non-polar solvent akin to olive °V.". Thus, the 'Meyer-Overton correlation revealed that the anesthetic potency of a gas molecule correlates with its solubility in a non-polar, 'lipid-like', hydrophobic (i.e. water exclud- ing) medium (Fig. 2A, Table SI). Surprisingly, this correlation holds for general anesthetic activity in many organ- isms" from paramecia to humans", and even plants30-22. Furthermore, anesthesia is completely reversible. The Meyer-Overton correlation suggests a common, unitary mechanism of anesthetic action. Initially this was taken to imply critical effects on the excitability oflipid bilayers in neuronal membranes. However, anesthetic effects on lipids cannot account for differences between mirror image chiral' anesthetic molecules", the 'cut-off' effect (lack of anesthetic effect of molecules which follow Meyer-Overton but are too large, e.g. increasing length of n-alcohols" or n-alkanes"), or the lack of anesthesia by temperature induced re-ordering in lipids which mimics anesthetic effects therein'''. Finally, there are exceptions to the Meyer-Overton rule. Agents, such as 1,3,5 tris(trifluoromethyl)senzene (TFMB) and 1,2 dichlorohexafluorocyclobutane (F6), which are predicted by their lipid solubility to have significant anesthetic potency but do not, even at higher concentrations (represented by an estimated MAC of 1000% atmospheres (atm)) (Fig. 2A). Consequently, these agents are called non-anesthetics. Other gases, such as flurothyl (indoldan), show unresponsiveness at their Meyer-Overton predicted anesthetic potency, but cause seizures at lower concentrations and are thus denoted as anesthetidconvulsants". Discerning how non-anesthetics differ from anesthetics may be an important clue to understanding anesthesia. It is well accepted that the effect of anesthetics is related to their hydrophobicity, as well as their permanent dipole strength, and polarizability"• E9. Considering the problems with the lipid bilayer theories of anesthesia, Franks and Lieb demonstrated that the Meyer-Overton correlation was consistent with anesthetics acting directly on proteins, specifically in non-polar, lipid-like 'hydrophobic pockets' within protein interiors". These non-polar intra-protein regions provide for a chiral environment, and therefore, the orientation of the pocket-bound anes- thetic is determined by its structure and permanent dipole moment. Furthermore, these regions are composed largely of highly polarizable, it-resonance clouds of aromatic amino acids such as tryptophan, tyrosine, and phe- nylalanine. Polarizability, which refers to the degree to which a molecule can be instantaneously polarized by nearby charge distributions, correlates strongly with anesthetic potency"•" (Fig. 2B, Table SI). However, the polarizability correlation also implies that the predicted MAC of the non-anesthetics F6 and TFMB are less than predicted by the Meyer-Overton relation suggesting an even higher anesthetic potency for these agents than pre- viously believed, but this is clearly not the case as they have no anesthetic effect even at significantly higher doses. What this does indicate is that there is a clear deviation from the correlation between the physical parameters SCIENTIFIC REPORTS17: 9877 IDC810.1038/s41598-017-09992-7 2 EFTA00606069  a IDDr . T 1000 WO. Tr146 100 • Flue I 10 Des 16 I ). • 126MAC in It' = 0.990 p • 50-7 0 0.10 10.00 100.00 1000.00 Minimum Alveolar Conomitratien, MAC pi atm) b 20 16 10 TFMS 14 Ia F6 10 412\ Ca 5.4. " . ",„„%ss • Flue • DE 4 a • -2.813log,o(MAC) • 9.21 R' • 0.957 p • 30-5 0 0.001 0.01 0.10 1.00 10.00 100.00 1000.00 Minimum Alveolar Concentration, MAC 416 atm) C 20.00 a • 2.40log,o0.1 • 4.18 Ri • 0.972 p•70-5 TIPIA• 16.00 s.:6 t 10.00 E• Meth Nele Fke• 'so Des DEDI li st, 0.00 0.10 1.00 10.00 100.00 1.000.00 011:Gas Partition Coefficient, A Figure 2. Correlation of anesthetic properties with anesthetic potency. (a) Meyer-Overton correlation of oil:gas partition coefficient versus MAC (Blue points - anesthetics; Red points - non-anesthetics; Green points - anesthetic/convulsant Red line - difference between non-anesthetic predicted and estimated (-1000% atm) MAC). (b) Correlation of polarizability versus MAC, with MAC for non-anesthetics determined from the Meyer-Overton correlation. (c) Correlation of polarizability versus solubility shows a difference in the relation between these properties for non-anesthetics and anesthetics. Trend lines and equations based on anesthetics alone, without the contributions from the non-anesthetics and anesthetic/convulsant. SCIENTIFIC RE PORTS17: 9877 IDOI:10.1038N41598-017-09992-7 3 EFTA00606070  for lipid solubility and molecular polarizability for these non-anesthetic agents compared to anesthetics (Fig. 2C, Table Si). Thus, it appears that non-polar 'lipid' solubility determines anesthetic binding at the site of action, i.e. within hydrophobic pockets, but how does this cooperate with molecular polarizability to act in the brain and cause loss of consciousness? Anesthetic binding within protein interiors is thought to block critical conformational changes or other spe- cific dynamic protein functions, but the question of which proteins are critically involved remains unclear. Franks and Lieb, and many others, assumed that anesthetics target membrane receptor and ion channel proteins, as these directly govern dendritic-somatic membrane excitability and depolarization, which govern axonal firing rates. Specifically, post-synaptic GABAA, acetylcholine, serotonin, glutamate, glycine, n2-adrenergic, acetylcholine, or adenosine receptors have been the presumptive membrane protein targets, and anesthetics have been shown to have a high binding affinity for these sites. However, under anesthesia, there's more anesthetic in the peripheral fat stores of the patient's body than in their brain, yet anesthetics act in the brain. Thus, anesthetics clearly bind to these receptors and channels at their MAC value, but as anesthetic actions on them are highly variable and inconsistent, they have been deemed fruitless in terms of a common mechanism of action". Yet, while membrane- bound receptors and ion channels continue to be considered the primary sites of anes- thetic action, the microtubule cytoskeleton inside neurons remains overlooked". In 1968, Allison and Nunn showed that the anesthetic halothane caused depolymerization of microtubules, although at high anesthetic concentrations of about 5 times their MAC value". While this is beyond clinical concentrations, a systematic approach by the Eckenhoff lab using radiolabeled halothane in mice showed that at clinically relevant concentra- tions (—the MAC value for mice), anesthetics bind to —70 different neuronal proteins, halfin membranes and half in the cytoplasm". Among these was cytoskeletal tubulin, the component subunit protein of microtubules. Many studies since have indicated direct binding of several anesthetics directly to tubulin including halothaner"• 3s, 6-Azi-pregnanolone36, and 1-azidoanthracene". Furthermore, proteomic analysis ofgenetic expression following exposure to the volatile anesthetics halothane, isoflurane, desfiurane, and sevollurane show alterations in tubulin gene expression several days after treatment'" 40 with no changes observed in the expression of membrane pro- teins". Clearly, anesthetics do bind to membrane receptors and channels as well as lipids at their MAC value, but they also bind to tubulin in microtubules. These findings are of relevance because learning, memory, cognition, and the long-term potentiation para- digm specifically require a cytoskeleton capable of complex reorganization to accommodate changes in synaptic activity and strength"-". This further suggests that anesthetic-induced changes in cytoskeletal stability may be a common mechanism for anesthesia". Neurodegenerative diseases share the pathology of a dysfunctional neu- ronal cytoskeleton (e.g. Alzheimer's, Parkinson's etc.)", and since the architecturally complex cytoskeletal matrix within neurons is responsible for neuron morphology and intracellular transport, the interaction of anesthetics with tubulin and microtubules is important for understanding the effects of anesthesia-induced post-operative cognitive dysfunction (POCD). While this is clearly an issue, the mechanisms leading to cognitive impairment after anesthesia and surgery are not yet fully understood. It has been previously hypothesized that anesthetics can alter resonance in it-electron cloud oscillations among highly polarizable non-polar amino acids in tubulin". Overall, dipoles can be induced within these electron clouds by nearby charges, dipoles, or other polarizable structures. When in proper orienta- tion it-resonance structures (exemplified in simplest form by benzene) attract each other by van der Waals-type London dispersion forces, which then couple and oscillate. As dispersion forces tend to be stronger between mol- ecules that are easily polarized, and as these dispersion forces contribute to protein folding and protein-protein interactions", the effect of anesthetic polarizability on tubulin has implications for the dynamics of microtubule stability during and after surgery. As any mechanism ofanesthesia is expected to discriminate between true anesthetics and non-anesthetics, we aim to further test this hypothesis by assessing the effects of several volatile anesthetics, the non-anesthetics F6 and TFMB, as well as the convulsant flurothyl on the London dispersion interactions between highly polarizable aromatic amino acids in tubulin. To investigate how molecular solubility and polarizability may contribute to anesthetic action we use results of previous anesthetic binding site predictions on tubulin with molecular dock- ing, quantum chemistry calculations, and theoretical modeling of collective London dispersion interactions to investigate the effects of this group of gases on tubulin function. As this mechanism has direct bearing on the link between anesthesia, post-operative cognitive disorder (POCD) and its effect on neurodegenerative disease, it has the potential to provide new insights on the site and mechanism of anesthetic action, and may in the future contribute to the design and development of new anesthetics with fewer potentially harmful side effects. Below, we discuss the results obtained in our study. Results Anesthetic Docking to Tubulin. To computationally predict the effect of anesthetics on the tubulin protein we ran docking simulations of 8 anesthetic molecules, 2 non-anesthetic molecules, and the convulsant flurothyl (Fig. 1), on previously predicted high affinity sites of binding to tubulin". The results of anesthetic docking to tubulin are summarized in Table S2. Successful docking was completed for all 9 high-affinity binding sites for all l l agents except for the non-anesthetic F6 to tubulin site 5 and non-anesthetic TFMB to site 7. Most likely these agents failed to dock due to the binding site being too small or containing unfavorable functional groups, donor/acceptor interac- tions, hydrogen bonds or hydrophobic effects. Whether this relates to their non-anesthetic effect is unknown. All binding energy values range between -4 and — 12ki/mol, which is indicative of weak non-covalent interactions consistent with the known binding action of anesthetic agents by van der Waals-type London dispersion forces. To determine if there is a preferred binding site on tubulin for all anesthetics, ANOVA analysis was run to compare anesthetic binding at each site. Figure S1 provides a graphical illustration of the distribution of binding SCIENTIFIC RE PORTS17: 9877 IDOI:181038N41598-017-09992-7 4 EFTA00606071  sites of the various anesthetics on tubulin. On average, the preference for anesthetics to bind at a tubulin site are ordered as site 7, 5, 38, 4, 1, 23, 37, and finally 21. However, statistical difference (p < 0.05) was only found between site 21 and sites 4, 5, 7 and 38. No other significant differences were found, suggesting that there is no overall preferred binding site for anesthetics to tubulin. Still, it is worth noting that sites 7 and 38 are within 5 angstroms of key residues of the colchicine-binding pocket which is consistent with the volatile anesthetic hal- othane being shown to reduce colchicine binding to tubulini9. Furthermore, sites 5, 7, and 38 are all within 5 angstroms of residues that interact with either the intradimer non-exchangeable GTP molecule or the magnesium ion required for the intradimer alpha-beta tubulin stability, and may be a mechanism by which volatile anesthet- ics disrupt microtubule structureS0. Quantum Chemical Estimate of Polarizability and MAC. We calculated the mean polarizability and polarizability tensors for all It agents listed in Fig. 1 using a density functional theory approach. The results of our quantum chemical estimate of mean polarizability are shown in Fig. 2B and listed in Table SI, along with experimental measures of anesthetic MAC and predicted MAC for non-anesthetics and convulsants. However, these estimates are based on the mean polarizabilities of the molecules and does not account for the directions of polarization, permanent dipole moments, or docking orientation of agents to a given protein. To investigate the full effect of anesthetic, non-anesthetic, and convulsant polarizabilities and permanent dipole moments on the collective electronic behavior of the tubulin dimer, we calculated the molecular dipole components induced by London dispersion forces between the highly polarizable aromatic amino acids tryp- tophan, tyrosine, and phenylalanine based on their molecular polarizability tensors, and evaluated the change between collective dipole oscillations in the presence and absence of the agent molecules. In the absence ofagent molecules the aromatic amino acids of tubulin set up normal oscillatory modes that range in frequency between 480 and 700 THz (1THz = 1012 Hz). This result provides a first-order description of the collective oscillation in tubulin. A full quantum mechanical parametrization of the coupled atomic dipolar fluctuations in valence elec- tronic response, as done in the many-body dispersion approach to describe collective wavelike charge density fluctuations", would provide a more refined estimate of this behavior. The presence of an agent molecule creates another normal mode of dipole oscillation for the aromatic-agent network In Fig. 3 we plot these new normal modes created by the addition ofan agent as a function of their MAC. A clear polynomial trend can be seen for anesthetics alone, with a very high degree of correlation (R.2= 0.995), which is slightly greater than that found for the Meyer-Overton relation (Fig. 2A). While the anesthetidconvul- sant tlurothyl also follows this trend, the non-anesthetics both do not. Rather, due to the polynomial nature of the relation the minimum possible frequency that would lie on the curve is 594 THz, which is marginally greater than both of the frequencies introduced by the non-anesthetics investigated, suggesting the non-anesthetics fall below a cutoffrequired for anesthetic action. The biological importance of this range is not fully understood at this time. The introduction of an agent also shifts the normal oscillatory modes of the aromatic dipoles by an order of to 100 GHz (1GHz = 10°Hz). Figure 4A shows specific plots illustrating the shifts in the oscillation frequencies of tubulin's aromatic amino acids for agents docked at each of the predicted tubulin binding sites. No correlation between peak shift in frequency and binding site energy was found. The most prominent shift observed for all the anesthetic agents was observed as a decrease in the normal mode of oscillation in the range of (613 ± 8) THz. Both the biological significance of this range and the relevance of a decrease in oscillation frequency to anesthetic action, versus an increase, require further investigation. The maximum frequency decrease in this range induced by anesthetics correlates with MAC (R2= 0.999) (Fig. 4B). Flurothyl again followed the same trend as the anes- thetics, while F6 and TFMB did not show any decreases in this range, but rather showed increases in oscillation frequency. Of note is that the predicted MAC of the non-anesthetics is greater than 1000% atm well beyond phys- iologically relevant concentrations, correctly predicting their lack of anesthetic action. Discussion Anesthesia is one of the great achievements of modern medicine, yet the mechanisms by which anesthetics selec- tively block consciousness and memory formation, while sparing non-conscious brain activities, remain unclear in spite of a century and a half of clinical use. Understanding anesthetic mechanisms may shed light on several problems in consciousness studies, which would be an important scientific and philosophical achievement. More practically, a better understanding of anesthesia can aid in clinical decisions to reduce the risk of adverse effects including POCD, particularly in those suffering from neurodegenerative conditions, and/or lead to the rational design and development of anesthetics with fewer deleterious side effects. Yet how do anesthetic gases, a disparate group of volatile chemical compounds, exert a common, unitary effect in all animals at concentrations specific to each gas? The Meyer-Overton correlation implies a unifying factor related to the solubility of a gas in lipid-like, non-polar hydrophobic solvents. Binding in these solvents occurs by very weak London dipole dispersion, a type of weak van der Waals force, but the significance of the Meyer-Overton correlation remains elusive. Solubility in lipids alone does not account for the cut-off' effect2azs anesthetic differences due to chirality", the lack of other lipid-altering effects to cause anesthesia", or the role of membrane proteins in neuron excitability, leading to the conclusion that anesthetics act in non-polar, hydropho- bic regions within proteins. Furthermore, there are exceptions to the Meyer-Overton rule where an agent's lipid solubility predicts it to have a significant anesthetic potency, but it does not (i.e. P6, TFMB). These exceptions, however, may provide insight into the mechanisms of anesthetics. Examining how molecular polarizability correlates with anesthetic potency reveals a discontinuity with the Meyer-Overton rule, specifically for the non-anesthetics F6 and TFMB. What is the difference in relation between non-polar, 'lipid' solubility and polarizability such that these gases don't cause anesthesia? In this paper we investi- gate this relationship and present a computational study aimed at examining the effect ofanesthetic, non-anesthetic, and anesthetic/convulsant agent polarizabilities and permanent dipole moments on protein function. SCIENTIFIC RE PORTS17: 9877 IDOI:181038N41598-017-09992-7 5 EFTA00606072  Flur En a 7rm I a F6 Moth Halo 11 .22/ Sewn DEM Deo SOO 600 700 SOO 900 1000 1100 Frequency, f (714z) b 1200 X 1000 0 800 DEth Des 0 Haloi " Moth Li. En Setronu 000 0 F6 400 True 7.1 • 0 200 f s 69(i0910(6160))2 87.51O91n(MAC) + 643 Ra a 0.995 p = 2e-6 0 0.10 1.00 10.00 100.00 1,000.00 Illabouon Mimes Concontraeon, MAC (% atm) Figure 3. Collective dipole modes ofoscillation in tubulin. (a) Average energies of the collective dipole modes of oscillation in tubulin. Gray - normal modes predicted for tryptophan, tyrosine and phenylalanine in tubulin in the absence of agents. (Blue - additional normal modes introduced due to the presence of an anesthetic agent; Red - additional normal modes introduced due to the presence of a non-anesthetic agent; Green - additional normal mode introduced to the presence of the anesthetic/convulsant agent flurothyl). (b) Agent-induced new frequency modes ofoscillation versus MAC. As the non-anesthetics fall below the trend line minimum there is no predicted MAC for non-anesthetics available at any value. (Blue points - anesthetics; Red points - non- anesthetics; Green points - anesthetidconvulsant; Red line - difference between non-anesthetic predicted and actual (- I 00096 atm) MAC). While membrane receptors and ion channel proteins are the assumed target of anesthetic action, genomic and proteomic studiesm4s point to microtubules as a functional target of anesthetic action. Relating anesthetic action to microtubules is relevant clinically because learning, memory, cognition, and the long-term potentia- tion paradigm specifically require a cytoskeleton capable of complex reorganization to accommodate changes in synaptic activity and strength''"". Furthermore, this suggests that anesthetic effects on microtubules which may affect cytoskeleton stability may be a common mechanism for anesthesiaJ6. As neurodegenerative diseases share the pathology of a dysfunctional neuronal cytoskeleton (e.g. Alzheimer's, Parkinson's etc.)°, and since the archi- tecturally complex cytoskeletal matrix within neurons is responsible for neuron morphology and intracellular transport, the interaction of anesthetics with tubulin and microtubules may be important for understanding the effects of anesthesia-induced POCD. As dispersion forces tend to be stronger between molecules that are easily polarized, and as these dispersion forces contribute to protein folding and protein-protein interactionsi8, the effect of anesthetic polarizability on tubulin has implications for dynamics and stability of microtubules during and after surgery. As such we focused our investigation on tubulin, the constituent protein of microtubules, as it is a direct target of anesthetics, has explicit relevance to POCD in cytoskeleton-compromised neurodegenerative disorders, and may be the site of a common, unitary mechanism of anesthesia. Our results indicate that for anesthetics there is a very strong correlation between their potency and the shifts they induce in the characteristic dipole collective modes in the tubulin dimer. Specifically we found tubulin aro- matic amino acids alone to possess collective oscillations in networks of London-force dipoles among pi electron resonance clouds of aromatic amino adds in the range of 480 to 700THz. The presence of an anesthetic (repre- sented as an additional polarizable molecule in the aromatic network) creates another normal mode of the dipolar oscillations. The introduction of an anesthetic also shifts the normal oscillatory modes of the dipoles downward (slower) by an order of 1 to 100GHz. The most prominent shift observed for the anesthetics is situated around a specific normal mode ofoscillation at (613 ± 8) THz. These new oscillatory modes introduced by anesthetics also highly correlate with their anesthetic potency. Interestingly, non-anesthetics follow this trend with a predicted SCIENTIFIC RE PORTS 17: 9877 IDOI:10.1038N41598-017-09992-7 6 EFTA00606073  a 0 DOSOVISIIS Dielhyl ESA 0 NSMUYas -10 40 15 400 Frequency Shift , At ( GHz) 20 400 100 ISO 500 WA 700 AM MX 700 WA 000 700 WA 600 700 listkoxylleans NknosOmMe Sesmilwas so 0 r 50 0 0 400 -50 4 40 •1000 -100 4500 4 400 500 SOO 700 500 GOO 700 500 SOO 700 500 SOO 700 FS WIAS Plurethyl 500 30 so — W1 - srne 4 0 0 S to - sis .500 40 — W al - W23 10 —537 .1000 -100 —W» — W39 •1500 0 G-W-JIIIL ISO 500 600 700 500 SOO 700 500 OM 700 Frequimcy, t (THz) b 0 En S:vFolur Des Nitr Halo DEtlI •O -200 "" -400 • -600 1- g -800 Af6, 3 = -83.6(MAC)-' 6'-22.4 — -1000 R2 = 0.999 .t p = le -3 tj-1200 Meth 1.1400 O U. 4600 0.10 1.00 10.00 100.00 1,000.00 Minimum Alwoolar Concentrallon, MAC (% alan) Figure 4. Change in tubulin collective dipole modes due to the addition of anesthetic/non-anesthetic molecules for different binding sites. (a) Site specific changes for anesthetics and anesthetic/convulsant flurothyl shows a prominent downwards shift at (613 ± 8) THz, while non-anesthetics F6 and TFMB show an increase at this frequency band. (b) Maximum agent induced change in tubulin normal mode oscillation frequency at (613 ± 8) THz versus agent MAC (Blue points - anesthetics; Red points - non-anesthetics; Green points - anesthetic/ convulsant). MAC of well over 100% atmospheres, suggesting that this mechanism correctly distinguishes anesthetic and non-anesthetic agents, a requirement for a common anesthetic mechanism. As the anesthetic/convulsant gas fiurothyl also lies on this trend line with a clinically relevant MAC it must be noted that flurothyl can indeed SCIENTIFIC REPORTS l 7: 9877 I DOI10.10313/s41598-017-09992-7 7 EFTA00606074  produce anesthesia in mice at a M