key: cord-0920852-t0t0m9rz
authors: Hernández-Arteaga, A.C.; Ojeda-Galván, H.J.; Rodríguez-Aranda, M.C.; Toro Vazquez, J.F.; Sánchez, J.; José-Yacamán, M.; Navarro-Contreras, H.R.
title: Determination of the Denaturation Temperature of the Spike Protein S1 of SARS-CoV-2 (2019 nCoV) by Raman Spectroscopy
date: 2021-08-12
journal: Spectrochim Acta A Mol Biomol Spectrosc
DOI: 10.1016/j.saa.2021.120269
sha: 5f257a82db27439b27c5b23d732ca90418ef7782
doc_id: 920852
cord_uid: t0t0m9rz

In the present work the temperature response of the constitutive S1 segment of the SARS-CoV-2 Spike Glycoprotein (GPS) has been studied. The intensity of the Raman bands remained almost constant before reaching a temperature of 133 °C. At this temperature a significant reduction of peak intensities was observed. Above 144 °C the spectra ceased to show any recognizable feature as that of the GPS S1, indicating that it had transformed after the denaturation process that it was subjected. The GPS S1 change is irreversible. Hence, Raman Spectroscopy (RS) provides a precision method to determine the denaturation temperature (T(D)) of dry powder GPS S1. The ability of RS was calibrated through the reproduction of T(D) of other well studied proteins as well as those of the decomposition temperature of some amino acids (AA). Through this study we established a T(D) of 139 ± 3 °C for powder GPS S1 of SARS-CoV-2.

• The Raman spectra before protein denaturation show characteristic Raman bands of Spike Glycoprotein S1, which disappear when the denaturation temperature onset is reached.

• The Raman resultant denaturation temperature of Spike Glycoprotein S1 are completely reproducible and easily accurate within 2 °C.

• Raman spectroscopy allowed us to obtain a precise determination of the denaturation onset temperature of the dry powder of Spike Glycoprotein S1 as 133 °C.

• Raman temperature dependence studies are applied to determine the denaturation temperature of proteins.

to show any recognizable feature as that of the GPS S1, indicating that it had transformed 1 after the denaturation process that it was subjected. The GPS S1 change is irreversible. 2

Hence, Raman Spectroscopy (RS) provides a precision method to determine the 3 denaturation temperature (TD) of dry powder GPS S1. The ability of RS was calibrated 4 through the reproduction of TD of other well studied proteins as well as those of the 5 decomposition temperature of some amino acids (AA). Through this study we established 6 a TD of 139 ± 3 °C for powder GPS S1 of SARS-CoV-2. The SARS-CoV-2 virus binds to the target cells via the ACE2 receptors protruding 8 through the membrane cells (ACE2: angiotensin-converting enzyme 2) [2, 3] . This 9 bonding is mediated by the Spike Glycoprotein, henceforth referred as GPS, which is 10 formed by homotrimers projected from the viral lipid bilayer. 11

The GPS of SARS-CoV-2 is composed by two subunits, S1 and S2, as shown in 12 Figure 1 . S1 subunit lies mainly at the head or flattened top of the spike, and conforms an 13 important portion of this segment, and S2 on the stem. The GPS S1 subunit contains a 14 receptor binding domain (RBD) that binds to the host cell ACE2 receptors. This process 15 results in conformational changes in the S2 subunit, which allows the fusion peptide to 16 insert into the host target cell membrane [4] . 17 1 Figure 1 Ribbon diagram of SARS-CoV-2 (2019-nCoV) Spike Glycoprotein GPS: a) S Trimmer, 2 b) S Protomer, c) S1 Subunit, and d) S2 Subunit.

The three-dimensional structure of the spike glycoprotein was determined using 4 cryo-electron microscopy [5] . This study confirmed that GPS couples to the ACE2 protein 5 of human cells with a higher affinity than that of the SARS-CoV coronavirus [6] . Thence, 6

it is now well established that the coronavirus GPS S1 subunit is essential in the viral 7

binding to the host cell at the onset of the infection process [7]. 8

Given the crucial role that this GPS protein plays in the infection process has 9

converted the study of its chemical and physical properties a highly relevant scientific and 10 priority subject. The aim of the present work is that having determined and understood its 1 physical, chemical, and biological functions as much as possible, these advances in 2 scientific knowledge may lead to the development of successive generations of 3 biotechnologically designed vaccines, as well as of medicaments, to inhibit the access of 4 the SARS-CoV-2 virus to infect human cells [8, 9] . 5 A second particularly important reason to characterize all the physical properties 6 of the GPS (and thence of subunits S1 and S2) is that it constitutes an abundant 7 (protruding) surface protein on the SARS-CoV-2, Consequently, it becomes a primary 8 target for neutralizing antibodies. The GPS undergo several structural rearrangements to 9

fuse virus and host cell membranes, thus enabling the delivery of the virus genome into 10 the target cells [10] . Thence, it is a primary target for virus-inactivating antibodies, and a 11 natural target in vaccine development [10] . 12 RS has successfully been applied to study several viruses, and in particular the 13 SARS family of viruses [11] . In this study, conformational changes in glycoproteins were 14 observed, and therefore RS was proposed as an effective and sensitive tool in the 15 As all proteins are sensitive to the temperature, which may cause structure and 1 function changes, up to the protein unfolding, i.e. protein denaturation, it is necessary to 2 study, among many other influencial parameters (applied pressure, for instance), the 3 temperature behavior of the GPS and separately that of the S1 subunit, which plays the 4 primordial role in viral-human cell binding. A very comprehensive study on the temperature 5 response of the GPS S1 subunit in contact with a wet environment such as that inside of 6 an infected subject, has been performed by the molecular dynamics modeling by S. Rath 7 and K. Kumar [16] . That study found that above 40 °C the RBD closes inhibiting the 8 possibility of any binding to host cells, therefore, inactivating the SARS-CoV-2. 9

Thence, RS is evidently an analytical tool that helps to establish important 10 properties of viruses. Within this context, we present in this work a study of the Raman 11 spectrum response of the GPS S1 subunit from this virus, as a function of the temperature. 12

In addition, the temperature response allowed us to reproducibly determine the 13 denaturation onset temperature at 133 °C ± 2 o C and a final temperature of 144 ± 2 °C, 14

where irreversible protein changes were clearly observed in the Raman spectra indicating 15 that the denaturation was terminated. This result was contrasted with the application of 16 RS to a study in the process of denaturation of well-known proteins, and of the 17 decomposing temperatures of AA, which allowed us to ascertain that RS provides reliable 18 results in the determination of the TD when compared with those measured by differential 19 scanning calorimetry (DSC) [17] , the preferred technique in biochemistry studies to 20 determine these temperatures [18, 19] , even in the case of proteins in which the Amide I 21 band is silent, for example in the case of the GPS S1. The study of the temperature 22 response of the main constitutive proteins of the SARS-COVID-2 virus, or of any similar 23 virus is nowadays a priority subject. Mainly because its infectivity and total inactivation 24 (destruction) largely depends on the temperature, along with other physical or chemical 1 agents. The present study is providing new knowledge about the GPS S1 subunit 2 response to temperature. This because the S1 subunit is the main element involved in the 3 mechanism for the virus binding easing the infection of human cells. 4

The of GPS S1 subunit (> 95 % purity as determined by the manufacturer through 6 size exclusion HPLC) refined from SARS-CoV-2 (2019-nCoV) viruses was obtained from 7

Sino-Biological Incorporated in powder form. The protein consists of 670 AA with a 8 molecular weight of 76.5 kDa. The GPS S1 samples were gently compressed to flat, thin, 9 compact discs, before the Raman analysis. 10

The Raman spectra as a function of temperature for the GPS S1 were obtained 12 using a HORIBA Xplora Plus Micro Raman, equipped with a solid-state infrared laser (785 13 nm wavelength) with an estimated power of 5 mW. The spectrometer grating conditions 14 were 1800 grooves/mm, the laser wavelength and focal distance of 0.5 cm, the spectral 15 resolution was 0.8 cm -1 per pixel. Each spectrum had an acquisition time of 20 sec, and 16 five consecutives were taken and averaged to provide the final spectrum. In order to 17 acquire the temperature dependence measurements, we used an environmental cell with 18 a transparent optical window and an electronic heating system. Moreover, to provide 19 uniformly heat to the compact disc protein sample, was placed inside the chamber at a 6 20 mm depth. The temperature controller was set up to increase in 5 °C steps, remaining for 21 5 min at each temperature before recording the Raman spectra at the preset temperature. 22

The whole temperature measurements were performed in three different occasions with 1 several days between them, to check for reproducibility. 2

The deconvolution process was performed using the Fityk program (V 1.3.0) [20] , 4 which offers an assortment of functions to fit the Raman bands. The choice of fitting 5 function does not alter the wavenumber position of a given band, but it may result in 6 slightly different full-widths at half maximum (FWHM). Fitting the baseline of the Raman 7 spectra was carefully established before data fitting. In order to do this, the fluorescence 8 background was removed by fitting a polynomial curve to the measured spectrum where 9

no Raman signal was expected. The treated spectra were used on data fitting. However, 10 it is necessary to mention that the samples exhibited a very weak fluorescence, i.e, the 11 subtraction performed is relatively small. Also, the background signal to noise ratio 12 essentially appeared of the same magnitude for all spectra, similar to that shown and 13 examined in Fig. 2 , which is generally very good, better than 10 2 . 14

The modeling process was performed using the UCSF chimera® program (V 1.15) which 16 is feedbacked with the GPS data obtained from the Protein Data Bank, PDB: 7kdi [21] . 17

The denaturation profile of the proteins was determined by differential scanning 19 calorimetry using a TA Instruments Calorimeter Model Discovery (New Castle, USA). To 20 obtain the profile decomposition, 5 to 10 mg of the corresponding protein was sealed in 21

Tzero aluminum pans (TA Instruments, New Castle, USA). After 3 min at 25 °C the system 22 was heated at a rate of 5°C/min until achieving the full ending of the denaturation 23 endotherm was achieved. The TD was determined at the maximum heat flow of the 24 corresponding endotherm, using the first derivative of the heat flow and the equipment 1 software (Trios V 3.3.0.4055; TA Instruments-Waters LLC, New Castle, DE). 2

The interpretation of the Raman spectra of proteins has been a subject of study at 4 least for the last two decades. In Table I the number and percentage contribution of the 5 AA-residues present in the GPS S1, is listed [22] . The aim has been to understand the with Gly as initial and terminal residues (GlyAAGly) produced better agreements than the 14 single AA-residue side chains, between the Raman bands of tripeptides and those 15 measured on three model proteins: bovine serum albumin, -lactoglobulin, and lysozyme. 16

The single AA-residue side-chains spectra present in some instance's wavenumber 17 displacements of almost 5-10 cm -1 from those expressed in the protein either to higher or 18 lower wavenumbers, but for high intensity bands in many instances the deviations of 19 single AA-residue side-chains emissions were very modest. Those of tripeptides studied 20 in [27] presented systematically modest deviations of ± 1 cm -1 , and seldom up to 5 cm -1 , 21

when present in those three model proteins. In Table II Figure 2 Raman spectrum of the GPS subunit S1. Upper spectrum, region of AA Raman 2 expression, grey lines: contributing Raman bands as determined from deconvolution process. Red 3 line: result of all contributing Raman bands. Spectrum of GPS S1 from 100 to 3600 cm -1 (bottom).

The full Raman spectrum (20 °C) for the compressed solid powder of the GPS S1 is 6 shown in Figure 2 , near bottom axis, black thick line as it can be seen there are two 7 spectral regions clearly separated, by a broad wavenumber expanse where no Raman 8 signal is observed, from just above 1600 to 2800 cm −1 . Although the Raman signal was 9 collected in the spectral interval from 100 cm −1 and 3600 cm −1 , only the 400 to 1600 cm -1 10 region is analyzed in this report. We also observe 1) a Raman band from 2800 to 3000 11 cm -1 , which in the literature has been identified as the signal from C-H bonds stretching 12 modes, which have been studied by other authors to determine if the protein is either 13 hydrophobic or hydrophilic in nature [28], and 2) the O-H broad band from 3100 to 3550 14 cm -1 , which similarly reflects information of the hydroxyl anions of the water molecules 15 within the proteins. This region covers most of the Raman bands used to identify the 1 fingerprints of the 20 constitutive AA-residues of all proteins [24] [25] [26] [27] . The red line shows 2 the data fitting using Lorentzian functions to deconvolute the 33 obtained Raman bands. 3

In Table II we with some proteins as mentioned above. In Figure 3 the expected relative contributions of 12 specific AA-residues to each Raman band of the Glycoprotein Spike S1 have been plotted. 1

In Figure 2 , the 870 cm -1 band (B11) stands out notoriously. Two constitutive amino 2 acids have strong Raman signals that are very close to this wavenumber, threonine (872 3 cm -1 ) and glutamic acid (873 cm -1 ) [23]. However, aspartic acid, arginine, cysteine, 4 isoleucine, methionine, and tryptophan have medium intensity Raman bands at 873 cm -1 5

[23]. Hence, it is expected that this strong band could be the collaborative signal of all 6 these AA-residues, as illustrated in Figure 3 , with colored and/or hatched bars. These 7 eight AA-residues comprise  29.85% of the total number of AA-residues constitutive of 1 the GPS S1 subunit, (TNA). 2 3 Figure 3 Contribution of specific AA to the Raman bands of the GPS subunit S1. 4

The data deconvolution indicates that a second band at 882 cm -1 (B12) is merged 5 into this strong band, producing the asymmetric resultant shape. This band most likely 6 corresponds to the fingerprints of lysine and histidine, both individually have strong 7

intensity Raman dispersions at 881 and 882 cm -1 , and tyrosine, which has medium 8 intensity emissions at 881 cm -1 . These constitute 12.09 % of TNA. Since this is a relatively 9 strong emission, this result suggested that the folded complex environment of the protein 10 might be enhancing the emission intensity of these amino acids. There were another five 11 bands of large intensity and standing out of the continuous spectral sections observed 12 from the spectral deconvolution. These bands are at 515, 1115, 1252, 1321 and 1454 cm -13 1 , corresponding to B14, B22, B27, B29 and B33, respectively. The B14 band occurring 14 at 515 cm -1 has been identified by [25] as originating by vibrations of the asparagine amide 1 terminal. The B22 band, occurring at 1115 cm -1 , coincides with the strong Raman signals 2 of histidine (at 1113 cm -1 ), and threonine (at 1116 cm -1 ). Alanine, aspartic acid, and 3 methionine present medium intensity bands at 1115, 1121, and 1122 cm -1 , hence, these 4 three are also expected to contribute to this emission. All five AA-residues constitute 20.00 5 % of TNA. At 1252 cm -1 (B27), there exists a plethora of Raman emissions in the following 6 7 AA-residues and the amide region emissions of the peptide bonds: Amide III, histidine, 7 isoleucine (medium intensity Raman intensity emissions); and the contribution with weak 8 free AA-residues emissions of aspartic acid, lysine, methionine, threonine, and 9

tryptophan. Once again, the complex folded protein structure might be enhancing the 10 intensity of the histidine and isoleucine vibrations located at 1252 and 1257 cm -1 , 11 respectively, in conjunction with the Amide III emissions, as well as of some of the weaker 12 expected contributions of the other 5 AA-residues. The B29 band at 1321 cm -1 13 corresponds to the amide III signal, present in all AA peptide bonds of proteins [25, 26] , 14 as the 1321 cm -1 signal from the -helix [24] . The Raman spectra for glycine, isoleucine 15 and serine histidine (very strong signal), presented strong emissions at 1327, 1329, and 16 1327 cm -1 , respectively, very likely corresponding to their amide III regions [25, 26] . 17

Arginine, glutamic acid, methionine tyrosine and valine also exhibited medium intensity or 18 weak emissions around 1328 cm -1 , for a TNA of 42.59%. A final-well resolved band, B33 19 at 1454 cm -1 , stood out at the higher wavenumber end of the spectrum. This band is most 20 likely associated to alanine, isoleucine, leucine and lysine that produce strong strength 21 signals in the isolated AA at 1461, 1450, 1457 and 1456 cm -1 , respectively. Additionally, 22

the following AA-residues should also contribute, glutamic acid, glycine, leucine, and 23 valine, all of which present medium intensity Raman scatterings as isolated AA, as well 24 as tryptophan which has a weak emission, all of them around this wavenumber (TNA 1

In Figure 4 , the Raman spectra recorded at varying increasing temperatures are 4

shown. This figure shows that the spectra exhibit almost identical features from ambient 5 temperature to 130 °C. In the spectra shown recorded at temperatures 135, 138, 140 and 6 144 °C, it is observed that the dominant spectral band at 870 cm -1 rapidly diminishes in 7 intensity, a situation that applies to the other spectral features. At 144 °C, hardly any 8 recognizable GPS S1 subunit spectral features are observed, and at 148 and 150 °C, the 9 GPS S1 spectra are totally deformed, i.e., all traces of the previous predominant 10 transitions have disappeared. The spectra are now dominated by what seems to be 11 interference bands. 12 1 Figure 4 Raman spectrum of the GPS S1 observed at increasing temperatures. 2

In Figure 5a a plot of the wavenumber positions dependence with temperature is 3 presented for the 9 Raman bands of larger intensities (medium and large). It may be 4 observed that B4, B11, B12, B19, B22, B24 and B27, display shifts toward lower 5 wavenumbers (redshift) with increasing temperatures, B30 displays a very moderate 6 tendency for blue shifting, as well as B33, but this last band has almost null shifts. The 7 wavenumber/temperature slopes (w/t) slopes are summarized below in Table III . 8

According to the literature we found similar w/t slopes in a Raman study on the 9 thermal response of two amino acids, L-alanine and L-threonine, as well as the related 10 compound taurine [29] . Their magnitudes are very similar, although for some Raman band 1 these slopes are larger. With respect to changes in relative intensities to those at 20 °C for the same bands, 7 it can be observed that for most of them their relative intensities suffer small decreasing 8 variations with increasing temperatures. However, the Raman band B11 presents a 9 noticeable diminution in the relative intensity above 80 °C. B11 is the strongest intensity 10 Raman peak. This variation may correspond to the ongoing protein changes that antecede 11 the final denaturized state, of several of the contributing AA to this collaborative signal. 12

Nevertheless, for all these 9 bands at 132 °C, their relative intensities start to vary widely, 13 with no discernible pattern for them a few degrees higher, followed by even more dramatic 14 variations if the plot is extended to include temperatures above 144 °C, of the remaining 15 spectral features at the wavenumbers at which these Raman bands appeared at 20 °C. 

These dramatic changes of the GPS S1 subunit Raman spectra above 130 and 8 144 °C are clearly the fingerprint of the denaturation of this protein in a process that starts 9

at 133 ± 2 °C, which is finished at 144 ± 2 °C. 10

The denaturation processes and TD's are standardly measured using differential 11 scanning calorimetry (DSC). Moreover, DSC is the preferred technique used in 12 biochemistry and food studies for determining denaturation and decomposing 13 temperatures of proteins and amino acids [18, 19] . Due to our GPS S1 limited availability, 14 and the amount of materials needed for DSC (milligrams to grams) we performed a 15 subrogate experiment using the well-known proteins bovine serum albumin (BSA), 1 lisozyme, ovalbumin, and of the amino acids L-glutamine, L-Cysteine, and L-Alanine. 2

In Figure 6 , we show as a comparative example our results for spectra of a standard 3 protein in dry powder form, Bovine Serum Albumin (BSA), a) at room temperature and b) 4 spectra after the protein BSA has been completely denaturized at 235 °C. The BSA 5 spectrum at ambient temperature agrees well that reported for BSA under wet conditions 6

[30]. However, the TD's are different for wet and dry conditions, being once again higher 7 than that of the dry form [31] . In Table IV , we summarize the DSC results measured for 8 these proteins and amino acids, in dry powder form, their denaturation or decomposing 9

temperatures obtained, compared with the temperature response of the Raman bands of 10 those three proteins and three amino acids, which disappeared at those temperatures, 11 are presented. The almost total coincidence of the denaturation (proteins) or decomposing 12 (amino acids) temperatures measured by DSC and Raman was very satisfactory. In fact, 13 the linear regression of the temperatures measured by Raman on the temperatures 14 measured by DSC, resulted in a regression coefficient of 0.986 (P < 0.01) with a slope of 15 1.04. The magnitude of this slope was not significantly different from 1.0, a value that we 16 would expect if the denaturation or decomposing temperature measured by Raman and 17 the ones measured by DSC were statistically the same. Further details of the complete 18 study will be presented elsewhere [17] . These results support our contention that the 19 dramatic changes observed in the Raman spectrum at a given onset temperature of these 20 three proteins, as well as in the GPS S1 subunit corresponds to the actual denaturation 21 onset temperature of this very important protein. 

In DSC the TD is reported from the maximum of the endothermic peak measured 8 during the denaturation process. This is usually located almost halfway between the onset 9 and terminal temperatures of this endothermic process. Hence it may be adopted and 10

reported as the TD of powder of the GPS S1 of SARS-CoV-2 a TD  139 ± 3 o C. 11

It is important to remember that the TD of all proteins depend on the medium and 1 several chemical and physical aspects of the surrounding media in which they are 2 immersed. The TD of the dry solid protein is always substantially larger than when it is 3 immersed in a given medium, in the GPS S1 case almost nominally a factor of 2, as 4 expressed in centigrade degrees, i.e., from 70 °C in wet conditions [16] to 140 °C (a 20% 5 increase in absolute Kelvin degrees). For instance, the surrounding medium PH, pressure, 6 salinity, as well as the constitutive chemistry of the solvent or medium it resides affect, 7 among many other factors, in an important magnitude the effective denaturation process 8 and temperature of any protein. Hence, the actual TD's of the S1 subunit attached to the 9 spike and ultimately to the encapsulating membrane of the SARS-CoV-2 depends on the 10 environment it resides. Therefore, they are expected to substantially vary from the one 11 determined for the protein in dry powder conditions. But for the fundamental 12 understanding of GPS S1 subunit, it is important to determine as a reference magnitude Also, it has been well stablished that protein denaturation strongly correlates with cell 22

death and a similar expectation could be physically expected to be valid for the complete 23 virus [35], although for the virus the denaturation of the envelope protein N for instance 24 may play a larger role in the virus disintegration than that of the constitutive subunits S1 1 and S2 of the GPS, but the S1 subunit determines the temperature range of this virus 2 infectivity. 3 4 CONCLUSIONS 5

In this work we report the Raman spectra response to controlled heating of the 6 GPS S1 subunit of the SARS-CoV-2 (2019 nCoV). A careful, detailed and complete 7 assignment of Raman bands in terms of the contribution of specific AA-residues on the 8

Spike Glycoprotein S1 subunit have been performed. The study on the temperature 9 response of this protein is necessary to understand its effect on the inactivation of the 10 virus infectivity above 40-50 °C, as GPS S1 subunit contains the receptor binding domain 11 that constitutes the entrance gate for the predominant mechanism for viral-human cells 12 attachment in order for the SARS-CoV-2 to transfer its genetic code to the attacked cell. 13

All recorded spectra showed similar spectral features and almost constant relative 14 intensities to those at the spectrum recorded at 20 °C, but at 133 °C significant variations 15 in these relative intensities, as well as decline in their intensities, are observed. Above 16 temperatures of 144 °C, all distinctive spectral features disappear. These results are the 17 fingerprint of the denaturation of the GPS S1 subunit in a process that starts at 133 ± 2 18 °C, which is finished at 144 ± 2 °C. The study results in a TD of solid GPS S1 subunit of 19 SARS-CoV-2 as TD = 139 ± 3 °C. The result is supported by a parallel study by RS and 20 DSC of three powder proteins and three amino acids, which indicates that Raman 21 dependent spectroscopy provides reliable denaturation (proteins) or decomposing (amino 22 acids) temperatures. 23 1

The authors acknowledge the access to Laboratorio Nacional de Análisis Físicos, 3

Químicos y Biológicos-UASLP (LANAFQB), during the course of this research. We also 4 acknowledge the NSF-NCI-SW center for financial support in using ASU cores and the 5 MIRA-NAU institute for grant on Metallic nanoparticles. We acknowledge the helpful and 6 graceful assistance of Marisol Dávila Martínez, for her very helpful assistance with the 7 DSC measurements. We wish to thank to José Sauli Ortiz Chávez, Natanael Cruz 8

González and Alondra Hernández Cedillo for some technical assistance. 

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