Molecular Simulations of the Lid Motion in Chimeras of Candida Antarctica Lipase B in Organic Solvents

Molecular Simulations of the Lid Motion in Chimeras of Candida Antarctica Lipase B in Organic Solvents | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular Simulations of the Lid Motion in Chimeras of Candida Antarctica Lipase B in Organic Solvents Zuzana Sochorová Vokáčová, Karolína Fárníková, Eva Pluhařová This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5737623/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The lid motion and the overall structure of Candida antarctica lipase B (CALB) and its mutants CALB-G. zeae and CALB-N. crassa in water, acetonitrile and toluene were investigated by molecular dynamics simulations employing all-atom force fields. This study very likely represents the first systematic application of the umbrella sampling to the lid motion of CALB and its mutants in various solvents. The simulations show that their structures are stable in all solvents. The lid-constituting helixes of the CALB – wild type adopt parallel arrangement, but they tend to be tilted for the mutants and can even change direction of the orientation in case of CALB-N. crassa. Water favors closed lid with contacts between the non-polar side chains of the helixes, mutations shift the corresponding free energy minimum towards slightly larger distance. Toluene causes the lid opening, the open structure of the wild type is stabilized by a salt bridge between the charged Asp145 and Lys308 or Arg309. The effect of the polar organic solvent acetonitrile on the lid opening is less pronounced and seems to be more force field dependent. These detailed insights into the lid opening and specific interactions are relevant for protein and medium engineering of a widely used lipase. Lipase lid swapping organic solvents molecular dynamics umbrella sampling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Lipases are biocatalysts that under natural aqueous conditions hydrolyze triacylglycerols. Interestingly, their substrate specificity for the hydrolytic reaction is very broad and, in addition, there are able to catalyze different reactions such as aldol addition, racemization, epoxidation and aminolysis.[1–3] Therefore, lipases are widely exploited in industry,[4–6] where they are used not only in conventional aqueous media, but also in organic solvents as efficient catalysts of esterification and transesterification reactions.[1] Due to the low water solubility of the natural substrates of lipases, the reaction normally occurs at the organic-water interface where the lipases usually exhibit increased catalytic activity. This phenomenon called interfacial activation is explained by movement of the flexible protein domain (lid) located in the vicinity of the active site. The lid covers the active site in water and opens upon contact with the hydrophobic phase which facilitates the entry of the substrate.[7,8] The relationship among lipolytic activity, interfacial activation and the lid are still debated, as there are lipases exhibiting no interfacial activation at all [8], some of them despite the presence of a large lid.[9] Other possesses it only towards certain substrates.[10] Nevertheless, the lid is an interesting target for protein engineering and its modifications can alter the enzymatic thermostability [11], reactivity [12–18] or enantioselectivity [19,20]. The atomistic details of the lid motion have been investigated by classical molecular dynamics (MD) simulations with empirical force fields (FFs). Conventional MD simulations revealed an existence of the open lipase conformation in water [21,22], but they indeed demonstrated that such arrangement is more stable at the water-hydrophobic interface [23–25] or in organic solvents [23,26–28]. Analysis of collected trajectories provides rationalization of the lid motion, but the long time-scale at which the structural rearrangement occurs suggests that it is desirable to go beyond the conventional MD to obtain converged results. So far, there are only a handful of studies employing enhanced sampling techniques [10] or applying external force [25,29–32] and, to the best of our knowledge, only one of them reports a free energy profile [31] for the lid opening. Here, we systematically investigate the lid motion of Candida antarctica lipase B (CALB) and its mutants CALB-G. zeae and CALB-N. crassa in water and in two commonly used organic solvents (acetonitrile and toluene). We have chosen CALB due to its extensive commercial use which indeed motivated simulation [33–38] and crystallographic [39] studies of the interactions of various mixed solvents or substrates with the lipase. Apart from these practical reasons, it represents a suitable model system as its interfacial activation is still debated [22,24,40] and the mutations in the lid region cause interesting changes in the catalytic properties.[20] Our aims are: (i) assess the structure and stability of the mutants in different environments as their crystal structures are not available, (ii) obtain free energy profile for the lid opening by combination of steered MD and umbrella sampling, (iii) analyze and interpret the differences cause by protein engineering as well as by change of the solvent. COMPUTATIONAL METHODS 2. 1 Model systems The initial structure of CALB was obtained from the 1TCA [41] entry in the Protein Data Bank. The two sugar units (NAG) were removed [42], the 286 crystal water molecules were kept and three disulfide bonds between Cys22 and Cys64, Cys216 and Cys258, and Cys293 and Cys311 were defined. The protonation states of titratable side chains correspond to pH = 7 except for Asp134 which is protonated (neutral) [41–43], thus the overall charge of the system is zero. The amino acid sequence of CALB-G. zeae (CALB-G) was obtained from CALB by performing the following mutations (V139I, G142N, P143I, L144G, D145G, L147T, A148G, V149L, S150IN, A151T, S153A, W155V) in the VMD 44 software. CALB-N. crassa (CALB-N) contains a larger lid than CALB (Y135F, K136H, V139M, G142Y, P143G, D145C, L147G, A148N, V149F, S150G, KVAKAGAPC, A151P, W155L). The protein structures and mutated regions are shown in green in Fig. 1 . The point mutations in CALB-N were again performed in VMD and the secondary structure of the additional amino acid sequence was estimated by I-TASSER[45–47]. The sequence containing the mutated parts (residues 139–156 of CALB-G and 135–164 of CALB-N) were minimized using the steepest descent algorithm and CHARMM36 FF [48] and the rest of the structure was kept fixed. The titratable side chains are in their standard protonation states except for Asp134 resulting in + 1 charge of CALB-G and + 2 charge of CALB-N which was compensated by one or two chloride ions, respectively. Resulting structures were solvated in neat water, acetonitrile or toluene in approx. 9nm simulation cell. The exact composition of the model systems are summarized in Table S1 in the Supporting Information (SI). It also includes the average length of the box in the NpT simulations described below. Two FF parameter sets (CHARMM36 [48] and AMBER03 [49]) were used to describe the enzymes and counterions in order to judge the robustness of the simulation results. TIP3P [50] FF was employed for water; acetonitrile [51] and toluene [48] parametrization was summarized and tested earlier [52]. 2. 2 Simulation Details Classical molecular dynamics simulations with a 1fs time step in the NpT ensemble were conducted using the Gromacs program package (versions 2018.6 and 2020. 3). [53] The temperature was kept at 300 K by the CSRV thermostat [54] with a coupling constant of 1 ps, the pressure was maintained at 1 bar by Berendsen barostat [55] with a coupling constant of 2 ps. The isothermal compressibility of each system was set to the experimental value of the pure solvent. The bonds containing hydrogen atoms were constrained by the LINCS algorithm [56]. The 3D periodic boundary conditions were used, the short-range electrostatic and van der Waals interactions were truncated at 1.2 nm and the long-range electrostatic interactions were treated by the particle mesh Ewald method. First, we performed a 10ns equilibration for each system with the backbone atoms and crystal water molecules restrained with a force constant of 1000 kJ mol -1 nm -2 and stronger thermostat coupling (0.1 ps). As the relative position of the helixes, as well as the orientation of the inserted amino acid sequence with respect to the helixes is unknown, we performed another two sets of equilibration for CALB-N. During the first one, the structure of the helixes was kept fixed, during the second one, the helixes were restrained and their separation kept around 1.1 nm. For CALB-N, this was followed by another 50 ns simulations without restraints which resulted by 3 different initial conditions for each FF and solvent. Then we collected a 200 ns trajectory for each of the systems (30 trajectories in total). As a coordinate for monitoring the lid motion, we have chosen the distance between the centers of mass of the backbone atoms of the following CALB residues of 142–146 (a part of the α 5 helix) and 282–286 (a part of the α 10 helix). The exact choice of the residues varies in the literature, ours lies in between the one in Refs. 11 and 14. The residue range of α 5 is the same also for the mutants, but it differs in case of α 10 : 283–287 (CALB-G) and 291–295 (CALB-N) because of the alteration of the sequence. To further characterize the mutual orientation of the helixes, we picked the vector between the Cα atoms at the beginning and at the end of each helix and calculated the angle between them. Next, to better quantify the probability of the different interhelical distances, we constructed the free energy profiles for the lid opening for the CALB-W and CALB-G. We picked ten configurations separated by at least 20 ns from each of the direct simulations and performed two steered MD to drive shortening and prolonging the interhelical distance for CALB-W and CALB-G. The pulling rate was 0.2 nm ns -1 and the length of the simulation was adjusted to span the range of interhelical distances of 0.9–2.1 nm for CALB-W and CALB-G. To prevent unfolding of the helixes because of the external force applied during pulling, distance restraints were introduced (see topology files in the national data repository https://doi.org/10.48700/datst.df023-eya04 ). These pulling trajectories provided initial conditions for umbrella sampling simulations without additional structural restraints. The sampling windows were equally spaced by 0.1 nm, the same force constant of 2000 kJ mol -1 nm -2 was employed and the simulation was conducted for 10 ns in each of the windows. The free energy profiles were constructed by the WHAM algorithm [57] as implemented in Gromacs [53]. RESULTS AND DISCUSSION 3. 1 Overall Structure CALB is well known to catalyze variety of chemical reactions in water and in organic solvents. Its secondary structure is not dramatically influenced by the surrounding medium, as is illustrated by comparison the DSSP analysis [58] in water and in acetonitrile (Fig. 2 ). The α helixes are slightly looser in water than in organic solvents, see, e.g., the time evolution of the color coding of the part of α 2 (residues around 45) or α 5 (residues 142–146). This observation is robust with respect to the force field (Figures S1 – S3 in the SI). The influence of solvent on flexibility as has been also pointed out in the previous studies. [21,59] The secondary structure of CALB-G has not been experimentally reported yet. Our simulations independently of the choice of FF suggest that it should be similar to the CALB-W, the stability in organic solvents is also preserved (Figures S4 – S6). The fact that α 5 is looser in water is predicted by the CHARMM36 FF. CALB-N with larger lid represents a more peculiar case, because the simulation predictions of the unknown structure of the newly inserted part vary from an unstructured loop to β -sheet (Figures S7 – S15). The more structured motifs occasionally appear in toluene. The position of the newly inserted part with respect to the entrance to the active site also varies a lot, as can be seen in Fig. 3 . The α 5 and α 10 helixes might be in contact with each other, not necessarily in the parallel arrangement, and the new part can cover them (a) or point outside from the protein (b). The helixes might also be pulled apart from each other and adopt different angles (c, d). The arrangements a) and b) are dominant in the aqueous systems independently on the FF. Arrangement d) is the most common in organic solvent. The protein stability is conventionally monitored by the root mean square displacement (rmsd) (Fig. 4 and Figure S16). The overall secondary structure does not unfold which is better manifested by the previous DSSP analysis. However, there can be huge variations with respect to the reference structure especially for initially restrained initial conditions of the CALB-N (panels d) and e). For example, the helixes move apart from each other during the course of the simulation and newly inserted part rearranges. The relatively large values of rmsd (above 0.3 nm) for the wild type are not related to protein unfolding or the lid motion in our simulations, but to large fluctuations of the residues 1–30. 3. 2 Lid Motion Next, we characterize the motion of α 5 and α 10 helixes in conventional MD simulations by monitoring the interhelical distance and angle (Figs. 5 and 6 and S17 and S18). The helixes of the wild type prefer aligned arrangement in all solvents (top panel of Figs. 5 and S17), the interhelical angle spans region around 30 degrees. Mutating from CALB-W to CALB-G leads to more tilted arrangement in water, as is illustrated by the difference between the first row and second row plots in Fig. 5 Insertion of a longer fragment in the CALB-N promotes nearly perpendicular arrangement of the helixes or even change of the direction of the α 5 helix (angle exceeding 90 degrees). The interhelical angles in CALB-N posses the highest values in water independently on the FF (Figs. 6 and S18). The observed interhelical distances can vary from 0.9 to 2.2 nm. Generally, all three CALB variants in water sample short distances around 1.1 nm. AMBER03 FF predicts somewhat more open structure for CALB-W and CALB-G (compare Fig. 5 and Fig. S17), but the distances do not exceed 1. 5 nm. The aqueous CALB-N systems have the helixes close to each other, even though the interhelical angles are span broad range of values. Toluene unequivocally promotes opening of the lid, the effect of more polar acetonitrile is less clear and it seems to depend on the mutant. Helixes of CALB-W and CALB-G have a slight preference to adopt short distances, but the open-lid structures are also possible (Fig. 5 and S17). 3. 4 Nonbonding Interactions of the Lid Residues To better understand the stabilization of the different lid conformations, we analyzed in detail the non-bonding interactions involving the amino acids composing the lid. In case of CALB-W, we focused on the charged amino acids of α 5 helix (Asp145) and Lys290 nearby the α 10 helix. CALB-G does not contain charge amino acid in the α 5 helix, but two polar amino acids (Asn142 and Thr147) instead. The cutoff for interactions involving N and O atoms of polar or charged groups was set to 0.35 nm. The hydrophobic interaction between non-polar amino acids of the helixes can stabilize the closed conformation, thus we evaluated number of contacts between carbon atoms of non-polar side chains of α 5 and α 10 helixes within 1.0 nm. Figure 9 depicts selected interesting interactions, Tables 1 , S2 and Table 2 , S3 summarize the results for CALB-W and CALB-G, respectively The charged Asp145 and Lys290 amino acid side chains of CALB-W (Table 1 ) keep about two or one water molecules, resp. in their vicinity independently of the bulk solvent. Note, that we kept the crystallographic water molecules in the system (see Methods). In organic solvents, Asp145 can form hydrogen bond with the OH groups of Ser150 or Thr158. Interestingly, a salt bridge between Asp145 and Lys308 or Arg309 appears in toluene. This prevents the lid from fully closing, as is indicate by the large decrease of interactions between the nonpolar residues of the α 5 and α 10 helixes. The polar groups of Asn142 and Thr147 in CALB-G possess significant interactions with water just in the aqueous system. In organic solvents, they from hydrogen bonds with polar parts of the α 5 helix or acetonitrile. Regarding the number of contacts between non-polar parts of the helixes, it is the smallest in toluene compared to water and acetonitrile. In contrast to CALB-W, we did not pinpoint any other very specific interaction of the polar amino acids in toluene, which would be similar to the salt bridge. This might be the reason why is the free energy minimum of CALB-G located at smaller separation than in CALB-W. The observed trends are similar for the two FF with a few exceptions. For example, for CALB-G in toluene, the interaction between the Thr147 and the polar residues of α5 helix in the trajectories with CHARMM36 FF is missing for the AMBER03 FF. Table 1 The average number of non-bonding interactions of selected parts of CALB-W with its surroundings analyzed in conventional MD simulation employing CHARMM36 FF. The interactions involving the polar atoms of the amino acid side chains (N, O), all polar atoms of the α5 helix (α5pol), oxygen atoms of water molecules (OW) and nitrogen atoms of acetonitrile (ACN) were evaluated. For the non-polar α5-α10 interaction all carbon atoms were taken into account. The cutoff for all polar interactions was 0.35 nm, for the interhelical interaction α5- α10 it was set to 1 nm. Water Acetonitrile Toluene Asp145 OW 2.00 ± 0.03 OW 1.93 ± 0.26 OW 2.00 ± 0.07 α5pol 1.15 ± 0.54 α5pol 1.69 ± 0.52 α5pol 1.16 ± 0.39 Ser150 0.001 ± 0.032 Ser150 0.98 ± 0.17 Thr158 1.00 ± 0.38 ACN 0.07 ± 0.27 Lys308,Arg309 1.28 ± 0.60 Lys290 OW 1.00 ± 0.04 OW 0.74 ± 0.44 OW 1.00 ± 0.02 α5pol 0.02 ± 0.013 ACN 0.84 ± 0.37 α5-α10 12.70 ± 5.95 13.66 ± 3.65 3.37 ± 1.06 Table 2 The average number of non-bonding interactions of selected parts of CALB-G. zeae with its surroundings analyzed in conventional MD simulation employing CHARMM36 FF. The interactions involving the polar atoms of the amino acid side chains (N, O), all polar atoms of the α5 helix (α5pol), oxygen atoms of water molecules (OW) and nitrogen atoms of acetonitrile (ACN) were evaluated. For the non-polar α5-α10 interaction all carbon atoms were taken into account. The cutoff for all polar interactions was 0.35 nm, for the interhelical interaction α5- α10 it was set to 1 nm. Water Acetonitrile Toluene Asn142 OW 1.95 ± 0.22 OW 0.09 ± 0.31 OW 0.35 ± 0.55 α5pol 0.91 ± 0.35 α5pol 0.96 ± 0.21 α5pol 1.07 ± 0.48 ACN 0.98 ± 0.33 Thr147 OW 0.96 ± 0.19 OW 0.04 ± 0.20 OW 0.01 ± 0.08 α5pol 0.44 ± 0.50 α5pol 0.96 ± 0.20 α5pol 0.98 ± 0.15 ACN 0.18 ± 0.38 α5-α10 7.51 ± 2.76 1.37 ± 1.25 0.57 ± 0.99 CONCLUSIONS This paper represents a systematic simulations study on the lid motion of CALB – wild type and its two mutants CALB-G. zeae and CALB-N. crassa with interesting catalytic properties. Our simulations show that their structure is stable in water, acetonitrile and toluene. There are slight variations in the simulated secondary structure of the mutated part of CALB-N. crassa , especially in organic solvents. Its position with respect to the lid of the wild type varies too. Therefore, it would be interesting to obtain crystal structures of both mutants. The lid-constituting helixes of CALB-W adopt parallel arrangement, but they tend to be tilted for the mutants. Already conventional simulations show that toluene promotes the lid opening, the effect of the more polar acetonitrile is less pronounced. To further quantify such phenomena, we performed the first series of umbrella sampling simulations of the lid opening in different solvents, as far as we know. The closed structure of CALB-W in water represents the global minimum, according to the CHARMM36 FF, the minimum is quite flat. AMBER03 FF predicts a slightly deeper closed minimum. The free energy minimum of CALB-G in water, very likely corresponding to a closed state, is located at a bit large interhelical separation than for the wild type. Toluene deepens the free energy minimum of both mutants by more than 50 kJ mol -1 and shifts its position towards the open structure. The lid CALB-W is a bit more open in comparison with CALB-G. The effect of the polar organic solvent acetonitrile on the lid opening is less pronounced and seems to be more force field dependent. The stability of the lid conformation of both CALB-W and CALB-G is related to the non-bonding interactions between amino acids of the two helixes and their environment. In CALB-W, there is a specific salt bridge between the charged Asp145 and Lys308 or Arg309, which forms only in toluene. This interaction explains a more open conformation of CALB-W in the nonpolar solvent in comparison with CALB-G. Polar side chains of the CALB-G lid form hydrogen bonds with other polar protein groups in their vicinity or with polar solvent (both acetonitrile and water). Finally, with increasing polarity of the solvent, the number of non-polar interaction between the amino acid residues of the two helixes increased, stabilizing a more closed lid conformation. In summary, our simulations give new insight into the structure and lid-motion of a CALB and its mutants in various solvents Presented results have implications for protein and medium engineering of technologically used lipases. Declarations ASSOCIATED CONTENT Supporting Information. Additional tables and figures are provided in the Supporting Information. The topology files and the input files for MD simulations are available at the national data repository https://doi.org/10.48700/datst.df023-eya04. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was started thanks to the support of the Czech Science Foundation, Grant No. 17-01982Y and finished during the course of the project No. 21-15936S. K. F. was funded solely by Grant No. 21-15936S. Part of the computational resources was provided by the e-INFRA CZ project (ID:90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic. References Adlercreutz, P. Immobilisation and Application of Lipases in Organic Media. Chem. Soc. Rev. 2013 , 42 (15), 6406. https://doi.org/10.1039/c3cs35446f. Galmés, M. À.; García-Junceda, E.; Świderek, K.; Moliner, V. 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Bussi, G.; Donadio, D.; Parrinello, M. Canonical Sampling through Velocity Rescaling. J. Chem. Phys. 2007 , 126 (1), 014101. https://doi.org/10.1063/1.2408420. Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. Molecular Dynamics with Coupling to an External Bath. J. Chem. Phys. 1984 , 81 (8), 3684–3690. https://doi.org/10.1063/1.448118. Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. LINCS: A Linear Constraint Solver for Molecular Simulations. J. Comput. Chem. 1997 , 18 (12), 1463–1472. https://doi.org/10.1002/(SICI)1096-987X(199709)18:123.3.CO;2-L. Hub, J. S.; de Groot, B. L.; van der Spoel, D. G_wham—A Free Weighted Histogram Analysis Implementation Including Robust Error and Autocorrelation Estimates. J. Chem. Theory Comput. 2010 , 6 (12), 3713–3720. https://doi.org/10.1021/ct100494z. Kabsch, W.; Sander, C. Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-Bonded and Geometrical Features. Biopolymers 1983 , 22 (12), 2577–2637. https://doi.org/10.1002/bip.360221211. Trodler, P.; Pleiss, J. Modeling Structure and Flexibility of Candida Antarctica Lipase B in Organic Solvents. BMC Struct. Biol. 2008 , 8 (1), 9. https://doi.org/10.1186/1472-6807-8-9. Additional Declarations No competing interests reported. Supplementary Files LidSI.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5737623","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":396233027,"identity":"4a72e603-d21f-411a-a444-aff4cc4510d2","order_by":0,"name":"Zuzana Sochorová Vokáčová","email":"","orcid":"","institution":"Heyrovský Institute of Physical Chemistry of The Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zuzana","middleName":"Sochorová","lastName":"Vokáčová","suffix":""},{"id":396233028,"identity":"2bcac96b-eb47-45b1-b955-b9a32cfa84a6","order_by":1,"name":"Karolína Fárníková","email":"","orcid":"","institution":"Heyrovský Institute of Physical Chemistry of The Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Karolína","middleName":"","lastName":"Fárníková","suffix":""},{"id":396233029,"identity":"a8ad4511-e60b-4100-bce6-ae041567460d","order_by":2,"name":"Eva Pluhařová","email":"data:image/png;base64,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","orcid":"","institution":"Heyrovský Institute of Physical Chemistry of The Czech Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Eva","middleName":"","lastName":"Pluhařová","suffix":""}],"badges":[],"createdAt":"2024-12-30 19:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5737623/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5737623/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72842404,"identity":"59b65d7a-1f7e-437e-bbde-31d08d42cfec","added_by":"auto","created_at":"2025-01-02 18:37:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2070129,"visible":true,"origin":"","legend":"\u003cp\u003eStructures of CALB – wild type (center), CALB-G.\u003cem\u003ezeae\u003c/em\u003e mutant (left) and CALB-N.\u003cem\u003ecrassa\u003c/em\u003e mutant (right). The parts of the mutated proteins that are identical with CALB are colored in gray, the mutations are highlighted in green.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5737623/v1/3acd4a6193dd14425dc0c31b.png"},{"id":72842202,"identity":"acd9ea3c-d1ef-4b87-92a5-91b3c478a52e","added_by":"auto","created_at":"2025-01-02 18:29:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":347903,"visible":true,"origin":"","legend":"\u003cp\u003eThe secondary structure of CALB-W described by CHARMM36 force field in water (top) and in acetonitrile (bottom) during the conventional MD simulation. Different colors correspond to different motifs of secondary structure as determined by DSSP utility.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5737623/v1/18cd1cb2bca7d2d7360318cf.png"},{"id":72842405,"identity":"5ca7e400-904e-48e7-a0c7-392c1dc981a4","added_by":"auto","created_at":"2025-01-02 18:37:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":301674,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of different arrangements that can be adopted by the newly inserted part of CALB-N (green) with respect to the \u003cem\u003ea\u003c/em\u003e\u003csub\u003e5\u003c/sub\u003e and \u003cem\u003ea\u003c/em\u003e\u003csub\u003e10\u003c/sub\u003e helixes (violet).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5737623/v1/63fe91181f1e1d87030c867d.png"},{"id":72842874,"identity":"3127672b-ec06-4359-baf8-1500b3b08f27","added_by":"auto","created_at":"2025-01-02 18:45:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":318218,"visible":true,"origin":"","legend":"\u003cp\u003eRoot mean square deviation from the reference structure of the backbone atoms of CALB-W (a), CALB-G (b) and three independent simulations of CALB-N (c - e) described by CHARMM36 force field in water (blue), acetonitrile (red), and toluene (green).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5737623/v1/07cc6b596be1d0ae642181c4.png"},{"id":72842406,"identity":"8aab14de-05ea-4333-94f5-ad0f039fb9cd","added_by":"auto","created_at":"2025-01-02 18:37:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":350891,"visible":true,"origin":"","legend":"\u003cp\u003eHistograms of sampled interhelical distances and interhelical angles in conventional MD simulations of CALB-W (top), CALB-G (bottom) described by CHARMM36 FF in water (left), acetonitrile (center) and toluene (right).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5737623/v1/d3d46aa9fe297c3023de0c0a.png"},{"id":72842209,"identity":"d1b903d4-8112-45a1-ac19-c68ff7dc8f48","added_by":"auto","created_at":"2025-01-02 18:29:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":457565,"visible":true,"origin":"","legend":"\u003cp\u003eHistograms of sampled interhelical distances and interhelical angles in conventional MD simulations of 3 independent simulations of CALB-N (rows) described by CHARMM36 FF in water (left), acetonitrile (center) and toluene (right).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5737623/v1/4b35bb2c31b9938063778429.png"},{"id":72842206,"identity":"be3984d6-e3b1-4781-bfb5-ba002c736862","added_by":"auto","created_at":"2025-01-02 18:29:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":170772,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of the lid opening (from red to blue) in a steered MD simulation of CALB-W in water.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5737623/v1/623372641ac45d09022cb150.png"},{"id":72842410,"identity":"3b2bf560-2338-4018-ac1e-024ca81969f7","added_by":"auto","created_at":"2025-01-02 18:37:14","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1595845,"visible":true,"origin":"","legend":"\u003cp\u003eFree energy profile for opening of the lid of CALB-W (a, c), CALB-G (b, d) in water (blue), acetonitrile (red) and toluene (green). Each lines corresponds to the average of ten independent profiles obtained by umbrella sampling. Dotted lines represent the respective standard deviations. The top (bottom) panels show results obtained by the CHARMM36 (AMBER03) FF.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5737623/v1/ba1d5fc4c0276d5c9b9fe8ba.png"},{"id":72842233,"identity":"47c78cfc-289a-4315-b80a-d7e343a53d16","added_by":"auto","created_at":"2025-01-02 18:29:15","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":12649283,"visible":true,"origin":"","legend":"\u003cp\u003eLipase with highlighted lid (α\u003csub\u003e5\u003c/sub\u003e helix red, α\u003csub\u003e10\u003c/sub\u003e helix blue) and interesting nonbonding interactions: a) Hydrogen bond between Asp145 and Ser150 in CALB-W, b) salt bridges between Asp145 and Lys308 or Arg309 are visualized, together with the potential hydrogen bond donor Thr158. Panel c) shows the interactions in the CALB-G.zeae (Asn142 and polar residues of α\u003csub\u003e5\u003c/sub\u003e helix and molecules of acetonitrile and the interaction between Thr147 and the polar residues of the α\u003csub\u003e5\u003c/sub\u003e helix). The residues of interest are visualized by balls and sticks and the rest of the protein by ribbons.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5737623/v1/6bef6fb6f27956c850ff3959.png"},{"id":76187184,"identity":"ff0e2fab-58be-4869-8daf-b337575333fc","added_by":"auto","created_at":"2025-02-13 08:47:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20514266,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5737623/v1/566cd76c-e762-41d9-88a8-550ec69f410d.pdf"},{"id":72842408,"identity":"25c652b1-e3f2-4eca-a391-641f6c5db578","added_by":"auto","created_at":"2025-01-02 18:37:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7185872,"visible":true,"origin":"","legend":"","description":"","filename":"LidSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-5737623/v1/a8a1acc52b6b0685e1a14a5e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular Simulations of the Lid Motion in Chimeras of Candida Antarctica Lipase B in Organic Solvents","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLipases are biocatalysts that under natural aqueous conditions hydrolyze triacylglycerols. Interestingly, their substrate specificity for the hydrolytic reaction is very broad and, in addition, there are able to catalyze different reactions such as aldol addition, racemization, epoxidation and aminolysis.[1\u0026ndash;3] Therefore, lipases are widely exploited in industry,[4\u0026ndash;6] where they are used not only in conventional aqueous media, but also in organic solvents as efficient catalysts of esterification and transesterification reactions.[1]\u003c/p\u003e \u003cp\u003eDue to the low water solubility of the natural substrates of lipases, the reaction normally occurs at the organic-water interface where the lipases usually exhibit increased catalytic activity. This phenomenon called interfacial activation is explained by movement of the flexible protein domain (lid) located in the vicinity of the active site. The lid covers the active site in water and opens upon contact with the hydrophobic phase which facilitates the entry of the substrate.[7,8] The relationship among lipolytic activity, interfacial activation and the lid are still debated, as there are lipases exhibiting no interfacial activation at all [8], some of them despite the presence of a large lid.[9] Other possesses it only towards certain substrates.[10] Nevertheless, the lid is an interesting target for protein engineering and its modifications can alter the enzymatic thermostability [11], reactivity [12\u0026ndash;18] or enantioselectivity [19,20].\u003c/p\u003e \u003cp\u003eThe atomistic details of the lid motion have been investigated by classical molecular dynamics (MD) simulations with empirical force fields (FFs). Conventional MD simulations revealed an existence of the open lipase conformation in water [21,22], but they indeed demonstrated that such arrangement is more stable at the water-hydrophobic interface [23\u0026ndash;25] or in organic solvents [23,26\u0026ndash;28]. Analysis of collected trajectories provides rationalization of the lid motion, but the long time-scale at which the structural rearrangement occurs suggests that it is desirable to go beyond the conventional MD to obtain converged results. So far, there are only a handful of studies employing enhanced sampling techniques [10] or applying external force [25,29\u0026ndash;32] and, to the best of our knowledge, only one of them reports a free energy profile [31] for the lid opening.\u003c/p\u003e \u003cp\u003eHere, we systematically investigate the lid motion of Candida antarctica lipase B (CALB) and its mutants CALB-G. \u003cem\u003ezeae\u003c/em\u003e and CALB-N. \u003cem\u003ecrassa\u003c/em\u003e in water and in two commonly used organic solvents (acetonitrile and toluene). We have chosen CALB due to its extensive commercial use which indeed motivated simulation [33\u0026ndash;38] and crystallographic [39] studies of the interactions of various mixed solvents or substrates with the lipase. Apart from these practical reasons, it represents a suitable model system as its interfacial activation is still debated [22,24,40] and the mutations in the lid region cause interesting changes in the catalytic properties.[20] Our aims are: (i) assess the structure and stability of the mutants in different environments as their crystal structures are not available, (ii) obtain free energy profile for the lid opening by combination of steered MD and umbrella sampling, (iii) analyze and interpret the differences cause by protein engineering as well as by change of the solvent.\u003c/p\u003e"},{"header":"COMPUTATIONAL METHODS","content":"\n\u003ch3\u003e2. 1 Model systems\u003c/h3\u003e\n\u003cp\u003eThe initial structure of CALB was obtained from the 1TCA [41] entry in the Protein Data Bank. The two sugar units (NAG) were removed [42], the 286 crystal water molecules were kept and three disulfide bonds between Cys22 and Cys64, Cys216 and Cys258, and Cys293 and Cys311 were defined. The protonation states of titratable side chains correspond to pH\u0026thinsp;=\u0026thinsp;7 except for Asp134 which is protonated (neutral) [41\u0026ndash;43], thus the overall charge of the system is zero.\u003c/p\u003e \u003cp\u003eThe amino acid sequence of CALB-G. \u003cem\u003ezeae\u003c/em\u003e (CALB-G) was obtained from CALB by performing the following mutations (V139I, G142N, P143I, L144G, D145G, L147T, A148G, V149L, S150IN, A151T, S153A, W155V) in the VMD\u003csup\u003e44\u003c/sup\u003e software. CALB-N. \u003cem\u003ecrassa\u003c/em\u003e (CALB-N) contains a larger lid than CALB (Y135F, K136H, V139M, G142Y, P143G, D145C, L147G, A148N, V149F, S150G, KVAKAGAPC, A151P, W155L). The protein structures and mutated regions are shown in green in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The point mutations in CALB-N were again performed in VMD and the secondary structure of the additional amino acid sequence was estimated by I-TASSER[45\u0026ndash;47]. The sequence containing the mutated parts (residues 139\u0026ndash;156 of CALB-G and 135\u0026ndash;164 of CALB-N) were minimized using the steepest descent algorithm and CHARMM36 FF [48] and the rest of the structure was kept fixed. The titratable side chains are in their standard protonation states except for Asp134 resulting in +\u0026thinsp;1 charge of CALB-G and +\u0026thinsp;2 charge of CALB-N which was compensated by one or two chloride ions, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResulting structures were solvated in neat water, acetonitrile or toluene in approx. 9nm simulation cell. The exact composition of the model systems are summarized in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e in the Supporting Information (SI). It also includes the average length of the box in the NpT simulations described below.\u003c/p\u003e \u003cp\u003eTwo FF parameter sets (CHARMM36 [48] and AMBER03 [49]) were used to describe the enzymes and counterions in order to judge the robustness of the simulation results. TIP3P [50] FF was employed for water; acetonitrile [51] and toluene [48] parametrization was summarized and tested earlier [52].\u003c/p\u003e\n\u003ch3\u003e2. 2 Simulation Details\u003c/h3\u003e\n\u003cp\u003eClassical molecular dynamics simulations with a 1fs time step in the NpT ensemble were conducted using the Gromacs program package (versions 2018.6 and 2020. 3). [53] The temperature was kept at 300 K by the CSRV thermostat [54] with a coupling constant of 1 ps, the pressure was maintained at 1 bar by Berendsen barostat [55] with a coupling constant of 2 ps. The isothermal compressibility of each system was set to the experimental value of the pure solvent. The bonds containing hydrogen atoms were constrained by the LINCS algorithm [56]. The 3D periodic boundary conditions were used, the short-range electrostatic and van der Waals interactions were truncated at 1.2 nm and the long-range electrostatic interactions were treated by the particle mesh Ewald method.\u003c/p\u003e \u003cp\u003eFirst, we performed a 10ns equilibration for each system with the backbone atoms and crystal water molecules restrained with a force constant of 1000 kJ mol\u003csup\u003e-1\u003c/sup\u003e nm\u003csup\u003e-2\u003c/sup\u003e and stronger thermostat coupling (0.1 ps). As the relative position of the helixes, as well as the orientation of the inserted amino acid sequence with respect to the helixes is unknown, we performed another two sets of equilibration for CALB-N. During the first one, the structure of the helixes was kept fixed, during the second one, the helixes were restrained and their separation kept around 1.1 nm. For CALB-N, this was followed by another 50 ns simulations without restraints which resulted by 3 different initial conditions for each FF and solvent. Then we collected a 200 ns trajectory for each of the systems (30 trajectories in total).\u003c/p\u003e \u003cp\u003eAs a coordinate for monitoring the lid motion, we have chosen the distance between the centers of mass of the backbone atoms of the following CALB residues of 142\u0026ndash;146 (a part of the \u003cem\u003eα\u003c/em\u003e\u003csub\u003e\u003cem\u003e5\u003c/em\u003e\u003c/sub\u003e helix) and 282\u0026ndash;286 (a part of the \u003cem\u003eα\u003c/em\u003e\u003csub\u003e\u003cem\u003e10\u003c/em\u003e\u003c/sub\u003e helix). The exact choice of the residues varies in the literature, ours lies in between the one in Refs. 11 and 14. The residue range of \u003cem\u003eα\u003c/em\u003e\u003csub\u003e\u003cem\u003e5\u003c/em\u003e\u003c/sub\u003e is the same also for the mutants, but it differs in case of \u003cem\u003eα\u003c/em\u003e\u003csub\u003e\u003cem\u003e10\u003c/em\u003e\u003c/sub\u003e: 283\u0026ndash;287 (CALB-G) and 291\u0026ndash;295 (CALB-N) because of the alteration of the sequence. To further characterize the mutual orientation of the helixes, we picked the vector between the Cα atoms at the beginning and at the end of each helix and calculated the angle between them.\u003c/p\u003e \u003cp\u003eNext, to better quantify the probability of the different interhelical distances, we constructed the free energy profiles for the lid opening for the CALB-W and CALB-G. We picked ten configurations separated by at least 20 ns from each of the direct simulations and performed two steered MD to drive shortening and prolonging the interhelical distance for CALB-W and CALB-G. The pulling rate was 0.2 nm ns\u003csup\u003e-1\u003c/sup\u003e and the length of the simulation was adjusted to span the range of interhelical distances of 0.9\u0026ndash;2.1 nm for CALB-W and CALB-G. To prevent unfolding of the helixes because of the external force applied during pulling, distance restraints were introduced (see topology files in the national data repository \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.48700/datst.df023-eya04\u003c/span\u003e\u003cspan address=\"10.48700/datst.df023-eya04\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ). These pulling trajectories provided initial conditions for umbrella sampling simulations without additional structural restraints. The sampling windows were equally spaced by 0.1 nm, the same force constant of 2000 kJ mol\u003csup\u003e-1\u003c/sup\u003e nm\u003csup\u003e-2\u003c/sup\u003e was employed and the simulation was conducted for 10 ns in each of the windows. The free energy profiles were constructed by the WHAM algorithm [57] as implemented in Gromacs [53].\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\n\u003ch3\u003e3. 1 Overall Structure\u003c/h3\u003e\n\u003cp\u003eCALB is well known to catalyze variety of chemical reactions in water and in organic solvents. Its secondary structure is not dramatically influenced by the surrounding medium, as is illustrated by comparison the DSSP analysis [58] in water and in acetonitrile (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The \u003cem\u003eα\u003c/em\u003e helixes are slightly looser in water than in organic solvents, see, e.g., the time evolution of the color coding of the part of \u003cem\u003eα\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e (residues around 45) or \u003cem\u003eα\u003c/em\u003e\u003csub\u003e5\u003c/sub\u003e (residues 142\u0026ndash;146). This observation is robust with respect to the force field (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e \u0026ndash; S3 in the SI). The influence of solvent on flexibility as has been also pointed out in the previous studies. [21,59]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe secondary structure of CALB-G has not been experimentally reported yet. Our simulations independently of the choice of FF suggest that it should be similar to the CALB-W, the stability in organic solvents is also preserved (Figures S4 \u0026ndash; S6). The fact that \u003cem\u003eα\u003c/em\u003e\u003csub\u003e5\u003c/sub\u003e is looser in water is predicted by the CHARMM36 FF. CALB-N with larger lid represents a more peculiar case, because the simulation predictions of the unknown structure of the newly inserted part vary from an unstructured loop to \u003cem\u003eβ\u003c/em\u003e-sheet (Figures S7 \u0026ndash; S15). The more structured motifs occasionally appear in toluene. The position of the newly inserted part with respect to the entrance to the active site also varies a lot, as can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The α\u003csub\u003e5\u003c/sub\u003e and α\u003csub\u003e10\u003c/sub\u003e helixes might be in contact with each other, not necessarily in the parallel arrangement, and the new part can cover them (a) or point outside from the protein (b). The helixes might also be pulled apart from each other and adopt different angles (c, d). The arrangements a) and b) are dominant in the aqueous systems independently on the FF. Arrangement d) is the most common in organic solvent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe protein stability is conventionally monitored by the root mean square displacement (rmsd) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Figure S16). The overall secondary structure does not unfold which is better manifested by the previous DSSP analysis. However, there can be huge variations with respect to the reference structure especially for initially restrained initial conditions of the CALB-N (panels d) and e). For example, the helixes move apart from each other during the course of the simulation and newly inserted part rearranges. The relatively large values of rmsd (above 0.3 nm) for the wild type are not related to protein unfolding or the lid motion in our simulations, but to large fluctuations of the residues 1\u0026ndash;30.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e3. 2 Lid Motion\u003c/h3\u003e\n\u003cp\u003eNext, we characterize the motion of \u003cem\u003eα\u003c/em\u003e\u003csub\u003e5\u003c/sub\u003e and \u003cem\u003eα\u003c/em\u003e\u003csub\u003e10\u003c/sub\u003e helixes in conventional MD simulations by monitoring the interhelical distance and angle (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and S17 and S18). The helixes of the wild type prefer aligned arrangement in all solvents (top panel of Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and S17), the interhelical angle spans region around 30 degrees. Mutating from CALB-W to CALB-G leads to more tilted arrangement in water, as is illustrated by the difference between the first row and second row plots in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e Insertion of a longer fragment in the CALB-N promotes nearly perpendicular arrangement of the helixes or even change of the direction of the \u003cem\u003eα\u003c/em\u003e\u003csub\u003e5\u003c/sub\u003e helix (angle exceeding 90 degrees). The interhelical angles in CALB-N posses the highest values in water independently on the FF (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and S18).\u003c/p\u003e \u003cp\u003eThe observed interhelical distances can vary from 0.9 to 2.2 nm. Generally, all three CALB variants in water sample short distances around 1.1 nm. AMBER03 FF predicts somewhat more open structure for CALB-W and CALB-G (compare Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig. S17), but the distances do not exceed 1. 5 nm. The aqueous CALB-N systems have the helixes close to each other, even though the interhelical angles are span broad range of values. Toluene unequivocally promotes opening of the lid, the effect of more polar acetonitrile is less clear and it seems to depend on the mutant. Helixes of CALB-W and CALB-G have a slight preference to adopt short distances, but the open-lid structures are also possible (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and S17).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e3. 4 Nonbonding Interactions of the Lid Residues\u003c/h3\u003e\n\u003cp\u003eTo better understand the stabilization of the different lid conformations, we analyzed in detail the non-bonding interactions involving the amino acids composing the lid. In case of CALB-W, we focused on the charged amino acids of \u003cem\u003eα\u003c/em\u003e\u003csub\u003e5\u003c/sub\u003e helix (Asp145) and Lys290 nearby the \u003cem\u003eα\u003c/em\u003e\u003csub\u003e10\u003c/sub\u003e helix. CALB-G does not contain charge amino acid in the \u003cem\u003eα\u003c/em\u003e\u003csub\u003e5\u003c/sub\u003e helix, but two polar amino acids (Asn142 and Thr147) instead.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe cutoff for interactions involving N and O atoms of polar or charged groups was set to 0.35 nm. The hydrophobic interaction between non-polar amino acids of the helixes can stabilize the closed conformation, thus we evaluated number of contacts between carbon atoms of non-polar side chains of \u003cem\u003eα\u003c/em\u003e\u003csub\u003e5\u003c/sub\u003e and \u003cem\u003eα\u003c/em\u003e\u003csub\u003e10\u003c/sub\u003e helixes within 1.0 nm. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e depicts selected interesting interactions, Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, S2 and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, S3 summarize the results for CALB-W and CALB-G, respectively\u003c/p\u003e \u003cp\u003eThe charged Asp145 and Lys290 amino acid side chains of CALB-W (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) keep about two or one water molecules, resp. in their vicinity independently of the bulk solvent. Note, that we kept the crystallographic water molecules in the system (see Methods). In organic solvents, Asp145 can form hydrogen bond with the OH groups of Ser150 or Thr158. Interestingly, a salt bridge between Asp145 and Lys308 or Arg309 appears in toluene. This prevents the lid from fully closing, as is indicate by the large decrease of interactions between the nonpolar residues of the \u003cem\u003eα\u003c/em\u003e\u003csub\u003e5\u003c/sub\u003e and \u003cem\u003eα\u003c/em\u003e\u003csub\u003e10\u003c/sub\u003e helixes.\u003c/p\u003e \u003cp\u003eThe polar groups of Asn142 and Thr147 in CALB-G possess significant interactions with water just in the aqueous system. In organic solvents, they from hydrogen bonds with polar parts of the \u003cem\u003eα\u003c/em\u003e\u003csub\u003e5\u003c/sub\u003e helix or acetonitrile. Regarding the number of contacts between non-polar parts of the helixes, it is the smallest in toluene compared to water and acetonitrile. In contrast to CALB-W, we did not pinpoint any other very specific interaction of the polar amino acids in toluene, which would be similar to the salt bridge. This might be the reason why is the free energy minimum of CALB-G located at smaller separation than in CALB-W. The observed trends are similar for the two FF with a few exceptions. For example, for CALB-G in toluene, the interaction between the Thr147 and the polar residues of α5 helix in the trajectories with CHARMM36 FF is missing for the AMBER03 FF.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe average number of non-bonding interactions of selected parts of CALB-W with its surroundings analyzed in conventional MD simulation employing CHARMM36 FF. The interactions involving the polar atoms of the amino acid side chains (N, O), all polar atoms of the α5 helix (α5pol), oxygen atoms of water molecules (OW) and nitrogen atoms of acetonitrile (ACN) were evaluated. For the non-polar α5-α10 interaction all carbon atoms were taken into account. The cutoff for all polar interactions was 0.35 nm, for the interhelical interaction α5- α10 it was set to 1 nm.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eAcetonitrile\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eToluene\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eAsp145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eα5pol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eα5pol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eα5pol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSer150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.001\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSer150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThr158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eACN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLys308,Arg309\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLys290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eα5pol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eACN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα5-α10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e12.70\u0026thinsp;\u0026plusmn;\u0026thinsp;5.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e13.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e3.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe average number of non-bonding interactions of selected parts of CALB-G.\u003cem\u003ezeae\u003c/em\u003e with its surroundings analyzed in conventional MD simulation employing CHARMM36 FF. The interactions involving the polar atoms of the amino acid side chains (N, O), all polar atoms of the α5 helix (α5pol), oxygen atoms of water molecules (OW) and nitrogen atoms of acetonitrile (ACN) were evaluated. For the non-polar α5-α10 interaction all carbon atoms were taken into account. The cutoff for all polar interactions was 0.35 nm, for the interhelical interaction α5- α10 it was set to 1 nm.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eAcetonitrile\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eToluene\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAsn142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eα5pol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eα5pol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eα5pol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eACN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eThr147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eα5pol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eα5pol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eα5pol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eACN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα5-α10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e7.51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis paper represents a systematic simulations study on the lid motion of CALB \u0026ndash; wild type and its two mutants CALB-G.\u003cem\u003ezeae\u003c/em\u003e and CALB-N.\u003cem\u003ecrassa\u003c/em\u003e with interesting catalytic properties. Our simulations show that their structure is stable in water, acetonitrile and toluene. There are slight variations in the simulated secondary structure of the mutated part of CALB-N.\u003cem\u003ecrassa\u003c/em\u003e, especially in organic solvents. Its position with respect to the lid of the wild type varies too. Therefore, it would be interesting to obtain crystal structures of both mutants.\u003c/p\u003e \u003cp\u003eThe lid-constituting helixes of CALB-W adopt parallel arrangement, but they tend to be tilted for the mutants. Already conventional simulations show that toluene promotes the lid opening, the effect of the more polar acetonitrile is less pronounced. To further quantify such phenomena, we performed the first series of umbrella sampling simulations of the lid opening in different solvents, as far as we know. The closed structure of CALB-W in water represents the global minimum, according to the CHARMM36 FF, the minimum is quite flat. AMBER03 FF predicts a slightly deeper closed minimum. The free energy minimum of CALB-G in water, very likely corresponding to a closed state, is located at a bit large interhelical separation than for the wild type. Toluene deepens the free energy minimum of both mutants by more than 50 kJ mol\u003csup\u003e-1\u003c/sup\u003e and shifts its position towards the open structure. The lid CALB-W is a bit more open in comparison with CALB-G. The effect of the polar organic solvent acetonitrile on the lid opening is less pronounced and seems to be more force field dependent.\u003c/p\u003e \u003cp\u003eThe stability of the lid conformation of both CALB-W and CALB-G is related to the non-bonding interactions between amino acids of the two helixes and their environment. In CALB-W, there is a specific salt bridge between the charged Asp145 and Lys308 or Arg309, which forms only in toluene. This interaction explains a more open conformation of CALB-W in the nonpolar solvent in comparison with CALB-G. Polar side chains of the CALB-G lid form hydrogen bonds with other polar protein groups in their vicinity or with polar solvent (both acetonitrile and water). Finally, with increasing polarity of the solvent, the number of non-polar interaction between the amino acid residues of the two helixes increased, stabilizing a more closed lid conformation.\u003c/p\u003e \u003cp\u003eIn summary, our simulations give new insight into the structure and lid-motion of a CALB and its mutants in various solvents Presented results have implications for protein and medium engineering of technologically used lipases.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003eASSOCIATED CONTENT\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupporting Information.\u003c/strong\u003e Additional tables and figures are provided in the Supporting Information. The topology files and the input files for MD simulations are available at the national data repository https://doi.org/10.48700/datst.df023-eya04.\u003c/p\u003e\n\u003cp\u003eNotes\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was started thanks to the support of the Czech Science Foundation, Grant No. 17-01982Y and finished during the course of the project No. 21-15936S. K. F. was funded solely by Grant No. 21-15936S. Part of the computational resources was provided by the e-INFRA CZ project (ID:90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdlercreutz, P. Immobilisation and Application of Lipases in Organic Media. \u003cem\u003eChem. Soc. 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Biol.\u003c/em\u003e \u003cstrong\u003e2008\u003c/strong\u003e, \u003cem\u003e8\u003c/em\u003e (1), 9. https://doi.org/10.1186/1472-6807-8-9.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lipase, lid swapping, organic solvents, molecular dynamics, umbrella sampling","lastPublishedDoi":"10.21203/rs.3.rs-5737623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5737623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe lid motion and the overall structure of Candida antarctica lipase B (CALB) and its mutants CALB-G.\u003cem\u003ezeae\u003c/em\u003e and CALB-N.\u003cem\u003ecrassa\u003c/em\u003e in water, acetonitrile and toluene were investigated by molecular dynamics simulations employing all-atom force fields. This study very likely represents the first systematic application of the umbrella sampling to the lid motion of CALB and its mutants in various solvents. The simulations show that their structures are stable in all solvents. The lid-constituting helixes of the CALB \u0026ndash; wild type adopt parallel arrangement, but they tend to be tilted for the mutants and can even change direction of the orientation in case of CALB-N.\u003cem\u003ecrassa.\u003c/em\u003e Water favors closed lid with contacts between the non-polar side chains of the helixes, mutations shift the corresponding free energy minimum towards slightly larger distance. Toluene causes the lid opening, the open structure of the wild type is stabilized by a salt bridge between the charged Asp145 and Lys308 or Arg309. The effect of the polar organic solvent acetonitrile on the lid opening is less pronounced and seems to be more force field dependent. These detailed insights into the lid opening and specific interactions are relevant for protein and medium engineering of a widely used lipase.\u003c/p\u003e","manuscriptTitle":"Molecular Simulations of the Lid Motion in Chimeras of Candida Antarctica Lipase B in Organic Solvents","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-02 18:29:09","doi":"10.21203/rs.3.rs-5737623/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"634f2562-a1ee-4eb4-91bd-5b8e53a0190f","owner":[],"postedDate":"January 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-13T08:39:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-02 18:29:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5737623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5737623","identity":"rs-5737623","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}