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Insulin conformational changes under high pressure in structural studies and molecular dynamics simulations
Journal Pre-proof Insulin conformational changes under high pressure in structural studies and molecular dynamics simulations
Katarzyna Kurpiewska, Anna Miłaczewska, Krzysztof Lewiński PII:
S0022-2860(19)31360-2
DOI:
https://doi.org/10.1016/j.molstruc.2019.127251
Reference:
MOLSTR 127251
To appear in:
Journal of Molecular Structure
Received Date:
19 July 2019
Accepted Date:
17 October 2019
Please cite this article as: Katarzyna Kurpiewska, Anna Miłaczewska, Krzysztof Lewiński, Insulin conformational changes under high pressure in structural studies and molecular dynamics simulations, Journal of Molecular Structure (2019), https://doi.org/10.1016/j.molstruc.2019.127251
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Journal Pre-proof
Insulin conformational changes under high pressure in structural studies and molecular dynamics simulations Katarzyna Kurpiewska1,2*, Anna Miłaczewska1*, Krzysztof Lewiński2 1Jerzy
Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences,
Niezapominajek 8, 30-239 Kraków, Poland 2Jagiellonian
University, Faculty of Chemistry, Department of Crystal Chemistry and Crystal
Physics, Gronostajowa 2, 30-387 Kraków, Poland
*Corresponding authors: Katarzyna Kurpiewska, Anna Miłaczewska phone +48 12 639 51 01, fax +48 12 425 19 23 mail: [email protected], [email protected]
Highlights:
For the first time insulin crystal structures were determined under pressure of 60, 100 and 200 MPa.
Experimental and theoretical approaches revealed high pressure insulin conformations.
Diverse susceptibility of the insulin molecule regions to compression were identified.
Under high pressure both terminal fragments of chain B were recognized as the most vulnerable regions. 1
Journal Pre-proof Graphical abstract
Keywords: insulin, high-pressure structure, molecular dynamic simulations, protein crystallography Abstract To study the mechanisms underlying protein misfolding and aggregation, therapeutic proteins can be successfully used as a model. Currently, insulin is widely tested as a useful model in this field, since it has been proved in both in vivo and in vitro studies that this small protein aggregates. In this article, exploiting the optimal coupling between high pressure protein crystallography and dynamic simulations, we probe the insulin conformations observed under high pressure, namely over the ranges 0-200 MPa for crystallographic experiments and 0-500 MPa for simulations. Crystal structures of insulin determined with diamond anvil cell technique present a step forward in understanding how pressure can modify protein conformation. Obtained results show different responses to volume compression of different fragments of the insulin molecule. For the first time, we have structurally proved that pressure noticeably modifies fragments of insulin molecules, especially terminal fragments of chain B. The observed structural modifications of insulin molecule in crystal state under pressure were compared to the results of insulin pressurization investigated by the molecular dynamic simulations. Comparing the crystallographic results and MD simulations, we were able to draw important considerations about the role of specific amino acids in pressure-induced insulin conformations.
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Journal Pre-proof 1. Introduction Insulin (ins) is a small polypeptide hormone that regulates glucose level in higher organisms [1]. Clinical relevance of insulin misfolding and its high tendency to aggregate into amyloid fibrils justify the fact that it is often considered as a model protein in the study of misfolding. Although insulin exists in several oligomeric forms, it is monomeric insulin that is responsible for its biological activity. The most stable oligomer is formed by six insulin molecules and this hexameric state serves as its storage unit. High temperature, low pH or contact with hydrophobic media promote partial destabilization of insulin and a series of structural changes, which result in the formation of the ordered aggregates [2]. High hydrostatic pressure due to its effect on the energy of the system that only involves change of the volume contribution to Gibbs free energy has also proved to be a very powerful tool in the study of misfolding and aggregation [3]. Previous studies showed that insulin is indeed susceptible to fibrillation under high pressure and according to Piccirilli et al. low degree assemblies are more susceptible to pressurization than oligomers [4]. Insulin is a 5800 Da molecule composed of two chains (A-chain of 21 residues; B-chain of 30 residues) and has three α-helices, two in the A chain (A2-A8 and A13-A19) and one in the B chain (B9-B19). Three disulfide bonds take part in protein stability and functionality: two inter-chain bridges are formed between A7-B7 and A20-B19. One intra-chain disulfide bond binds cysteines A6 and A11. The hydrophobic core that is essential for correct folding of insulin [5] is formed by the residues Gly8, Leu11, Val12, Leu15, Phe24, Tyr26 of chain B and two sidechains of the residues located in chain A: Ile2 and Val3. The first crystal structure of insulin determined in 1969 [5] was followed by other structures of insulin as well as its analogs in the storage form [6]. The latter form of insulin refers to a Zn stabilized hexamer, which is not active and provides the organism with the hormone when required. Native insulin can be crystallized in cubic, monoclinic and rhombohedral space group. The cubic form of insulin crystallizes in the absence of Zn ions (space group I213, a = 78.9 Å). These crystals have much larger solvent content (64%) in comparison to the Zn form (only 35% solvent content). Large solvent content and high symmetry of crystals are advantageous for high pressure crystallographic experiments, thus the cubic insulin crystals provide a good model system for exploring structural changes under high pressure conditions and were selected for our structural studies, and as a model in MD simulation.
3
Journal Pre-proof Even though there are increasing data regarding insulin folding, assembly and dynamics the precise study of the structural basis for this phenomenon is still missing. It was reported that insulin aggregation must proceed through a bulky, non-native monomer [7], which quickly forms larger oligomers. Therefore, our analyses of insulin structure determined for crystals under high hydrostatic pressure is followed by an investigation of the mechanical properties of the most sensitive to pressure fragments performed with MD simulations. Considering the physiological and therapeutic importance of insulin [8] and the fact that high hydrostatic pressure was proved to evoke aggregation-prone intermediate states in insulin molecule [9] our studies aim in structural investigation of conformations present at the beginning of the aggregation process, which are crucial for the formation of non-native insulin conformations. 2. Methods 2.1. Chemicals Liofilizated protein was purchased from Sigma (I6634, Sigma-Aldrich Co, St. Louis, Mo) and used without purification. All the other chemical reagents used were of analytical grade. 2.2. Crystallization Crystals were grown at room temperature using the hanging drop vapor diffusion method according to Yu & Caspar [5]. Protein was dissolved in 0.01 M Na2HPO4, 0.1 M Na2EDTA, pH 10 to the final concentration of 10 mg/ml. A crystallization drop containing 3 μl of the protein and 2 μl of reservoir solution was equilibrated against 0.01 M Na3EDTA, 0.03 M Na2HPO4 pH 10.4 (298K, atmospheric pressure). Average size of insulin crystals selected for diffraction experiments was 0.25 x 0.40 x 0.50 mm. Size and quality of obtained crystals were suitable for experiments using Merrill-Bassett type of diamond anvil cell (DAC). 2.3. Data collection and processing X-ray diffraction data at ambient pressure (ap) were collected for insulin crystal mounted in MicroRTTM from MiTeGen. For high pressure (hp) conditions single crystals were mounted inside the screw driven DAC. Steel gaskets (0.3 mm thickness) with a hole diameter of 0.5 mm were used according to the procedure previously described in Kurpiewska et al. [10,11]. Protein crystals were transferred into the chamber previously filled with the crystallization mother liquor (Figure 1). Determination of pressure was performed at room 4
Journal Pre-proof temperature using the ruby fluorescence method according to protocol described by Pirermarini et al. [12] and calculated as Δp= 247*Δλ (where p [MPa], λ [nm]; pressure vs Δλ shift is measured with an accuracy better than approximately 1%). Data were collected at room temperature using SuperNova (Rigaku Oxford Diffraction) four-circle diffractometer equipped with a double microfocus X-ray source and multilayer X-ray optics. CuKα radiation was used for reference structure at ambient pressure while MoKα radiation was used for high pressure experiments. The data sets were processed with Mosflm [13] and AIMLESS [14]. Preliminary experiments were performed for crystals compressed to 0.1, 27, 60, 100, 173 and 200 MPa in order to determine the unit cell parameters. Complete data collections were performed for crystal at ambient pressure (ap_ins), at 60 MPa (hp_ins_1), at 100 MPa (hp_ins_2) and at 200 MPa (hp_ins_3), respectively. The structures were solved by molecular replacement method with PHASER [15] using the coordinates of insulin PDBid: 2BN3 as a starting model [16]. Crystallographic refinement was carried out using maximum-likelihood target functions implemented in PHENIX [17]. Each round of the refinement with automatic weighting was interspersed with a round of the model building using WinCOOT [18]. About 10% of the data were randomly excluded from the refinement and used as a test data set to monitor Rfree. As phases improved ordered solvent molecules were added with the protocol implemented in WinCOOT and then manually. Finally, the structures were validated using MolProbity [19] and deposited to PDB as: ins_ap (6QQ7), hp_ins_1 (6QQG), hp_ins_2 (6QRH) and hp_ins_3 (6QRK). 2.4. Analysis of the structures Superposition of all structures and calculation of the root-mean-square deviation for atomic coordinates (r.m.s. deviation) was performed using LSQKAB [20] program from CCP4 suite (Collaborative Computational Project, 1994) [21]. Molecular volume and area using a radius equal to 1.4 Å and difference distance matrices (DDM) were calculated with Chimera. For calculation of an accessible surface area, AreaIMol (CCP4 suite) was used. The energy computation was done with GROMOS96 43B1 parameters implemented in SPDBV_4.1 [22]. Figures were prepared using PyMOL [23] and Chimera [24]. 2.5. MD simulations The molecular dynamic simulation was carried out for insulin monomer using Amber14 software with ff03 force field [25]. Protonation states of residues were checked by PDB2PQR 5
Journal Pre-proof server, version 2.1.1 [26] with implemented PROPKA [27,28] software for pKa calculations at pH 7.0; the latter predicted no unexpected protonation states. Starting geometry was taken from the crystal structure deposited under PDB code: 2BN3. The protein was placed inside a cube filled with water molecules modeled by the TIP3P model with at least a 10 Å distance between the protein and the edge of the cube [29]. The system was then minimized in three steps: in the first run, the complex was restrained with a 500 kcal/(mol·Å2) harmonic potential, while the positions of water and the Na+ ions were optimized. Next, the protein was restrained with a 10 kcal/(mol·Å2) potential, and in the third step, the whole system was minimized with no restraints. Subsequently, in the following step, the system was heated to 300 K under constant volume conditions over 100 ps. Next, a density of the system was equilibrated during 1 ns constant pressure dynamics simulations employing a restraining potential of 1 kcal/(mol·Å2) for protein backbone. The final production MD simulations of 50 ns were carried out in triplicate with constant temperature (300 K) and ambient pressure (0.1 MPa) under periodic boundary conditions using a 2 fs time step performed with Langevin dynamics, isotropic position scaling, SHAKE algorithm to constrain bonds involving hydrogen atoms, and the Particle Mesh Ewald method for the long-range electrostatics. The last geometry was used to run 6 ns simulations under high pressure (200 MPa, 500 MPa) in triplicate. All trajectories were clustered into 5 clusters each. For 50 ns simulations, only last 10 ns with stable RMSD were considered. A representative of the most populated cluster was taken as the reference to determine backbone displacement parameters. 3. Results and discussion The symmetry of cubic space group I213 is preserved by compressed crystals. Diffraction data at ambient and high pressure were collected to maximum resolution 1.65 Å and 2.0-2.15 Å, respectively. Details of the data collection and refinement are summarized in Table 1. The noticeable loss of the diffraction was observed for insulin crystals compressed to a pressure above 220 MPa. The compressibility β of the unit cell parameter (Figure 2) was calculated as β= 1/Vu(∂Vu/∂p). An increase of compressibility was observed up to 100 MPa and then β started to decrease with pressure. This is the first time we discover transition in compressibility for protein crystal. For insulin crystal compressed to 100 MPa the calculated compressibility of the unit cell parameter a and volume V are 50 MPa-1 and 148 MPa-1, respectively, which corresponds to the unit cell volume reduction of 1.4% at 100 MPa. For experiments under 200 MPa the calculated compressibility of the corresponding parameters a and V are 40 MPa-1 and 120 MPa-1, respectively, that refers to the unit cell 6
Journal Pre-proof volume reduction of 0.9% at 200 MPa. Taking into account that the protein in the crystalline state is a homogeneous system filled with water that freely diffuses into and out of the crystal, the compression can be distributed within it and by this means protein molecules can be uniformly influenced by hydrostatic pressure. According to the approach discussed by Kharakoz [30] the nonlinear protein response to mechanical disturbance can lead to the positive and negative changes in the compressibility of the protein upon compression. Our findings are the first experimental example that supports this hypothesis of nonmonotonically pressure influence. The cubic insulin crystals compressibility increases with pressure up to 100 MPa, but at elevated pressure trend toward lower compressibility is observed. From the structural point of view it is clear that under 100 MPa a noticeable compression within both A and B chains contributes to observed high compressibility, whereas under 200 MPa chain B is compressed to a greater extent, but chain A response to pressurization is less spectacular. The structural rearrangements will be further discussed in the section referring to the global changes altered by high pressure. Unit cell changes for insulin rhombohedral crystal (space group R3) treated with high pressure were reported by Nicholson [31]. In those studies, hydrostatic pressure was applied within the cell via nitrogen gas and data were recorded at pressures ranging from 0.1 to 3 MPa. Changes in the diffraction pattern were observed at pressures as low as 0.5 MPa and data analysis revealed unit cell volume reductions of 2.60% at 2 MPa. Experiments showed that above 25 bar (2.5 MPa) diffraction power is lost irretrievably and that upon depressurization the crystals do not return to their original cell parameters. These results are significantly different from the results obtained for cubic crystals and can be explained by the presence of pressure-induced phase transition in rhombohedral crystals. The large number of compressible voids present in insulin core as well as its globular arrangement, fortified both by covalent and non-covalent interactions, can be listed as factors contributing to its high stability. The previous results reported by Piccirilli et al. [32] for insulin in solution under pressure studied with FTIR revealed that insulin preserved its globular structure even at very high pressure (above 1500 MPa). Surprisingly, in our studies for insulin crystals compressed to a noticeable lower pressure (slightly above 200 MPa) no diffraction was observed, even though the crystal shape was maintained. Our previous results for proteins in crystal under high pressure i.e. RNase A and β-lactoglobulin showed that folded protein conformation can withstand significantly higher pressure in the crystal than in solution. Interestingly, Piccirilli et al. reported that growth of cubic crystals at pressure of about 400 MPa was observed for the solution of previously misfolded insulin [32,33], 7
Journal Pre-proof unfortunately no structural data are available for this experiment. Furthermore, in powder diffraction studies on insulin reported by Smith et al., the phase transition (TR transition) was induced by the pressure resulting from the mechanical grinding of the sample, but the sample returned to its original form after several days [34]. The overall architecture of the insulin molecule before and after pressurization is almost identical. A dimer stabilized by hydrogen bonds between the main chain atoms of both subunits related by the crystallographic 2-fold axis is retained. The r.m.s. deviation between the Cα-atom positions in ap and hp insulin structures for chain A are in range 0.098 to 0.139 Å while for chain B they are larger and were found in range 0.113 to 0.304 Å. Similar observation referring to larger changes in chain B are observed for all main chain atom positions, 0.112 to 0.155 Å for chain A and 0.332 to 0.524 Å for chain B. Analysis of side chains conformation reveals two groups of residues referring to different response to pressure elevation. The group in which the alternative conformations relate to pressurization includes Glu4(A), Arg22(A), Glu13(B) and Phe25(B). The preference for two rotamers of Arg22(A) side chain can be correlated with pressure, in hp_ins_1 two alternative conformations can be observed: the same as in ap_ins and second which corresponds to lower accessible surface area of insulin molecule. For structure compressed to 100 and 200 MPa only the second conformation is present. Similar rearrangement is observed for side chain of Glu13(B). Two alternative position of glutamic acid are present in structures ap, hp_ins_1 and hp_ins_2, while for hp_ins_3 only one conformation was identified, again the one which favors protein state with smaller accessible surface area. Phe25(B) side chain in all hp structures is in the same conformation, but different from the one observed in ap structure of insulin. Alternative conformations of Ser9(A), Val2(B), Gln15(B), Glu21(B), Lys29(B) do not show a clear correlation with changes in pressure and thus were assigned to the second group. It was previously reported by Gursky et al. that ~30% of residue conformations observed at pH 7 are different from those present in insulin form crystallized using alkaline solutions (pH 11) [35]. The side chains of residues that were proved to be influenced by pressure do not coincide with those found to be sensitive to pH changes. To investigate the behavior of terminal parts of insulin chains under pressure we have conducted a set of MD simulations in three different conditions: under 0.1 MPa, 200 MPa and 500 MPa. The pressure higher than applied in crystallographic studies was used to reveal possible changes more clearly. Comparison of obtained averaged displacement parameters (shown in Figure 3A) allowed us to determine the most pressure sensitive regions of the insulin molecule. The largest differences occur mainly in chain B (residues: 1-4, 7-9, 12-18 8
Journal Pre-proof and 25-29), while chain A is less affected by pressure (mostly region comprising residues 11-15) (Figure 4B). N- and C-terminus of chain B, as well as part of chain A, present the most pronounced effect (Figure 3C), which supports the experimental results described above. Additionally, to identify global rearrangements, shifts of Cα positions and difference distance matrices were calculated (Figure 4). Crystal structure of insulin compressed to 60 MPa showed no pronounced effect of pressure on the molecule conformation except shift of A1-A2 residues. When pressure was increased to 100 MPa and 200 MPa the entire insulin molecule is compressed and fragments of chain A (A1-A3, A12-A18) and chain B (B16-B24, B26-B28) are displaced as can be seen on Figure 5A-5C. Fragments A1-A3, A12-A18 correspond to part of -helix H1 and entire H2, while for chain B shift of larger fragments can be observed, including N-terminal part of -helix and C terminus. Interestingly, as a result of MD simulation for insulin modeled under 200 MPa an expansion of the molecule, which coincides with subtle rearrangements of the same fragments mentioned above, can be noticed (Figure 5E, 5F). However, a substantial shift regards N-terminal part of chain B (B1-B4). The response of the molecule to higher pressure (500 MPa) is clearly different and shift of chain A towards chain B in insulin molecule can be identified, without apparent movement of fragments within chains. The largest pressure-induced structural effects occur in terminal regions of both polypeptide chains, though noticeable positional changes, mainly of the N-terminal and C-terminal fragments of chain B, can be observed. Both approaches, experimental and theoretical, applied by us in presented studies revealed that chain B of insulin is much more sensitive to pressurization than chain A (Figure 5D). Observed shifts of B chain terminal regions refer to Jiménez et al. studies of the insulin derivatives which showed that the two B-chain's termini might play distinct roles in the amyloidogenesis: the C-terminal slows down the process, whereas the residues at the N-terminus are necessary for the lateral aggregation, i.e., enable protofilaments to form fibrils [36]. Within a given pressure range, only subtle rearrangements and positional shifts of helices, rather than any signs of their unfolding can be identified. Therefore, our crystallographic studies are unable to verify the scenario proposed by Jiménez, in which the α-helical structure of native insulin must unfold completely to form the amyloid cross-β structure [36]. Hydrophobic interactions appear to be one of the most important factor influencing aggregation process [37]. Such a mechanism was also suggested previously as a driving force for insulin aggregation [2,38,39]. In our work, no significant changes in insulin structure were observed which could explain possible hydrophobic fragments exposition. On the other hand, as it was pointed out by Gazit and co-workers [40] there are other structural factors that 9
Journal Pre-proof contribute to the formation of amyloid: 1) surface accessible area reduction, 2) hydrogen bonding saturation and 3) reaching an alternative non-native global free energy minimum. Structures determined by us under high pressure prove that in case of insulin molecule, all mentioned factors contribute to the formation of pressure-treated conformation. 4. Conclusions In this studies, we presented structural models of insulin obtained under high pressure in the scope of 0-200 MPa, which serve as a starting point to discussion about insulin conformational stability. Aggregation of insulin is an important issue as it inactivates its therapeutic role. The comparison of structural data determined under high pressure with results at ambient pressure condition together with molecular dynamics simulations indicates that terminal fragments of chain B are the most vulnerable regions and might contribute significantly in the insulin aggregation process. The phenomenon of protein propensity to form amyloid fibrils is expected to vary from protein to protein under a given set of conditions. Therefore, understanding the factors that govern the amyloidogenic propensity is a persisting and important issue. Taken together with the earlier reports, our study shows that high pressure protein crystallography combined with molecular dynamics simulations may serve as a powerful tool to gain insight into the role of pressure on promotion of amyloid formation. Authors contributions K.K. and K.L. initiated the study and directed the project. K.K. conducted crystallographic studies and computational analyses of structures. A.M. performed molecular dynamics simulations. K.K., K.L. and A.M. prepared the manuscript, which was revised and approved by all authors. Acknowledgments The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08). Authors would like to thank
Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of
Sciences for financial support of presented studies.
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Journal Pre-proof References: [1]
R. Tattersall, Bovine insulin., BMJ. 305 (1992) 831–831. doi:10.1136/bmj.305.6857.831.
[2]
L. Nielsen, R. Khurana, A. Coats, S. Frokjaer, J. Brange, S. Vyas, V.N. Uversky, A.L. Fink, Effect of Environmental Factors on the Kinetics of Insulin Fibril Formation: Elucidation of the Molecular Mechanism, Biochemistry. 40 (2001) 6036–6046. doi:10.1021/bi002555c.
[3]
J.L. Silva, A.C. Oliveira, T.C.R.G. Vieira, G.A.P. De Oliveira, M.C. Suarez, D. Foguel, HighPressure Chemical Biology and Biotechnology, (2014). doi:10.1021/cr400204z.
[4]
F. Piccirilli, S. Mangialardo, P. Postorino, S. Lupi, A. Perucchi, FTIR analysis of the high pressure response of native insulin assemblies, J. Mol. Struct. 1050 (2013) 159–165. doi:10.1016/J.MOLSTRUC.2013.07.028.
[5]
M.J. Adams, T.L. Blundell, E.J. Dodson, G.G. Dodson, M. Vijayan, E.N. Baker, M.M. Harding, D.M.C. Hodgkin, B. Rimmer, S. Sheat, Structure of Rhombohedral 2 Zinc Insulin Crystals, Nature. 224 (1969) 491–495. doi:10.1038/224491a0.
[6]
E.N. Baker, T.L. Blundell, J.F. Cutfield, S.M. Cutfield, E.J. Dodson, G.G. Dodson, D.M.C. Hodgkin, R.E. Hubbard, N.W. Isaacs, C.D. Reynolds, K. Sakabe, N. Sakabe, N.M. Vijayan, The Structure of 2Zn Pig Insulin Crystals at 1.5 A Resolution, Philos. Trans. R. Soc. B Biol. Sci. 319 (1988) 369–456. doi:10.1098/rstb.1988.0058.
[7]
A. Ahmad, I.S. Millett, S. Doniach, V.N. Uversky, A.L. Fink, Partially Folded Intermediates in Insulin Fibrillation †, Biochemistry. 42 (2003) 11404–11416. doi:10.1021/bi034868o.
[8]
P. Sonksen, J. Sonksen, Insulin: understanding its action in health and disease, Br. J. Anaesth. 85 (2000) 69–79. doi:10.1093/bja/85.1.69.
[9]
R. Jansen, W. Dzwolak, R. Winter, Amyloidogenic self-assembly of insulin aggregates probed by high resolution atomic force microscopy., Biophys. J. 88 (2005) 1344–53. doi:10.1529/biophysj.104.048843.
11
Journal Pre-proof [10]
K. Kurpiewska, K. Lewiński, High pressure macromolecular crystallography for structural biology: a review, Cent. Eur. J. Biol. 5 (2010) 531–542. doi:10.2478/s11535010-0044-y.
[11]
K. Kurpiewska, K. Dziubek, A. Katrusiak, J. Font, M. Ribò, M. Vilanova, K. Lewiński, Structural investigation of ribonuclease A conformational preferences using high pressure protein crystallography, Chem. Phys. 468 (2016) 53–62. doi:10.1016/j.chemphys.2016.01.010.
[12]
G.J. Piermarini, S. Block, J.D. Barnett, R.A. Forman, Calibration of the pressure dependence of the R1 ruby fluorescence line to 195 kbar, J. Appl. Phys. 46 (1975) 2774–2780. doi:10.1063/1.321957.
[13]
T.G.G. Battye, L. Kontogiannis, O. Johnson, H.R. Powell, A.G.W. Leslie, iMOSFLM : a new graphical interface for diffraction-image processing with MOSFLM, Acta Crystallogr. Sect. D Biol. Crystallogr. 67 (2011) 271–281. doi:10.1107/S0907444910048675.
[14]
P.R. Evans, G.N. Murshudov, How good are my data and what is the resolution?, Acta Crystallogr. Sect. D Biol. Crystallogr. 69 (2013) 1204–1214. doi:10.1107/S0907444913000061.
[15]
A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni, R.J. Read, Phaser crystallographic software, J. Appl. Crystallogr. 40 (2007) 658–674. doi:10.1107/S0021889807021206.
[16]
M.H. Nanao, G.M. Sheldrick, R.B.G. Ravelli, IUCr, Improving radiation-damage substructures for RIP, Acta Crystallogr. Sect. D Biol. Crystallogr. 61 (2005) 1227–1237. doi:10.1107/S0907444905019360.
[17]
P.D. Adams, P. V Afonine, G. Bunkóczi, V.B. Chen, I.W. Davis, N. Echols, J.J. Headd, L.W. Hung, G.J. Kapral, R.W. Grosse-Kunstleve, A.J. McCoy, N.W. Moriarty, R. Oeffner, R.J. Read, D.C. Richardson, J.S. Richardson, T.C. Terwilliger, P.H. Zwart, PHENIX : a comprehensive Python-based system for macromolecular structure solution, Acta Crystallogr. Sect. D Biol. Crystallogr. 66 (2010) 213–221. doi:10.1107/S0907444909052925. 12
Journal Pre-proof [18]
P. Emsley, K. Cowtan, Coot: Model-building tools for molecular graphics, Acta Crystallogr. Sect. D Biol. Crystallogr. 60 (2004) 2126–2132. doi:10.1107/S0907444904019158.
[19]
V.B. Chen, W.B. Arendall, J.J. Headd, D.A. Keedy, R.M. Immormino, G.J. Kapral, L.W. Murray, J.S. Richardson, D.C. Richardson, MolProbity: All-atom structure validation for macromolecular crystallography, Acta Crystallogr. Sect. D Biol. Crystallogr. 66 (2010) 12–21. doi:10.1107/S0907444909042073.
[20]
W. Kabsch, A solution for the best rotation to relate two sets of vectors, Acta Crystallogr. Sect. A. 32 (1976) 922–923. doi:10.1107/S0567739476001873.
[21]
M.D. Winn, C.C. Ballard, K.D. Cowtan, E.J. Dodson, P. Emsley, P.R. Evans, R.M. Keegan, E.B. Krissinel, A.G.W. Leslie, A. McCoy, S.J. McNicholas, G.N. Murshudov, N.S. Pannu, E.A. Potterton, H.R. Powell, R.J. Read, A. Vagin, K.S. Wilson, Overview of the CCP4 suite and current developments., Acta Crystallogr. D. Biol. Crystallogr. 67 (2011) 235– 42. doi:10.1107/S0907444910045749.
[22]
N. Guex, M.C. Peitsch, SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling, Electrophoresis. 18 (1997) 2714–2723. doi:10.1002/elps.1150181505.
[23]
W.L. DeLano, The PyMOL Molecular Graphics System, Version 0.99 Schrödinger, LLC., Schrödinger LLC. Version 1. (2002) http://www.pymol.org. doi:citeulike-articleid:240061.
[24]
E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E. Ferrin, UCSF Chimera--a visualization system for exploratory research and analysis., J. Comput. Chem. 25 (2004) 1605–12. doi:10.1002/jcc.20084.
[25]
Y. Duan, C. Wu, S. Chowdhury, M.C. Lee, G. Xiong, W. Zhang, R. Yang, P. Cieplak, R. Luo, T. Lee, J. Caldwell, J. Wang, P. Kollman, A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations, J. Comput. Chem. 24 (2003) 1999–2012. doi:10.1002/jcc.10349.
[26]
T.J. Dolinsky, J.E. Nielsen, J.A. McCammon, N.A. Baker, PDB2PQR: an automated 13
Journal Pre-proof pipeline for the setup of Poisson-Boltzmann electrostatics calculations, Nucleic Acids Res. 32 (2004) W665–W667. doi:10.1093/nar/gkh381. [27]
M.H.M. Olsson, C.R. Søndergaard, M. Rostkowski, J.H. Jensen, PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical p K a Predictions, J. Chem. Theory Comput. 7 (2011) 525–537. doi:10.1021/ct100578z.
[28]
C.R. Søndergaard, M.H.M. Olsson, M. Rostkowski, J.H. Jensen, Improved Treatment of Ligands and Coupling Effects in Empirical Calculation and Rationalization of p K a Values, J. Chem. Theory Comput. 7 (2011) 2284–2295. doi:10.1021/ct200133y.
[29]
W.L. Jorgensen, J. Chandrasekhar, J.D. Madura, R.W. Impey, M.L. Klein, Comparison of simple potential functions for simulating liquid water, J. Chem. Phys. 79 (1983) 926– 935. doi:10.1063/1.445869.
[30]
D.P. Kharakoz, Protein Compressibility, Dynamics, and Pressure, Biophys. J. 79 (2000) 511–525. doi:10.1016/S0006-3495(00)76313-2.
[31]
J. Nicholson, F. Körber, S. Lambert, Application of moderate hydrostatic pressure induces unit-cell changes in rhombohedral insulin, Acta Crystallogr. Sect. D Biol. Crystallogr. 52 (1996) 1012–1015. doi:10.1107/S0907444996004386.
[32]
S. Mangialardo, F. Piccirilli, A. Perucchi, P. Dore, P. Postorino, Raman analysis of insulin denaturation induced by high-pressure and thermal treatments, J. Raman Spectrosc. 43 (2012) 692–700. doi:10.1002/jrs.3097.
[33]
F. Piccirilli, S. Mangialardo, S. Lupi, P. Postorino, A. Perucchi, Infrared Microspectroscopy study of insulin crystals at high pressure, J. Phys. Conf. Ser. 359 (2012) 012014. doi:10.1088/1742-6596/359/1/012014.
[34]
G.D. Smith, W. a. Pangborn, R.H. Blessing, Phase changes in T3R3f human insulin: temperature or pressure induced?, Acta Crystallogr. Sect. D Biol. Crystallogr. 57 (2001) 1091–1100. doi:10.1107/S0907444901007685.
[35]
O. Gursky, J. Badger, Y. Li, D.L. Caspar, Conformational changes in cubic insulin crystals in the pH range 7-11., Biophys. J. 63 (1992) 1210–20. doi:10.1016/S00063495(92)81697-1. 14
Journal Pre-proof [36]
J.L. Jiménez, E.J. Nettleton, M. Bouchard, C. V Robinson, C.M. Dobson, H.R. Saibil, The protofilament structure of insulin amyloid fibrils., Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 9196–201. doi:10.1073/pnas.142459399.
[37]
J.P. Schmittschmitt, J.M. Scholtz, The role of protein stability, solubility, and net charge in amyloid fibril formation, Protein Sci. 12 (2003) 2374. doi:10.1110/PS.03152903.
[38]
J. Brange, G.G. Dodson, D.J. Edwards, P.H. Holden, J.L. Whittingham, A model of insulin fibrils derived from the x-ray crystal structure of a monomeric insulin (despentapeptide insulin), Proteins Struct. Funct. Genet. 27 (1997) 507–516. doi:10.1002/(SICI)1097-0134(199704)27:4<507::AID-PROT4>3.0.CO;2-7.
[39]
R.L. Millican, D.N. Brems, Equilibrium Intermediates in the Denaturation of Human Insulin and Two Monomeric Insulin Analogs, Biochemistry. 33 (1994) 1116–1124. doi:10.1021/bi00171a010.
[40]
E. Gazit, Mechanisms of amyloid fibril self-assembly and inhibition, FEBS J. 272 (2005) 5971–5978. doi:10.1111/j.1742-4658.2005.05022.x.
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Journal Pre-proof Table 1. Statistics of data collection, refinement and structural parameters. Values in parentheses are for the outer shell. ap_ins
hp_ins_1
hp_ins_2
hp_ins_3
6QQG 2.15 60 MPa 8352 (740)
6QRH 2.15 100 MPa 7347 (652)
6QRK 2.10 200 MPa 8472 (724)
R(I)merge
6QQ7 1.65 0.1 MPa 179532 (6434) 9941 (489) 0.80 18.1 (13.2) 15.45-1.65 (1.68-1.65) 0.118 (0.320)
Mean (I)/sd(I) CC1/2
20.0 (1.39) 1.000 (0.616)
4373 (382) 4467 (402) 0.23 0.24 1.9 (1.9) 1.6 (1.6) 16.78-2.15 16.00-2.15 (2.21-2.15) (2.22-2.15) 0.105 0.131 (0.329) (0.453) 7.7 (2.00) 4.7 (1.40) 0.996 0.973 (0.701) (0.656) 97.0 (99.9) 99.3 (98.7) I213
4702 (392) 0.24 1.8 (1.8) 16.66-2.10 (2.16-2.10) 0.127 (0.703) 5.5 (1.10) 0.980 (0.454) 99.0 (100)
Data collection and refinement PDBid Maximum resolution (Å) Pressure [MPa] Number of reflections Number of unique reflections Mosaicity (˚) Multiplicity Resolution range (Å)
Completeness (%) Space group Unit-cell parameters a (Å)
99.9 (100) 78.77
78.71
78.38
78.14
15.55-1.65
16.78-2.15
16.00-2.15
15.95-2.10
0.173 0.190 40
0.167 0.176 25
0.193 0.232 24
0.197 0.226 24
0.007 0.738
0.014 1.641
0.013 1.639
0.014 1.807
Structural parameters RMSDa Cα / main chain, A
-
RMSDa Cα / main chain, B
-
Molecular volume (V), [Å3] Molecular area (A), [Å2] Energy, [kJ/mol]
6207 2672 -1445
0.098 / 0.112 0.113 / 0.332 6168 2648 -1390
0.126 / 0.145 0.161 / 0.282 6150 2646 -1385
0.139 / 0.155 0.304 / 0.524 6146 2645 -1381
Refinement Refinement resolution range (Å) R (%) Rfree (%) Number of solvent molecules Stereochemical restraints, r.m.s. Bond distance (Å) Bond angles (°)
16
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Figure 1. Schematic of a typical diamond anvil cell setup (on the left) and sample chamber with insulin crystal and ruby chip inside the DAC (on the right).
Figure 2. Compressibility of the unit-cell parameter a (open diamond) and the unit-cell volume (open squares) as a function of pressure (Δp= 247*Δλ where p is given in MPa and λ in nm; pressure vs Δλ shift is measured with an accuracy better than approximately 1%).
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Figure 3. Displacement parameter B analysis: A) per residue obtained from MD simulations under atmospheric pressure, 200 MPa or 500 MPa; B) comparison of displacement parameters per residue obtained from MD simulations under atmospheric pressure (green) and 500 MPa (dark blue) Error bars specify standard deviation of values obtained in three independent simulations for each pressure condition; C) structural model of insulin with highlighted in green regions with the highest value of B.
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Figure 4. Shifts of Cα positions in insulin structures determined at 60 MPa, 100 MPa and 200 MPa vs. reference structure determined at room temperature, ambient pressure.
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Figure 5. Difference distance matrices for insulin molecule determined at: A) 60 MPa; B) 100 MPa and C) 200 MPa vs. reference structure (determined at room temperature, ambient pressure); D) superposition of structures compressed to 200 MPa (purple, pink). Results of molecular dynamics simulation for insulin molecule under E) 200 MPa and F) 500 MPa.
20
Katarzyna Kurpiewska, Anna Miłaczewska, Krzysztof Lewiński PII:
S0022-2860(19)31360-2
DOI:
https://doi.org/10.1016/j.molstruc.2019.127251
Reference:
MOLSTR 127251
To appear in:
Journal of Molecular Structure
Received Date:
19 July 2019
Accepted Date:
17 October 2019
Please cite this article as: Katarzyna Kurpiewska, Anna Miłaczewska, Krzysztof Lewiński, Insulin conformational changes under high pressure in structural studies and molecular dynamics simulations, Journal of Molecular Structure (2019), https://doi.org/10.1016/j.molstruc.2019.127251
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
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Insulin conformational changes under high pressure in structural studies and molecular dynamics simulations Katarzyna Kurpiewska1,2*, Anna Miłaczewska1*, Krzysztof Lewiński2 1Jerzy
Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences,
Niezapominajek 8, 30-239 Kraków, Poland 2Jagiellonian
University, Faculty of Chemistry, Department of Crystal Chemistry and Crystal
Physics, Gronostajowa 2, 30-387 Kraków, Poland
*Corresponding authors: Katarzyna Kurpiewska, Anna Miłaczewska phone +48 12 639 51 01, fax +48 12 425 19 23 mail: [email protected], [email protected]
Highlights:
For the first time insulin crystal structures were determined under pressure of 60, 100 and 200 MPa.
Experimental and theoretical approaches revealed high pressure insulin conformations.
Diverse susceptibility of the insulin molecule regions to compression were identified.
Under high pressure both terminal fragments of chain B were recognized as the most vulnerable regions. 1
Journal Pre-proof Graphical abstract
Keywords: insulin, high-pressure structure, molecular dynamic simulations, protein crystallography Abstract To study the mechanisms underlying protein misfolding and aggregation, therapeutic proteins can be successfully used as a model. Currently, insulin is widely tested as a useful model in this field, since it has been proved in both in vivo and in vitro studies that this small protein aggregates. In this article, exploiting the optimal coupling between high pressure protein crystallography and dynamic simulations, we probe the insulin conformations observed under high pressure, namely over the ranges 0-200 MPa for crystallographic experiments and 0-500 MPa for simulations. Crystal structures of insulin determined with diamond anvil cell technique present a step forward in understanding how pressure can modify protein conformation. Obtained results show different responses to volume compression of different fragments of the insulin molecule. For the first time, we have structurally proved that pressure noticeably modifies fragments of insulin molecules, especially terminal fragments of chain B. The observed structural modifications of insulin molecule in crystal state under pressure were compared to the results of insulin pressurization investigated by the molecular dynamic simulations. Comparing the crystallographic results and MD simulations, we were able to draw important considerations about the role of specific amino acids in pressure-induced insulin conformations.
2
Journal Pre-proof 1. Introduction Insulin (ins) is a small polypeptide hormone that regulates glucose level in higher organisms [1]. Clinical relevance of insulin misfolding and its high tendency to aggregate into amyloid fibrils justify the fact that it is often considered as a model protein in the study of misfolding. Although insulin exists in several oligomeric forms, it is monomeric insulin that is responsible for its biological activity. The most stable oligomer is formed by six insulin molecules and this hexameric state serves as its storage unit. High temperature, low pH or contact with hydrophobic media promote partial destabilization of insulin and a series of structural changes, which result in the formation of the ordered aggregates [2]. High hydrostatic pressure due to its effect on the energy of the system that only involves change of the volume contribution to Gibbs free energy has also proved to be a very powerful tool in the study of misfolding and aggregation [3]. Previous studies showed that insulin is indeed susceptible to fibrillation under high pressure and according to Piccirilli et al. low degree assemblies are more susceptible to pressurization than oligomers [4]. Insulin is a 5800 Da molecule composed of two chains (A-chain of 21 residues; B-chain of 30 residues) and has three α-helices, two in the A chain (A2-A8 and A13-A19) and one in the B chain (B9-B19). Three disulfide bonds take part in protein stability and functionality: two inter-chain bridges are formed between A7-B7 and A20-B19. One intra-chain disulfide bond binds cysteines A6 and A11. The hydrophobic core that is essential for correct folding of insulin [5] is formed by the residues Gly8, Leu11, Val12, Leu15, Phe24, Tyr26 of chain B and two sidechains of the residues located in chain A: Ile2 and Val3. The first crystal structure of insulin determined in 1969 [5] was followed by other structures of insulin as well as its analogs in the storage form [6]. The latter form of insulin refers to a Zn stabilized hexamer, which is not active and provides the organism with the hormone when required. Native insulin can be crystallized in cubic, monoclinic and rhombohedral space group. The cubic form of insulin crystallizes in the absence of Zn ions (space group I213, a = 78.9 Å). These crystals have much larger solvent content (64%) in comparison to the Zn form (only 35% solvent content). Large solvent content and high symmetry of crystals are advantageous for high pressure crystallographic experiments, thus the cubic insulin crystals provide a good model system for exploring structural changes under high pressure conditions and were selected for our structural studies, and as a model in MD simulation.
3
Journal Pre-proof Even though there are increasing data regarding insulin folding, assembly and dynamics the precise study of the structural basis for this phenomenon is still missing. It was reported that insulin aggregation must proceed through a bulky, non-native monomer [7], which quickly forms larger oligomers. Therefore, our analyses of insulin structure determined for crystals under high hydrostatic pressure is followed by an investigation of the mechanical properties of the most sensitive to pressure fragments performed with MD simulations. Considering the physiological and therapeutic importance of insulin [8] and the fact that high hydrostatic pressure was proved to evoke aggregation-prone intermediate states in insulin molecule [9] our studies aim in structural investigation of conformations present at the beginning of the aggregation process, which are crucial for the formation of non-native insulin conformations. 2. Methods 2.1. Chemicals Liofilizated protein was purchased from Sigma (I6634, Sigma-Aldrich Co, St. Louis, Mo) and used without purification. All the other chemical reagents used were of analytical grade. 2.2. Crystallization Crystals were grown at room temperature using the hanging drop vapor diffusion method according to Yu & Caspar [5]. Protein was dissolved in 0.01 M Na2HPO4, 0.1 M Na2EDTA, pH 10 to the final concentration of 10 mg/ml. A crystallization drop containing 3 μl of the protein and 2 μl of reservoir solution was equilibrated against 0.01 M Na3EDTA, 0.03 M Na2HPO4 pH 10.4 (298K, atmospheric pressure). Average size of insulin crystals selected for diffraction experiments was 0.25 x 0.40 x 0.50 mm. Size and quality of obtained crystals were suitable for experiments using Merrill-Bassett type of diamond anvil cell (DAC). 2.3. Data collection and processing X-ray diffraction data at ambient pressure (ap) were collected for insulin crystal mounted in MicroRTTM from MiTeGen. For high pressure (hp) conditions single crystals were mounted inside the screw driven DAC. Steel gaskets (0.3 mm thickness) with a hole diameter of 0.5 mm were used according to the procedure previously described in Kurpiewska et al. [10,11]. Protein crystals were transferred into the chamber previously filled with the crystallization mother liquor (Figure 1). Determination of pressure was performed at room 4
Journal Pre-proof temperature using the ruby fluorescence method according to protocol described by Pirermarini et al. [12] and calculated as Δp= 247*Δλ (where p [MPa], λ [nm]; pressure vs Δλ shift is measured with an accuracy better than approximately 1%). Data were collected at room temperature using SuperNova (Rigaku Oxford Diffraction) four-circle diffractometer equipped with a double microfocus X-ray source and multilayer X-ray optics. CuKα radiation was used for reference structure at ambient pressure while MoKα radiation was used for high pressure experiments. The data sets were processed with Mosflm [13] and AIMLESS [14]. Preliminary experiments were performed for crystals compressed to 0.1, 27, 60, 100, 173 and 200 MPa in order to determine the unit cell parameters. Complete data collections were performed for crystal at ambient pressure (ap_ins), at 60 MPa (hp_ins_1), at 100 MPa (hp_ins_2) and at 200 MPa (hp_ins_3), respectively. The structures were solved by molecular replacement method with PHASER [15] using the coordinates of insulin PDBid: 2BN3 as a starting model [16]. Crystallographic refinement was carried out using maximum-likelihood target functions implemented in PHENIX [17]. Each round of the refinement with automatic weighting was interspersed with a round of the model building using WinCOOT [18]. About 10% of the data were randomly excluded from the refinement and used as a test data set to monitor Rfree. As phases improved ordered solvent molecules were added with the protocol implemented in WinCOOT and then manually. Finally, the structures were validated using MolProbity [19] and deposited to PDB as: ins_ap (6QQ7), hp_ins_1 (6QQG), hp_ins_2 (6QRH) and hp_ins_3 (6QRK). 2.4. Analysis of the structures Superposition of all structures and calculation of the root-mean-square deviation for atomic coordinates (r.m.s. deviation) was performed using LSQKAB [20] program from CCP4 suite (Collaborative Computational Project, 1994) [21]. Molecular volume and area using a radius equal to 1.4 Å and difference distance matrices (DDM) were calculated with Chimera. For calculation of an accessible surface area, AreaIMol (CCP4 suite) was used. The energy computation was done with GROMOS96 43B1 parameters implemented in SPDBV_4.1 [22]. Figures were prepared using PyMOL [23] and Chimera [24]. 2.5. MD simulations The molecular dynamic simulation was carried out for insulin monomer using Amber14 software with ff03 force field [25]. Protonation states of residues were checked by PDB2PQR 5
Journal Pre-proof server, version 2.1.1 [26] with implemented PROPKA [27,28] software for pKa calculations at pH 7.0; the latter predicted no unexpected protonation states. Starting geometry was taken from the crystal structure deposited under PDB code: 2BN3. The protein was placed inside a cube filled with water molecules modeled by the TIP3P model with at least a 10 Å distance between the protein and the edge of the cube [29]. The system was then minimized in three steps: in the first run, the complex was restrained with a 500 kcal/(mol·Å2) harmonic potential, while the positions of water and the Na+ ions were optimized. Next, the protein was restrained with a 10 kcal/(mol·Å2) potential, and in the third step, the whole system was minimized with no restraints. Subsequently, in the following step, the system was heated to 300 K under constant volume conditions over 100 ps. Next, a density of the system was equilibrated during 1 ns constant pressure dynamics simulations employing a restraining potential of 1 kcal/(mol·Å2) for protein backbone. The final production MD simulations of 50 ns were carried out in triplicate with constant temperature (300 K) and ambient pressure (0.1 MPa) under periodic boundary conditions using a 2 fs time step performed with Langevin dynamics, isotropic position scaling, SHAKE algorithm to constrain bonds involving hydrogen atoms, and the Particle Mesh Ewald method for the long-range electrostatics. The last geometry was used to run 6 ns simulations under high pressure (200 MPa, 500 MPa) in triplicate. All trajectories were clustered into 5 clusters each. For 50 ns simulations, only last 10 ns with stable RMSD were considered. A representative of the most populated cluster was taken as the reference to determine backbone displacement parameters. 3. Results and discussion The symmetry of cubic space group I213 is preserved by compressed crystals. Diffraction data at ambient and high pressure were collected to maximum resolution 1.65 Å and 2.0-2.15 Å, respectively. Details of the data collection and refinement are summarized in Table 1. The noticeable loss of the diffraction was observed for insulin crystals compressed to a pressure above 220 MPa. The compressibility β of the unit cell parameter (Figure 2) was calculated as β= 1/Vu(∂Vu/∂p). An increase of compressibility was observed up to 100 MPa and then β started to decrease with pressure. This is the first time we discover transition in compressibility for protein crystal. For insulin crystal compressed to 100 MPa the calculated compressibility of the unit cell parameter a and volume V are 50 MPa-1 and 148 MPa-1, respectively, which corresponds to the unit cell volume reduction of 1.4% at 100 MPa. For experiments under 200 MPa the calculated compressibility of the corresponding parameters a and V are 40 MPa-1 and 120 MPa-1, respectively, that refers to the unit cell 6
Journal Pre-proof volume reduction of 0.9% at 200 MPa. Taking into account that the protein in the crystalline state is a homogeneous system filled with water that freely diffuses into and out of the crystal, the compression can be distributed within it and by this means protein molecules can be uniformly influenced by hydrostatic pressure. According to the approach discussed by Kharakoz [30] the nonlinear protein response to mechanical disturbance can lead to the positive and negative changes in the compressibility of the protein upon compression. Our findings are the first experimental example that supports this hypothesis of nonmonotonically pressure influence. The cubic insulin crystals compressibility increases with pressure up to 100 MPa, but at elevated pressure trend toward lower compressibility is observed. From the structural point of view it is clear that under 100 MPa a noticeable compression within both A and B chains contributes to observed high compressibility, whereas under 200 MPa chain B is compressed to a greater extent, but chain A response to pressurization is less spectacular. The structural rearrangements will be further discussed in the section referring to the global changes altered by high pressure. Unit cell changes for insulin rhombohedral crystal (space group R3) treated with high pressure were reported by Nicholson [31]. In those studies, hydrostatic pressure was applied within the cell via nitrogen gas and data were recorded at pressures ranging from 0.1 to 3 MPa. Changes in the diffraction pattern were observed at pressures as low as 0.5 MPa and data analysis revealed unit cell volume reductions of 2.60% at 2 MPa. Experiments showed that above 25 bar (2.5 MPa) diffraction power is lost irretrievably and that upon depressurization the crystals do not return to their original cell parameters. These results are significantly different from the results obtained for cubic crystals and can be explained by the presence of pressure-induced phase transition in rhombohedral crystals. The large number of compressible voids present in insulin core as well as its globular arrangement, fortified both by covalent and non-covalent interactions, can be listed as factors contributing to its high stability. The previous results reported by Piccirilli et al. [32] for insulin in solution under pressure studied with FTIR revealed that insulin preserved its globular structure even at very high pressure (above 1500 MPa). Surprisingly, in our studies for insulin crystals compressed to a noticeable lower pressure (slightly above 200 MPa) no diffraction was observed, even though the crystal shape was maintained. Our previous results for proteins in crystal under high pressure i.e. RNase A and β-lactoglobulin showed that folded protein conformation can withstand significantly higher pressure in the crystal than in solution. Interestingly, Piccirilli et al. reported that growth of cubic crystals at pressure of about 400 MPa was observed for the solution of previously misfolded insulin [32,33], 7
Journal Pre-proof unfortunately no structural data are available for this experiment. Furthermore, in powder diffraction studies on insulin reported by Smith et al., the phase transition (TR transition) was induced by the pressure resulting from the mechanical grinding of the sample, but the sample returned to its original form after several days [34]. The overall architecture of the insulin molecule before and after pressurization is almost identical. A dimer stabilized by hydrogen bonds between the main chain atoms of both subunits related by the crystallographic 2-fold axis is retained. The r.m.s. deviation between the Cα-atom positions in ap and hp insulin structures for chain A are in range 0.098 to 0.139 Å while for chain B they are larger and were found in range 0.113 to 0.304 Å. Similar observation referring to larger changes in chain B are observed for all main chain atom positions, 0.112 to 0.155 Å for chain A and 0.332 to 0.524 Å for chain B. Analysis of side chains conformation reveals two groups of residues referring to different response to pressure elevation. The group in which the alternative conformations relate to pressurization includes Glu4(A), Arg22(A), Glu13(B) and Phe25(B). The preference for two rotamers of Arg22(A) side chain can be correlated with pressure, in hp_ins_1 two alternative conformations can be observed: the same as in ap_ins and second which corresponds to lower accessible surface area of insulin molecule. For structure compressed to 100 and 200 MPa only the second conformation is present. Similar rearrangement is observed for side chain of Glu13(B). Two alternative position of glutamic acid are present in structures ap, hp_ins_1 and hp_ins_2, while for hp_ins_3 only one conformation was identified, again the one which favors protein state with smaller accessible surface area. Phe25(B) side chain in all hp structures is in the same conformation, but different from the one observed in ap structure of insulin. Alternative conformations of Ser9(A), Val2(B), Gln15(B), Glu21(B), Lys29(B) do not show a clear correlation with changes in pressure and thus were assigned to the second group. It was previously reported by Gursky et al. that ~30% of residue conformations observed at pH 7 are different from those present in insulin form crystallized using alkaline solutions (pH 11) [35]. The side chains of residues that were proved to be influenced by pressure do not coincide with those found to be sensitive to pH changes. To investigate the behavior of terminal parts of insulin chains under pressure we have conducted a set of MD simulations in three different conditions: under 0.1 MPa, 200 MPa and 500 MPa. The pressure higher than applied in crystallographic studies was used to reveal possible changes more clearly. Comparison of obtained averaged displacement parameters (shown in Figure 3A) allowed us to determine the most pressure sensitive regions of the insulin molecule. The largest differences occur mainly in chain B (residues: 1-4, 7-9, 12-18 8
Journal Pre-proof and 25-29), while chain A is less affected by pressure (mostly region comprising residues 11-15) (Figure 4B). N- and C-terminus of chain B, as well as part of chain A, present the most pronounced effect (Figure 3C), which supports the experimental results described above. Additionally, to identify global rearrangements, shifts of Cα positions and difference distance matrices were calculated (Figure 4). Crystal structure of insulin compressed to 60 MPa showed no pronounced effect of pressure on the molecule conformation except shift of A1-A2 residues. When pressure was increased to 100 MPa and 200 MPa the entire insulin molecule is compressed and fragments of chain A (A1-A3, A12-A18) and chain B (B16-B24, B26-B28) are displaced as can be seen on Figure 5A-5C. Fragments A1-A3, A12-A18 correspond to part of -helix H1 and entire H2, while for chain B shift of larger fragments can be observed, including N-terminal part of -helix and C terminus. Interestingly, as a result of MD simulation for insulin modeled under 200 MPa an expansion of the molecule, which coincides with subtle rearrangements of the same fragments mentioned above, can be noticed (Figure 5E, 5F). However, a substantial shift regards N-terminal part of chain B (B1-B4). The response of the molecule to higher pressure (500 MPa) is clearly different and shift of chain A towards chain B in insulin molecule can be identified, without apparent movement of fragments within chains. The largest pressure-induced structural effects occur in terminal regions of both polypeptide chains, though noticeable positional changes, mainly of the N-terminal and C-terminal fragments of chain B, can be observed. Both approaches, experimental and theoretical, applied by us in presented studies revealed that chain B of insulin is much more sensitive to pressurization than chain A (Figure 5D). Observed shifts of B chain terminal regions refer to Jiménez et al. studies of the insulin derivatives which showed that the two B-chain's termini might play distinct roles in the amyloidogenesis: the C-terminal slows down the process, whereas the residues at the N-terminus are necessary for the lateral aggregation, i.e., enable protofilaments to form fibrils [36]. Within a given pressure range, only subtle rearrangements and positional shifts of helices, rather than any signs of their unfolding can be identified. Therefore, our crystallographic studies are unable to verify the scenario proposed by Jiménez, in which the α-helical structure of native insulin must unfold completely to form the amyloid cross-β structure [36]. Hydrophobic interactions appear to be one of the most important factor influencing aggregation process [37]. Such a mechanism was also suggested previously as a driving force for insulin aggregation [2,38,39]. In our work, no significant changes in insulin structure were observed which could explain possible hydrophobic fragments exposition. On the other hand, as it was pointed out by Gazit and co-workers [40] there are other structural factors that 9
Journal Pre-proof contribute to the formation of amyloid: 1) surface accessible area reduction, 2) hydrogen bonding saturation and 3) reaching an alternative non-native global free energy minimum. Structures determined by us under high pressure prove that in case of insulin molecule, all mentioned factors contribute to the formation of pressure-treated conformation. 4. Conclusions In this studies, we presented structural models of insulin obtained under high pressure in the scope of 0-200 MPa, which serve as a starting point to discussion about insulin conformational stability. Aggregation of insulin is an important issue as it inactivates its therapeutic role. The comparison of structural data determined under high pressure with results at ambient pressure condition together with molecular dynamics simulations indicates that terminal fragments of chain B are the most vulnerable regions and might contribute significantly in the insulin aggregation process. The phenomenon of protein propensity to form amyloid fibrils is expected to vary from protein to protein under a given set of conditions. Therefore, understanding the factors that govern the amyloidogenic propensity is a persisting and important issue. Taken together with the earlier reports, our study shows that high pressure protein crystallography combined with molecular dynamics simulations may serve as a powerful tool to gain insight into the role of pressure on promotion of amyloid formation. Authors contributions K.K. and K.L. initiated the study and directed the project. K.K. conducted crystallographic studies and computational analyses of structures. A.M. performed molecular dynamics simulations. K.K., K.L. and A.M. prepared the manuscript, which was revised and approved by all authors. Acknowledgments The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08). Authors would like to thank
Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of
Sciences for financial support of presented studies.
10
Journal Pre-proof References: [1]
R. Tattersall, Bovine insulin., BMJ. 305 (1992) 831–831. doi:10.1136/bmj.305.6857.831.
[2]
L. Nielsen, R. Khurana, A. Coats, S. Frokjaer, J. Brange, S. Vyas, V.N. Uversky, A.L. Fink, Effect of Environmental Factors on the Kinetics of Insulin Fibril Formation: Elucidation of the Molecular Mechanism, Biochemistry. 40 (2001) 6036–6046. doi:10.1021/bi002555c.
[3]
J.L. Silva, A.C. Oliveira, T.C.R.G. Vieira, G.A.P. De Oliveira, M.C. Suarez, D. Foguel, HighPressure Chemical Biology and Biotechnology, (2014). doi:10.1021/cr400204z.
[4]
F. Piccirilli, S. Mangialardo, P. Postorino, S. Lupi, A. Perucchi, FTIR analysis of the high pressure response of native insulin assemblies, J. Mol. Struct. 1050 (2013) 159–165. doi:10.1016/J.MOLSTRUC.2013.07.028.
[5]
M.J. Adams, T.L. Blundell, E.J. Dodson, G.G. Dodson, M. Vijayan, E.N. Baker, M.M. Harding, D.M.C. Hodgkin, B. Rimmer, S. Sheat, Structure of Rhombohedral 2 Zinc Insulin Crystals, Nature. 224 (1969) 491–495. doi:10.1038/224491a0.
[6]
E.N. Baker, T.L. Blundell, J.F. Cutfield, S.M. Cutfield, E.J. Dodson, G.G. Dodson, D.M.C. Hodgkin, R.E. Hubbard, N.W. Isaacs, C.D. Reynolds, K. Sakabe, N. Sakabe, N.M. Vijayan, The Structure of 2Zn Pig Insulin Crystals at 1.5 A Resolution, Philos. Trans. R. Soc. B Biol. Sci. 319 (1988) 369–456. doi:10.1098/rstb.1988.0058.
[7]
A. Ahmad, I.S. Millett, S. Doniach, V.N. Uversky, A.L. Fink, Partially Folded Intermediates in Insulin Fibrillation †, Biochemistry. 42 (2003) 11404–11416. doi:10.1021/bi034868o.
[8]
P. Sonksen, J. Sonksen, Insulin: understanding its action in health and disease, Br. J. Anaesth. 85 (2000) 69–79. doi:10.1093/bja/85.1.69.
[9]
R. Jansen, W. Dzwolak, R. Winter, Amyloidogenic self-assembly of insulin aggregates probed by high resolution atomic force microscopy., Biophys. J. 88 (2005) 1344–53. doi:10.1529/biophysj.104.048843.
11
Journal Pre-proof [10]
K. Kurpiewska, K. Lewiński, High pressure macromolecular crystallography for structural biology: a review, Cent. Eur. J. Biol. 5 (2010) 531–542. doi:10.2478/s11535010-0044-y.
[11]
K. Kurpiewska, K. Dziubek, A. Katrusiak, J. Font, M. Ribò, M. Vilanova, K. Lewiński, Structural investigation of ribonuclease A conformational preferences using high pressure protein crystallography, Chem. Phys. 468 (2016) 53–62. doi:10.1016/j.chemphys.2016.01.010.
[12]
G.J. Piermarini, S. Block, J.D. Barnett, R.A. Forman, Calibration of the pressure dependence of the R1 ruby fluorescence line to 195 kbar, J. Appl. Phys. 46 (1975) 2774–2780. doi:10.1063/1.321957.
[13]
T.G.G. Battye, L. Kontogiannis, O. Johnson, H.R. Powell, A.G.W. Leslie, iMOSFLM : a new graphical interface for diffraction-image processing with MOSFLM, Acta Crystallogr. Sect. D Biol. Crystallogr. 67 (2011) 271–281. doi:10.1107/S0907444910048675.
[14]
P.R. Evans, G.N. Murshudov, How good are my data and what is the resolution?, Acta Crystallogr. Sect. D Biol. Crystallogr. 69 (2013) 1204–1214. doi:10.1107/S0907444913000061.
[15]
A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni, R.J. Read, Phaser crystallographic software, J. Appl. Crystallogr. 40 (2007) 658–674. doi:10.1107/S0021889807021206.
[16]
M.H. Nanao, G.M. Sheldrick, R.B.G. Ravelli, IUCr, Improving radiation-damage substructures for RIP, Acta Crystallogr. Sect. D Biol. Crystallogr. 61 (2005) 1227–1237. doi:10.1107/S0907444905019360.
[17]
P.D. Adams, P. V Afonine, G. Bunkóczi, V.B. Chen, I.W. Davis, N. Echols, J.J. Headd, L.W. Hung, G.J. Kapral, R.W. Grosse-Kunstleve, A.J. McCoy, N.W. Moriarty, R. Oeffner, R.J. Read, D.C. Richardson, J.S. Richardson, T.C. Terwilliger, P.H. Zwart, PHENIX : a comprehensive Python-based system for macromolecular structure solution, Acta Crystallogr. Sect. D Biol. Crystallogr. 66 (2010) 213–221. doi:10.1107/S0907444909052925. 12
Journal Pre-proof [18]
P. Emsley, K. Cowtan, Coot: Model-building tools for molecular graphics, Acta Crystallogr. Sect. D Biol. Crystallogr. 60 (2004) 2126–2132. doi:10.1107/S0907444904019158.
[19]
V.B. Chen, W.B. Arendall, J.J. Headd, D.A. Keedy, R.M. Immormino, G.J. Kapral, L.W. Murray, J.S. Richardson, D.C. Richardson, MolProbity: All-atom structure validation for macromolecular crystallography, Acta Crystallogr. Sect. D Biol. Crystallogr. 66 (2010) 12–21. doi:10.1107/S0907444909042073.
[20]
W. Kabsch, A solution for the best rotation to relate two sets of vectors, Acta Crystallogr. Sect. A. 32 (1976) 922–923. doi:10.1107/S0567739476001873.
[21]
M.D. Winn, C.C. Ballard, K.D. Cowtan, E.J. Dodson, P. Emsley, P.R. Evans, R.M. Keegan, E.B. Krissinel, A.G.W. Leslie, A. McCoy, S.J. McNicholas, G.N. Murshudov, N.S. Pannu, E.A. Potterton, H.R. Powell, R.J. Read, A. Vagin, K.S. Wilson, Overview of the CCP4 suite and current developments., Acta Crystallogr. D. Biol. Crystallogr. 67 (2011) 235– 42. doi:10.1107/S0907444910045749.
[22]
N. Guex, M.C. Peitsch, SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling, Electrophoresis. 18 (1997) 2714–2723. doi:10.1002/elps.1150181505.
[23]
W.L. DeLano, The PyMOL Molecular Graphics System, Version 0.99 Schrödinger, LLC., Schrödinger LLC. Version 1. (2002) http://www.pymol.org. doi:citeulike-articleid:240061.
[24]
E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E. Ferrin, UCSF Chimera--a visualization system for exploratory research and analysis., J. Comput. Chem. 25 (2004) 1605–12. doi:10.1002/jcc.20084.
[25]
Y. Duan, C. Wu, S. Chowdhury, M.C. Lee, G. Xiong, W. Zhang, R. Yang, P. Cieplak, R. Luo, T. Lee, J. Caldwell, J. Wang, P. Kollman, A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations, J. Comput. Chem. 24 (2003) 1999–2012. doi:10.1002/jcc.10349.
[26]
T.J. Dolinsky, J.E. Nielsen, J.A. McCammon, N.A. Baker, PDB2PQR: an automated 13
Journal Pre-proof pipeline for the setup of Poisson-Boltzmann electrostatics calculations, Nucleic Acids Res. 32 (2004) W665–W667. doi:10.1093/nar/gkh381. [27]
M.H.M. Olsson, C.R. Søndergaard, M. Rostkowski, J.H. Jensen, PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical p K a Predictions, J. Chem. Theory Comput. 7 (2011) 525–537. doi:10.1021/ct100578z.
[28]
C.R. Søndergaard, M.H.M. Olsson, M. Rostkowski, J.H. Jensen, Improved Treatment of Ligands and Coupling Effects in Empirical Calculation and Rationalization of p K a Values, J. Chem. Theory Comput. 7 (2011) 2284–2295. doi:10.1021/ct200133y.
[29]
W.L. Jorgensen, J. Chandrasekhar, J.D. Madura, R.W. Impey, M.L. Klein, Comparison of simple potential functions for simulating liquid water, J. Chem. Phys. 79 (1983) 926– 935. doi:10.1063/1.445869.
[30]
D.P. Kharakoz, Protein Compressibility, Dynamics, and Pressure, Biophys. J. 79 (2000) 511–525. doi:10.1016/S0006-3495(00)76313-2.
[31]
J. Nicholson, F. Körber, S. Lambert, Application of moderate hydrostatic pressure induces unit-cell changes in rhombohedral insulin, Acta Crystallogr. Sect. D Biol. Crystallogr. 52 (1996) 1012–1015. doi:10.1107/S0907444996004386.
[32]
S. Mangialardo, F. Piccirilli, A. Perucchi, P. Dore, P. Postorino, Raman analysis of insulin denaturation induced by high-pressure and thermal treatments, J. Raman Spectrosc. 43 (2012) 692–700. doi:10.1002/jrs.3097.
[33]
F. Piccirilli, S. Mangialardo, S. Lupi, P. Postorino, A. Perucchi, Infrared Microspectroscopy study of insulin crystals at high pressure, J. Phys. Conf. Ser. 359 (2012) 012014. doi:10.1088/1742-6596/359/1/012014.
[34]
G.D. Smith, W. a. Pangborn, R.H. Blessing, Phase changes in T3R3f human insulin: temperature or pressure induced?, Acta Crystallogr. Sect. D Biol. Crystallogr. 57 (2001) 1091–1100. doi:10.1107/S0907444901007685.
[35]
O. Gursky, J. Badger, Y. Li, D.L. Caspar, Conformational changes in cubic insulin crystals in the pH range 7-11., Biophys. J. 63 (1992) 1210–20. doi:10.1016/S00063495(92)81697-1. 14
Journal Pre-proof [36]
J.L. Jiménez, E.J. Nettleton, M. Bouchard, C. V Robinson, C.M. Dobson, H.R. Saibil, The protofilament structure of insulin amyloid fibrils., Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 9196–201. doi:10.1073/pnas.142459399.
[37]
J.P. Schmittschmitt, J.M. Scholtz, The role of protein stability, solubility, and net charge in amyloid fibril formation, Protein Sci. 12 (2003) 2374. doi:10.1110/PS.03152903.
[38]
J. Brange, G.G. Dodson, D.J. Edwards, P.H. Holden, J.L. Whittingham, A model of insulin fibrils derived from the x-ray crystal structure of a monomeric insulin (despentapeptide insulin), Proteins Struct. Funct. Genet. 27 (1997) 507–516. doi:10.1002/(SICI)1097-0134(199704)27:4<507::AID-PROT4>3.0.CO;2-7.
[39]
R.L. Millican, D.N. Brems, Equilibrium Intermediates in the Denaturation of Human Insulin and Two Monomeric Insulin Analogs, Biochemistry. 33 (1994) 1116–1124. doi:10.1021/bi00171a010.
[40]
E. Gazit, Mechanisms of amyloid fibril self-assembly and inhibition, FEBS J. 272 (2005) 5971–5978. doi:10.1111/j.1742-4658.2005.05022.x.
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Journal Pre-proof Table 1. Statistics of data collection, refinement and structural parameters. Values in parentheses are for the outer shell. ap_ins
hp_ins_1
hp_ins_2
hp_ins_3
6QQG 2.15 60 MPa 8352 (740)
6QRH 2.15 100 MPa 7347 (652)
6QRK 2.10 200 MPa 8472 (724)
R(I)merge
6QQ7 1.65 0.1 MPa 179532 (6434) 9941 (489) 0.80 18.1 (13.2) 15.45-1.65 (1.68-1.65) 0.118 (0.320)
Mean (I)/sd(I) CC1/2
20.0 (1.39) 1.000 (0.616)
4373 (382) 4467 (402) 0.23 0.24 1.9 (1.9) 1.6 (1.6) 16.78-2.15 16.00-2.15 (2.21-2.15) (2.22-2.15) 0.105 0.131 (0.329) (0.453) 7.7 (2.00) 4.7 (1.40) 0.996 0.973 (0.701) (0.656) 97.0 (99.9) 99.3 (98.7) I213
4702 (392) 0.24 1.8 (1.8) 16.66-2.10 (2.16-2.10) 0.127 (0.703) 5.5 (1.10) 0.980 (0.454) 99.0 (100)
Data collection and refinement PDBid Maximum resolution (Å) Pressure [MPa] Number of reflections Number of unique reflections Mosaicity (˚) Multiplicity Resolution range (Å)
Completeness (%) Space group Unit-cell parameters a (Å)
99.9 (100) 78.77
78.71
78.38
78.14
15.55-1.65
16.78-2.15
16.00-2.15
15.95-2.10
0.173 0.190 40
0.167 0.176 25
0.193 0.232 24
0.197 0.226 24
0.007 0.738
0.014 1.641
0.013 1.639
0.014 1.807
Structural parameters RMSDa Cα / main chain, A
-
RMSDa Cα / main chain, B
-
Molecular volume (V), [Å3] Molecular area (A), [Å2] Energy, [kJ/mol]
6207 2672 -1445
0.098 / 0.112 0.113 / 0.332 6168 2648 -1390
0.126 / 0.145 0.161 / 0.282 6150 2646 -1385
0.139 / 0.155 0.304 / 0.524 6146 2645 -1381
Refinement Refinement resolution range (Å) R (%) Rfree (%) Number of solvent molecules Stereochemical restraints, r.m.s. Bond distance (Å) Bond angles (°)
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Figure 1. Schematic of a typical diamond anvil cell setup (on the left) and sample chamber with insulin crystal and ruby chip inside the DAC (on the right).
Figure 2. Compressibility of the unit-cell parameter a (open diamond) and the unit-cell volume (open squares) as a function of pressure (Δp= 247*Δλ where p is given in MPa and λ in nm; pressure vs Δλ shift is measured with an accuracy better than approximately 1%).
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Figure 3. Displacement parameter B analysis: A) per residue obtained from MD simulations under atmospheric pressure, 200 MPa or 500 MPa; B) comparison of displacement parameters per residue obtained from MD simulations under atmospheric pressure (green) and 500 MPa (dark blue) Error bars specify standard deviation of values obtained in three independent simulations for each pressure condition; C) structural model of insulin with highlighted in green regions with the highest value of B.
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Figure 4. Shifts of Cα positions in insulin structures determined at 60 MPa, 100 MPa and 200 MPa vs. reference structure determined at room temperature, ambient pressure.
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Figure 5. Difference distance matrices for insulin molecule determined at: A) 60 MPa; B) 100 MPa and C) 200 MPa vs. reference structure (determined at room temperature, ambient pressure); D) superposition of structures compressed to 200 MPa (purple, pink). Results of molecular dynamics simulation for insulin molecule under E) 200 MPa and F) 500 MPa.
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