RSC Chemical Biology

The allosteric O 2 release of haemoglobin (Hb) allows for eﬃcient O 2 delivery from the lungs to the tissues. However, allostery is weakened in Hb-based O 2 carriers because the chemical modifications of the Lys- and Cys- b 93 residues prevent the quaternary transition of Hb. In this paper, we describe the synthesis and O 2 binding properties of a recombinant Hb [rHb( b K120C)]–albumin heterotrimer that maintains sufficient Hb allostery. The rHb( b K120C) core, with two additional cysteine residues at the symmetrical positions on its protein surface, was expressed using yeast cells. The mutations did not influence either the O 2 binding characteristics or the quaternary transition of Hb. Maleimide-activated human serum albumins (HSAs) were coupled with rHb( b K120C) at the two Cys- b 120 positions, yielding the rHb( b K120C)–HSA 2 trimer, in which the Cys- b 93 residues were unreacted. Molecular dynamics simulation demonstrated that the HSA moiety does not interact with the amino acid residues around the haem pockets and the a 1 b 2 surfaces of the rHb( b K120C) core, the alteration of which retards Hb allostery. Circular dichroism spectroscopy demonstrated that the quaternary transition between the relaxed (R) state and the tense (T) state of the Hb core occurred upon both the association and dissociation of O 2 . In phosphate-buffered saline solution (pH 7.4) at 37 1 C, the rHb( b K120C)–HSA 2 trimer exhibited a sigmoidal O 2 equilibrium curve with the O 2 affinity and cooperativity identical to those of native Hb ( p 50 = 12 Torr, n = 2.4). Moreover, we observed an equal Bohr effect and 2,3-diphosphoglycerate response in the rHb( b K120C)–HSA 2 trimer compared with naked Hb.


Introduction
Allosteric regulation plays a crucial role in controlling the protein function in biological systems through the binding of a small-molecule effector that induces a conformational change. Haemoglobin (Hb) is a well-known allosteric protein encapsulated in red blood cells (RBCs), [1][2][3][4][5] and its allostery has been extensively studied to understand its mechanism. 6-12 O 2 binding converts the quaternary structure of Hb from a deoxy tense (T) state with a low O 2 affinity to a relaxed (R) state with a high O 2 affinity. [1][2][3][4] The homotropic allosteric effect, which is known as cooperativity, allows Hb to efficiently transport O 2 from the lungs to the peripheral tissues. As a heterotropic allosteric effect, 2,3-diphospoglycerate (DPG) stabilizes the T-state conformation by creating salt bridges between the two b subunits to reduce O 2 affinity. 13,14 Furthermore, O 2 affinity decreases with decreasing pH (the Bohr effect), shifting the equilibrium toward the T state. 11,15,16 Artificial O 2 carriers are currently required as RBC substitutes for transfusion therapy because of worldwide shortages in blood supply. 17,18 However, the stroma-free Hb splits to an ab dimer (a 2 b 2 -2ab) in the bloodstream and is immediately expelled into urine. Moreover, Hb diffuses into the interstitial spaces between the endothelial cells and smooth muscle cells, causing hypertension by scavenging nitric oxide (NO; endothelial-derived vasodilator). 19,20 Therefore, naked Hb is not available for use in transfusion therapy. Since the 1980s, several types of Hb-based O 2 carriers have been developed, including cross-linked Hb, 21,22 polymerized Hb, 23,24 and PEGylated Hb. [25][26][27] However, these chemical modifications have markedly reduced cooperativity, the Bohr effect, and the DPG effect, 18 and a modified Hb that maintains the original allosteric effects of Hb has never been reported. 18 We previously synthesized a protein cluster as an RBC substitute consisting of an Hb molecule wrapped covalently in human serum albumins (HSAs), called an Hb-HSA m cluster (m = 2-4, average m = 3.0 AE 0.2). 28,29 This protein cluster shows a long circulation lifetime in the bloodstream and a superior biocompatibility. 30,31 The quaternary structural analysis of the Hb-HSA m cluster revealed that the chemical modification of the Lys and Cys-b93 residues of Hb by N-succinimidyl 3-maleimidopropionate as a crosslinker prevents the R-T quaternary transition and maintains the Hb core close to the R state. 32 Thus, the Hb-HSA m cluster demonstrated a low cooperativity, a low Bohr effect, and a low DPG effect. If the HSA shells are linked at appropriate positions on the Hb surface so that they do not affect the R-T quaternary motion, the resulting Hb-HSA m conjugate maintains the original O 2 binding properties of the native Hb. The genetically introduced cysteine could act as a cross-linking point for maleimide-activated HSA (MA-HSA). We assume that the mutation of Hb residues, excluding the mutation of (i) the a 1 b 2 interfaces, including the N-and C-terminals (which play important roles in the R-T quaternary transition), and (ii) the haem pockets (which directly affect O 2 affinity), would allow the resulting Hb variant to maintain its original O 2 binding capability. Based on this hypothesis, the Lys-b120 residues were chosen as the symmetrical mutation points (Fig. 1A). In this study, we describe a novel recombinant Hb variant, rHb(bK120C), in which Lys-b120 is replaced with Cys at symmetrical positions on the Hb surface, and we also present the rHb(bK120C)-HSA 2 heterotrimer, in which MA-HSAs are linked at the Cys-b120 positions (Fig. 1B). Ultraviolet-Visible (UV-vis) and circular dichroism (CD) spectroscopies revealed that the quaternary structure of the rHb(bK120C)-HSA 2 trimer is reversibly convertible between the R and T states. We also observed identical O 2 affinity and allosteric effects in the rHb(bK120C)-HSA 2 trimer compared with naked Hb.

Results and discussion
Expression and physicochemical properties of rHb(bK120C) We first expressed rHb(bK120C) using Pichia yeast (Pichia pastoris GS115) according to our previous reports. 33,34 The expressed rHb(bK120C) was purified using cation-and anion-exchange chromatographies (CEC and AEC). The sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis of rHb(bK120C) depicted two clear bands corresponding to the aand b-subunits ( Fig. 2A). The aand b-chain bands demonstrated near-identical mobility to those of native Hb. The matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum analysis of rHb(bK120C) exhibited molecular-related ion peaks at 15 122 and 15 841 Da. These were almost identical to the simulated masses of the amino acid sequences (15 127 Da for the a-chain and 15 842 Da for the b-chain), indicating that Lys-b120 was replaced with cysteine. The size-exclusion chromatography (SEC) chromatogram of rHb(bK120C) exhibited a peak with the same elution volume as that of native Hb, indicating that the tetramer (a 1 a 2 b 1 b 2 ) was formed in the rHb(bK120C) ( Fig. 2B; elution volume = 1.9 mL). Fortunately, the oligomeric rHb(bK120C) species created via the disulfide bonds of Cys-b120 was not observed in the SEC chromatogram. The fact that rHb(bK120C) and native Hb had identical CD spectra (l = 200-250 nm) indicates that their secondary structures were equivalent (Fig. 2C). The isoelectric point (pI) value of rHb(bK120C) was determined to be 6.8, which was slightly lower than that of native Hb (pI = 6.9) because the mutation (Lys-b120 -Cys) had negatively shifted the net surface charge (Fig. S1, ESI †). The results of the sulfhydryl group assay using 4,4 0 -dithiodipyridine (4,4 0 -DTP) 35 revealed the number of reactive sulfhydryl groups in rHb(bK120C) and native Hb as 4.0 and 2.0, respectively. This result strongly indicates that the two Lys-b120 residues of the b 1 and b 2 chains were replaced by Cys and that the introduced Cys-b120 residues were reactive with 4,4 0 -DTP, as they are located at the molecular surface of rHb(bK120C).

Preparation of the rHb(bK120C)-HSA 2 trimer
To prepare MA-HSA, we used 1,6-bis(maleimido)hexane (BMH) as the cross-linking agent. One of the two maleimide groups in BMH was reacted with the free sulfhydryl group of the Cys-34 in HSA (which is the only reduced-form of cysteine in HSA), yielding MA-HSA. The formation of the HSA dimer was not observed because of electrostatic repulsion between the negatively charged HSA surfaces present under our experimental conditions. The This journal is © The Royal Society of Chemistry 2020 sulfhydryl group assay using 4,4 0 -DTP revealed the complete disappearance of the reduced form.
Capping of the Cys-b93 residues of native Hb with maleimide derivatives causes a decline in O 2 binding cooperativity, the Bohr effect, and the influence of the allosteric effectors. The Cys-b93 residues of native Hb were able to react with maleimide derivatives, such as N-ethyl maleimide (NEM) and N-succinimidyl 3-maleimidopropionate. 32,36 We found that the Cys-b93 residues had not reacted with MA-HSA because of the steric hindrance between the native Hb and MA-HSA. In contrast, genetically introduced Cys-b120 located at the molecular surface of rHb(bK120C) had reacted with MA-HSA. The SEC chromatogram of the reaction mixture [rHb(bK120C) + MA-HSA] revealed a new peak in the high-molecular-weight region ( Fig. 3A; elution volume = 1.4 mL), and native PAGE analysis also revealed a new band above that of rHb(bK120C) (Fig. 3B). The highmolecular-weight component was purified using AEC followed by gel filtration chromatography (GFC). The ion peak of the b-chain at 15.8 kDa in the MALDI-TOF MS disappeared after the reaction of rHb(bK120C) with MA-HSA. A new peak then appeared at 82.5 kDa, a value almost identical to the simulated mass of a b-chain + MA-HSA (82.3 kDa), indicating that the HSA moiety was covalently linked to the b-chain of rHb(bK120C). Based on the [total protein]/[Hb unit] assays, the average HSA/Hb ratio of the product was determined to be 2.0. The heme loss was not observed during the preparation. We denoted this hybrid protein as rHb(bK120C)-HSA 2 (M w : 198 kDa). The CD spectrum of the rHb(bK120C)-HSA 2 trimer coincided perfectly with the sum of the rHb(bK120C) spectrum and a two-times-enlarged HSA spectrum (Fig. 3C). This result demonstrates that the secondary structures   of the individual proteins were unaltered by the protein coupling. The dynamic light scattering measurements revealed that the hydrodynamic diameter of the rHb(bK120C)-HSA 2 trimer (12.4 AE 2.8 nm, P.I. = 0.010) was significantly larger than that of rHb(bK120C) (6.9 nm AE 1.9 nm, P.I. = 0.004). The pI value of the trimer (pI = 5.4) was close to that of HSA (pI = 4.9), which further indicates the coupling of rHb(bK120C) (pI = 6.7) with HSAs ( Fig. S1, ESI †). The net negative surface charge and the large molecular size could allow the rHb(bK120C)-HSA 2 trimer to circulate in the bloodstream for long periods of time and prevent the undesirable vasopressor response through NO depletion. 19,20 Furthermore, the sulfhydryl group assay using 4,4 0 -DTP 35 demonstrated that the number of reactive sulfhydryl groups in the rHb(bK120C)-HSA 2 trimer was 1.8, indicating that the Hb core maintained the free Cys-b93 residues that are important for O 2 binding ability, as mentioned above. 36 We could not observe the dissociation of the a 2 b 2 tetramer into two ab dimers during the synthesis, purification, and measurements of rHb(bK120C)-HSA 2 , implying that the a 2 b 2 tetramer was sufficiently stable the same as native Hb under our experimental conditions. An MD simulation was used to predict the interactions between the rHb(bK120C) and HSA moieties. The Cys-b 1 120 and Cys-b 2 120 residues exist at symmetrical positions of the core Hb; therefore, a sufficient distance is maintained between the two HSA parts in the rHb(bK120C)-HSA 2 trimer. We performed MD simulation using a half model [ab(K120C)-HSA] constructed using the crystal structures of native Hb (PDB ID: 2DN1) 37 and HSA (PDB ID: 1AO6) 38 (Fig. S2, ESI †). The main chains (Ca and amide atoms) of the ab(K120C) moiety were frozen for the duration of a 50 ns simulation. The root-meansquare deviation (RSMD) and the distances of the salt bridges and hydrogen bonds between the ab(K120C) and HSA residues reached a plateau within 25 ns (Fig. S3, ESI †). The MD simulation structure was represented in the rHb(bK120C)-HSA 2 form which was prepared by overlapping the two ab(K120C) moieties of the models with the crystal structure of native Hb (a 1 b 1 a 2 b 2 ; Fig. 3D and Fig. S2B, ESI †). The distance between the sulfide atoms of Cys-b120 [rHb(bK120C)] and Cys-34 (HSA) was measured to be 13.8 Å, meaning that the BMH was long enough to cross-link the Cys residues (Fig. 3E). We found that the following salt bridges and hydrogen bonding interactions between rHb(bK120C) and HSA helped stabilize the conformation of the rHb(bK120C)-HSA 2 trimer: (i) Glu-a23, Glu-a27, and His-a112 of rHb(bK120C)-Arg-81 of HSA and (ii) Ser-a49 and Asp-a47 of rHb(bK120C)-Gln-94 of HSA. The HSA moiety did not interact with the amino acid residues around the haem pockets and the a 1 b 2 surfaces of the rHb(bK120C) core.

Quaternary structure analysis of the Hb core
We investigated the quaternary structure of the Hb core using UV-vis absorption and CD spectroscopies. The UV-vis spectral patterns and the absorbances of the oxy, deoxy, and carbonyl rHb(bK120C) as well as the rHb(bK120C)-HSA 2 trimer were indistinguishable from those of native Hb in PBS solution (pH 7.4; Fig. 4A and Table S1, ESI †). Generally, the chemical modification of the Lys and Cys-b93 residues of Hb decreases the Soret band absorption of the deoxy form, because the deoxy Hb remains near the R-state quaternary structure. 32 In contrast, the deoxy rHb(bK120C) and the rHb(bK120C)-HSA 2 trimer revealed the same Soret band absorption as that of deoxy native Hb, indicating that the identical T-state quaternary structures were formed. CD spectroscopy was also used to analyse the quaternary structure of the Hb core in the rHb(bK120C)-HSA 2 trimer. Generally, deoxy Hb (T state) exhibits a negative CD band at 287 nm, which is known as the T-state marker. [39][40][41] At the same time, this band is not usually observed in the R-state quaternary structure of oxy Hb. The CD spectral patterns and intensities of oxy and deoxy rHb(bK120C) both resembled those of native Hb, implying that the oxy and deoxy forms of these proteins are comparable (Fig. 4B). 39 CD also revealed similar spectral changes in the rHb(bK120C)-HSA 2 trimer upon deoxygenation. CD spectra for the oxy and deoxy Hb cores in the rHb(bK120C)-HSA 2 trimer were obtained by subtracting the two-times-enlarged HSA spectrum from the corresponding rHb(bK120C)-HSA 2 trimer spectra (Fig. 4B). The CD spectra of the oxy and deoxy Hb cores coincided with those of rHb(bK120C). These spectral analyses revealed that the mutation Lys-b120 -Cys and coupling of HSA at the Cys-b120 positions did  modified Hbs have demonstrated a low cooperativity because the quaternary transition of the Hb core is prevented. 32 Such modified Hbs include PEG-Hb (71% reduction), 18,42 polymerized Hb (41% reduction), 18,42 and Hb-HSA m clusters (71% reduction). 29,42 In contrast, the OECs of rHb(bK120C) and the rHb(bK120C)-HSA 2 trimer were found to have a sigmoidal shape completely consistent with that of native Hb (Fig. 5A).
The O 2 binding parameters of rHb(bK120C) ( p 50 = 12 Torr, n = 2.3) and the rHb(bK120C)-HSA 2 trimer ( p 50 = 12 Torr, n = 2.4) were also equivalent to those of native Hb (Table 1). Both rHb(bK120C) and the rHb(bK120C)-HSA 2 trimer were able to maintain identical O 2 affinity and cooperativity because the quaternary structure of the Hb units was interconvertible between the R and T states upon O 2 association and dissociation, as in native Hb. The capping of Cys-b93 with NEM in native Hb (NEM-Hb) reduced the p 50 and n values (p 50 = 8 Torr, n = 1.9). The NEM-rHb(bK120C)-HSA 2 trimer demonstrated O 2 binding parameters similar to those of NEM-Hb, clearly indicating that the Cys-b93 residues in the rHb(bK120C)-HSA 2 trimer were in a reduced form. This result is supported by the sulfhydryl group assay mentioned above. We concluded that the selective coupling of MA-HSA occurred at the two Cys-b120 positions of the rHb(bK120C), whereas it did not take place at the Cys-b93 residues because of steric hindrance. On the basis of these findings, we also concluded that the Lys-b120 -Cys mutation and the selective conjugation of MA-HSA at the Cys-b120 positions were impervious to O 2 affinity and cooperativity.
To the best of our knowledge, this is the first example of a chemically modified Hb showing an identical O 2 affinity and cooperativity to those of native Hb. 18 The autoxidation rate constant (k ox ) of the oxygenated Hb core in the rHb(bK120C)-HSA 2 trimer was measured in PBS solution (pH 7.4) at 37 1C. The k ox value of the rHb(bK120C)-HSA 2 trimer was ascertained as 0.027 h À1 , which was almost similar to the data of native Hb (0.020 h À1 ). The oxy form of the internal Hb maintains good stability even after being covered with HSAs.

Bohr effect on rHb(bK120C)-HSA 2 trimer
A high metabolic activity leads to an increase in the concentration of CO 2 and a decrease in the pH value within the tissues. Under low-pH conditions, the protonation of Hb occurs and the T-state quaternary structure is stabilized, reducing O 2 affinity (the Bohr effect). Thus, the OEC of native Hb was right-shifted to a p 50 value of 28 Torr at pH 6.2 compared with p 50 = 12 Torr at  a The Bohr coefficients were derived from the slopes obtained from plotting ÀDlog p 50 /DpH between pH 6.2 and 8.0 (Fig. 5B).

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This journal is © The Royal Society of Chemistry 2020 RSC Chem. Biol., 2020, 1, 128--136 | 133 pH 7.4 (Table 1 and Fig. 5A). The Bohr effect allows for Hb high O 2 -transport efficiency to the tissues. However, chemical modifications have tended to notably reduce the Bohr effect, such as in PEG-Hb (87% reduction) 18 and polymerized Hb (75% reduction). 18 We observed that the O 2 affinities of rHb(bK120C) and the rHb(bK120C)-HSA 2 trimer were reduced under lower pH conditions, similar to native Hb (p 50 : 12 -29 Torr (pH 7.4 -6.2)). The p 50 and n values of both rHb(bK120C) and the rHb(bK120C)-HSA 2 trimer were nearly identical to those of native Hb at pH 6.2 ( Table 1). The Bohr coefficients were derived from the slopes obtained from plotting ÀDlog p 50 /DpH between pH 6.2 and 8.0 ( Fig. 5B and Table S2, ESI †). The Bohr coefficients of both rHb(bK120C) and the rHb(bK120C)-HSA 2 trimer were identical to those of native Hb ( Table 1). The value measured for native Hb, however, was somewhat weaker than the result reported by Ho et al. 43 This difference is probably attributable to several factors, such as the difference in temperature (37 1C vs. 29 1C) and the difference in solvents (PBS with low phosphate and high NaCl concentrations vs. 0.1 M sodium phosphate buffer (PB)). Altogether, we conclude that neither the Lys-b120 -Cys mutation nor the HSA conjugation affected the Bohr effect.

Effects of DPG on O 2 binding affinity
Allosteric effectors bind to proteins, altering their activity. The most important allosteric regulator of Hb in RBCs is DPG, which reduces the O 2 binding affinity (the DPG effect). An X-ray crystallography analysis has previously revealed that a single DPG molecule binds to the cleft of Hb b chains, known as the DPG binding site. 13,14 The anionic groups of DPG form salt bridges with the cationic groups of the b-chains, including the His-b2, Lys-b82, and His-b143 residues, stabilizing the T-state conformation and reducing O 2 affinity. The T -R conformational change induced by oxygenation leads to the closure of the binding site and weakens the DPG binding affinity to oxy Hb. This DPG response allows Hb to efficiently deliver O 2 from the lungs to the tissues. We measured the OEC of native Hb in PBS (pH 7.4) solution containing 1 mM DPG at 37 1C, but a reduction in O 2 affinity was not observed because of the fact that phosphate anions in the PBS solution interrupt the binding of DPG to Hb. Thus, we used 0.1 M Tris-HCl buffer for DPG response experiments. Indeed, native Hb in Tris-HCl buffer demonstrated a higher O 2 affinity (p 50 = 9 Torr) than in PBS solution because of the exclusion of the Hb-phosphate anion interactions ( Table 2). The O 2 affinity of the native Hb was also reduced upon the addition of DPG to Tris-HCl buffer. Interestingly, rHb(bK120C) (p 50 = 26 Torr) and the rHb(bK120C)-HSA 2 trimer (p 50 = 26 Torr) demonstrated identical DPG responses to native Hb (p 50 = 27 Torr; Fig. 5C). These results indicate that the rHb(bK120C)-HSA 2 trimer maintained a DPG response similar to that of native Hb and that its O 2 affinity is controllable via the allosteric effector.

Conclusions
A novel Hb variant (Lys-b120 -Cys) was designed to prepare the rHb(bK120C)-HSA 2 trimer as an artificial O 2 carrier. The mutation did not affect the O 2 binding properties of Hb. Two MA-HSA molecules were selectively linked to Cys-b120 in rHb(bK120C), resulting in the rHb(bK120C)-HSA 2 trimer in which Cys-b93 remained free because of steric repulsion between the rHb(bK120C) core and the MA-HSA shell. The CD spectra revealed that the R-T quaternary motion of the Hb core in the rHb(bK120C)-HSA 2 trimer occurred upon O 2 association and dissociation. Thus, the rHb(bK120C)-HSA 2 trimer demonstrated sufficient allosteric O 2 binding properties with respect to cooperativity, the Bohr effect, and the DPG effect. The rHb(bK120C)-HSA 2 trimer is a unique artificial O 2 carrier with sufficient allostery to be an RBC substitute.
Transformed clone cells were grown in a buffered mineral glycerol complex (BMGY) medium (4 L total) in a shaking The mixture solution was diluted with the same volume of water and loaded onto the column equilibrated with PBS solution. The column was then washed using 10 mM PB (pH 7.4) solution. The rHb(bK120C)-HSA 2 trimer was eluted with a 10 mM PB (pH 6.0) + 120 mM NaCl solution. Under this condition, the HSA dimer remained in the column. The collected solution was concentrated and subjected to GFC using a Superdex 200 pg column (GE Healthcare UK Ltd) to isolate the rHb(bK120C)-HSA 2 trimer (yield: 50%). The total protein and Hb concentrations were measured using a protein assay kit (Pierce 660 nm; Thermo Fischer Scientific Inc.) and the extinction coefficient of cyano-metHb (e 541 = 4.4 Â 10 4 M À1 cm À1 ), respectively. The average [HSA]/[rHb(bK120C) unit] ratio of the product was estimated to be 2.0. The sulfhydryl group assay of MA-HSA was conducted via the reaction with 4,4 0 -DTP.

Molecular dynamics simulations
MD simulations were performed using the Desmond application implemented in a Maestro graphical interface. 44,45 We used the optimized potentials for liquid simulations (OPLS_2005) force field implemented in the Desmond software for all molecules in the system. 46 We constructed a half model of rHb(bK120C) [ab(K120C)-HSA model] because rHb(bK120C)-HSA 2 has a C 2 symmetry, and there has to be a sufficient distance between the HSAs for their interactions to be negligible. The crystal structure of native Hb (PDB ID: 2DN1) 37 was used for the ab(K120C) model. Lys-b120 was replaced with Cys using the PyMOL software (PyMOL Molecular Graphics System Version 2.0, Schrödinger, LLC.). For the HSA moiety, we used the crystal structure of HSA (PDB ID: 1AO6). 38 The missing atoms of the side chains in the HSA were added to the model using the PyMOL software. The water molecules in the model were removed. The bond orders were assigned, and the hydrogen atoms were added to the models using the Protein Preparation Wizard tool 47 in the Maestro software. The ab(K120C) and HSA models were covalently linked using the BMH cross-linker [Cys-b120(ab)-BMH-Cys-34(HSA)] and arranged so that sufficient distance was maintained between them (Fig. S2A, ESI †). The Cys-b120(Hb), Cys-34(HSA), and BMH moieties were minimized using the 3D builder in the Maestro software. The hydrogen networks and the protonated states were optimized to pH 7 using the Protein Preparation Wizard for which the pK a values of the protein residues were predicted using PROPKA. 48 The ab(K120C)-HSA model was solvated using explicit TIP3P water 49 with a buffering distance of 10 Å in an octahedral box. Sodium counterions were added to neutralize the charges. The model was relaxed using a default relaxation protocol implemented in the Desmond software. An isothermal-isobaric MD simulation was performed for 50 ns to maintain the 300 K temperature and 1.01325 bar pressure. The main chains (Ca and amide atoms) of the ab(K120C) moiety were frozen during the simulation. The time step was 2 fs, and the information for analysis was printed every 100 ps. A 10 Å cut off was used for non-bonded interactions.