AuNPs of different sizes were procured from Sigma Aldrich: 5 nm (Product number 756528), 10 nm (Product number 752584), and 20 nm (Product number 753610).

The plasmids used in this study are previously engineered, and the details are mentioned elsewhere (Panda et al., 2021). Mtu SufB wild type, splicing inactive double mutant (C1A/N359A) SufB (negative control), and SufB intein all carrying N-terminal 6X (His) tag were overexpressed in BL21 (DE3) E. coli cells via IPTG (sigma 367-93-1) induction. Cells were resuspended in lysis buffer (20 mM sodium phosphate, 0.5 M NaCl, pH 7.4) and lysed via a tip sonicator (Sonics vibra cell VCX-130). The overexpressed proteins were isolated from inclusion bodies (IBs) via centrifugation. The IB materials were solubilized in 8 M urea (Merck, 1084870500) buffer (lysis buffer, 8 M urea, 20 mM of imidazole (MP–biochemicals-288-32-4) and centrifuged at 16,500x g for 20 min to collect the supernatant (Friedel et al., 2019a). Solubilized test proteins were purified using a Ni-NTA affinity column (Ni-NTA His trap, HP GE healthcare life sciences- 17524802). Prior to sample application, columns were equilibrated with binding buffer (20 mM sodium phosphate, 0.5 M NaCl,40 mM imidazole) and after sample loading, the columns were washed several times (15 CV) in binding buffer. Finally, test proteins were eluted as purified fractions in elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole), followed by quantification via Bradford’s assay.

For splicing assay, 2.5 µM of denatured test protein was refolded in refolding buffer (20 mM sodium phosphate, 0.5 M NaCl, 0.5 M Arginine) and 2 mM TCEP-HCl (sigma- 51805-45-9) for 4 hours, in the presence and absence of AuNPs (0.25 and 0.5 ppm) at 25°C (Mills et al., 1998; Mills and Paulus, 2001; Gangopadhyay et al., 2003; Zhang et al., 2011a; Yamaguchi and Miyazaki, 2014; Chan et al., 2016a). The 0 h sample was obtained by collecting the sample prior to renaturation and abrupt addition of loading dye (0.1% bromophenol blue, 50% glycerol, β-mercaptoethanol, 10% SDS, and Tris 6.8), followed by rapid freezing at −20°C.

The N-terminal cleavage assay for the purified proteins was conducted following the aforementioned experimental steps. Denatured test proteins were allowed to refold in the presence of AuNPs at a concentration of 0.5 ppm for 1 h. Samples were taken out at different periods (10, 20, 30, and 60 mins) followed by abrupt addition of loading dye and boiling at 95°C for 5 mins. Products from splicing and cleavage reactions were resolved through 4–10% gradient SDS-PAGE. Protein bands were stained with Coomassie brilliant blue R-250, and densitometric analysis was performed using GelQuant.NET biochemical solutions software. The splicing and cleavage efficiencies were calculated as percentage of the ratio of total splicing product (LE and I) over total proteins (LE + I + P) and total N-terminal cleavage product (NE + NC) over total proteins (NE + NC + P), respectively. The 0 h splicing and cleavage value(s) were subtracted each time for baseline correction. The results were analyzed using one-way ANOVA and plotted using GraphPad Prism version 5.01 for Windows, GraphPad Software, San Diego, California, United States, www.graphpad.com.

Western blot analysis was performed by using an anti-His antibody (Invitrogen, LOT 1902132) to confirm the identity of splicing and cleavage products. The test proteins were transferred from SDS gel to PVDF membrane (Millipore, IPVH 00010); at 50V for 2 h. Next, the blot was blocked with 5% skim milk for 2 h at 25°C, washed with 1X TBST, and incubated with primary anti-His antibody (abgenex-32–6116) at 1:5,000 dilutions for 16 h at 4°C. In the final step, the blot was washed with 1XTBST and incubated with secondary antibody (anti-mouse, abgenex 11-301) at 1:8,000 dilutions for 2.5 h at 25°C. The N-extein band was detected using a higher dilution of the primary (1: 2,500) and secondary antibodies (1:6000). The blot was developed using enhanced chemiluminescence substrate (ECL) (abcam, ab65628).

Purified SufB intein was incubated with AuNPs (20, 10, and 5 nm) at a concentration of 0.5 ppm for 4 h. Untreated SufB intein was taken as a control against each treated sample. The samples were scanned via UV–visible spectroscopy (UV-1800 Shimadzu) at 25°C in the visible region of spectrum 200–800 nm. To eliminate the interference of buffer nanoparticle interaction, the refolding buffer (20 mM sodium phosphate, 0.5 M NaCl, 0.5 M arginine + 8 M urea) was incubated with the AuNPs under similar experimental conditions and then scanned against respective test samples. The sole buffer solution was considered as the blank and used for baseline correction. The data were analyzed by plotting an absorbance vs wavelength graph in Origin (Pro) 8.5, OriginLab Corporation, Northampton, MA, United States.

ITC measurement was carried out at 25°C on MicroCal ITC200, Malvern. SufB intein was overexpressed and purified under native conditions. The protein was buffer exchanged to 0.1 M PBS (pH 7.4) by dialysis as the ligands (AuNPs) were in the same buffer. The ligand concentration was maintained 10 times higher than that of the protein to get the proper saturation. In total, 2 μl of AuNP solution was injected into the sample cell and allowed to titrate for 4 s. Within every injection, a 120-s time interval was maintained for equilibration. Then AuNP sample was loaded in a syringe and titrated against SufB intein for 30 cycles. The heat released or absorbed during the reaction was measured by an instrument. The titration of AuNP to the buffer solution was carried out along with each experiment as a reference for baseline corrections.

The reaction parameters for nanoparticle–protein interactions were obtained by fitting the titration graph of each nanoparticle against the protein in Origin (Pro) 8.5, OriginLab Corporation, Northampton, MA, United States. A baseline correction was applied to each experiment by subtracting data from titration of the AuNP solution into the buffer blank correlating to the heat of dilution (Zhang et al., 2010a). The binding isotherm was fitted to the data using a linear fit. The detected change in enthalpy and binding constants were obtained from the best-fit parameters.

Protein corona formed by protein–AuNP interaction was visualized by using a transmission electron microscope (Jeol JEM-1400). SufB intein was overexpressed and purified under native conditions. The AuNP was allowed to interact with the protein for 4 h. The protein–AuNP complex was harvested and washed twice with 0.1 M PBS to remove the unbound protein from the reaction mixture. Bare AuNPs in 0.1 M PBS solution were used as controls against protein adsorption on the AuNP surface. The protein–AuNP complex pellet was resuspended and fixed with 2.5% glutaraldehyde for 1 h. For TEM analysis, 5 μl of the AuNP complex was placed onto the carbon-coated copper grid and imaged under a TEM, with an acceleration voltage of 120 kV (Sasidharan et al., 2015).

The structure of Mtu SufB precursor has been depicted in Figure 6, where the amino acid sequence, 251–254, has been shown as EGCL (Panda et al., 2021). According to the previous model (Dearden et al., 2013; Dearden, 2014), we have selected the active site (Gly-Cys) with capping of the –CH3 (carboxylic terminal) and –CONH₂ group (amide terminal), as shown in Figure 6. The selected active site (i.e., Gly-Cys) has been treated with the first principle–based density functional theory which is implemented on the Gaussian 09 computational code (Frisch et al., 2009). The Gly-Cys structure has been relaxed with full freedom of movement with the fixing of the dihedral angles at the B3LYP/6-311+G (d, p) level of theory. The dihedral angle constraints are necessary because of the removal of the surrounding protein system. During the geometry relaxations, average force and average changes over distance were fixed to the value 3 × 10^–4 Hartree/Bohr and 1.2 × 10^–3 Bohr, respectively. In the case of the induction of a Au atom, it was fixed at a distance of 2.3 Å away from the attacking sulfur (of Cys1), on the basis of the earlier experimental study, where zinc was found to be closer to the C-terminal catalytic cysteine (Sun et al., 2005). With the nearby Au atom, the active site system is relaxed with aforementioned constraints at the functional, B3LYP, and basis set LANL2DZ for Au, and 6-311+G(d, p) for other atoms. Furthermore, the optimized structure of the system in vacuum is treated with the polarized continuum model for solvent inclusion and calculation. In the case of PCM model, when a molecule is placed in the dielectric medium, spherical cavities are created around the atoms. These dielectric media are characterized by a dielectric constant (ε), for example, ε = 1, for vacuum as default and ε = 78.35 for water in solvent case. A previous study has found the dielectric constant of protein to be ∼10; therefore, in our calculation, we have used 10 as the dielectric constant for the subsequent investigations. For the reaction barrier calculations, the transition states were found using the synchronous transit and quasi-Newton method, which was then refined with the rational function optimization using the Berny algorithm. The presence of an imaginary frequency mode confirms the TS of both of the systems, that is, Au-induced and without Au system. For a detailed investigation of the reaction barrier, we perform the Mulliken charge analysis which is implemented in Gaussian 09.

It has been widely accepted that AuNPs interact with proteins in a biological medium to form protein corona (PC). Factors like local environment; concentration; and affinity of protein, size, shape, and surface charge of AuNPs affect the kinetics of protein adsorption (Kaur and Forrest, 2012; Wang et al., 2015; Nicoară et al., 2019). We performed in vitro refolding of Mtu SufB (Materials and Methods Section) in the presence of AuNPs (0.25 and 0.5 ppm) of various sizes (5, 10, and 20 nm), to check the effect on the splicing reaction. Optimum AuNP concentrations were selected after performing gradient assay to evaluate the effect of differing concentrations of AuNPs on SufB splicing (Supplementary Figure S1).

After 4 h of refolding, the protein was resolved through 4–10% gradient SDS-PAGE. Splicing and cleavage reactions gave rise to precursor (P; 95.98 kDa), N-cleavage (NC; 66 kDa) product, N-extein (NE; 29.9 kDa), ligated extein (LE; 55.7 kDa), and intein (I; 40.2 kDa) (Figure 1Bi). None of these products were observed in splicing inactive Mtu SufB double mutant (SI, C1A/N359A), which was used as a negative control for protein splicing and to check the effect of the AuNPs on the stability of the precursor protein (Figures 1Bi,ii, 2C, 3C; Supplementary Figure S2). Previous studies have reported that alanine substitution in place of catalytic residues Cys1 and Terminal Asn completely abolishes splicing activity (Zhang et al., 2015). C-terminal cleavage (CC; 70.12 kDa) products and C-extein (CE; 25.7 kDa) were not detected in SDS-PAGE possibly due to protein degradation. SufB precursor protein (P), splicing, and cleavage products (CC, LE, and NE) carrying the N-terminal 6X (His) tag were further confirmed via Western blot using anti-His antibodies (Figure 1Bii). Two additional bands were detected, possibly precursor degradation product below the precursor protein and splicing intermediate product above LE (Figure 1Bii). The aforementioned bands were not visible with Mtu SufB intein treated under similar experimental conditions (Supplementary Figure S3B). The identity of splicing and cleavage products were confirmed by MALDI TOF/TOF Mass spectrometry and the results were submitted to the ProteomeXchange Consortium via the PRIDE with data ID PXD015199 (Submission details are provided in the Supplementary Material (Page S7).) (Panda et al., 2021).

As shown earlier, binding of the proteins on the nanoparticle surface leads to conformational changes that affect protein function (Saptarshi et al., 2013; Lynch and Dawson, 2020; Park, 2020). Moreover, several parameters including size distribution, morphology, surface charge, and surface chemistry determine the biological activity of the NPs. Besides, the larger the nanoparticle, the more surface area is accessible for protein adsorption, and vice versa, resulting in efficient nanoparticle–protein interaction (Lynch and Dawson, 2008; Fei and Perrett, 2009; Woods et al., 2016; Tian et al., 2017).

In our study, AuNPs at 0.5 ppm concentration displayed an overall superior splicing efficiency than 0.25 ppm AuNPs. In the presence of 20 nm AuNPs, Mtu SufB exhibited 52.7% increase (p < 0.0001) in splicing efficiency. And 10 nm AuNPs induced 41.8% increase (p < 0.0001) in splicing, and 5 nm AuNPs had the least effect (24.6% increase, p = 0.004) on SufB splicing (Figure 1Biii). The observed size effect of AuNPs on SufB splicing is beyond the scope of our current study. However, the superior effect of the 20-nm AuNPs in the splicing reaction may be explained as follows. An efficient adsorption of SufB on the NP surface might have affected the active site residues, positively leading to splicing enhancement.

The N-terminal cleavage reaction is the first step of protein splicing which sometimes yields NC and NE as splicing by-products (Figure 1Aii). It is initiated with an N-S acyl shift by the first Cys residue (C1) of the intein, which converts the N-terminal extein–intein peptide bond to a (thio) ester linkage. As soon as transesterification via Cys+1 attack and resolution of branched chain intermediate occurs via Asn cyclization, splicing ensues, giving rise to LE and I (Figure 1Ai). Previous literature studies have described the affinity of AuNPs for cysteine thiols (Kreibig and Genzel, 1985; Aryal et al., 2006; Li et al., 2006; Petean et al., 2008; Li and Li, 2009; Ashjari et al., 2015); thus, it is likely that AuNPs interact with catalytic Cys1 to regulate N-terminal cleavage reaction, the first critical step of splicing. Further inferences were drawn by performing in vitro refolding and quantitative N-terminal cleavage analysis of Mtu SufB in the presence of AuNPs, over two different time intervals: 10–20 minutes and 30–60 minutes (Materials and Methods Section). Resultant cleavage products were resolved through 4–10% gradient SDS-PAGE and confirmed by Western blot (Figures 2, 3).

Wild-type SufB precursor gives rise to optimum N-cleavage reaction products until 60 mins after which splicing ensues (Supplementary Figure S3). Hence, the N-terminal cleavage efficiency was monitored over 10–60 mins (Figures 2, 3). After 10 mins of the refolding reaction, 20-, 10-, and 5-nm AuNP-treated Mtu SufB protein exhibited 42.1% (p < 0.001), 35.1% (p < 0.01), and 26.8% (p < 0.05) rise in the N-terminal cleavage reaction, respectively (Figure 2D). Similarly, the enhanced N-terminal cleavage reaction was noticed after 20 mins of in vitro SufB renaturation; 20 nm (48.8% rise, p < 0.001, two-way ANOVA), 10 nm (40.4% rise, p < 0.001, two-way ANOVA), and 5 nm (33.6% rise, p < 0.01, two-way ANOVA) (Figure 2D).

After 30 mins of in vitro refolding assay, it was observed that 20-nm AuNP-treated Mtu SufB had a 33.6% increase in the N-cleavage product as compared to untreated protein, which was further analyzed by two-way ANOVA (p < 0.0001) (Figure 3D). The 10-nm AuNP-treated SufB protein resulted in 26.4% enhancement of the N-cleavage product (p < 0.01, two-way ANOVA). We observed the least effect by 5-nm AuNPs (p < 0.05, two-way ANOVA) on the N-cleavage reaction of Mtu SufB protein after 30 mins of reaction. The 20- and 10-nm AuNPs maintained a significant enhancement on the N-cleavage reaction even after 60 mins of the renaturation reaction, where it resulted in 38.8% (p < 0.001, two-way ANOVA), and 33.2% (p < 0.001, two-way ANOVA) increase in the N-cleavage reaction, respectively. And 5-nm AuNP had the least effect on the N-cleavage reaction (22.1%, p < 0.05, two-way ANOVA) (Figure 3D). From the quantitative data, it is noticeable that the AuNPs of larger size display a consistently greater fold change in N-terminal cleavage reaction throughout 1 h. Similar experimental optimizations were maintained for all the in vitro refolding experiments. We hypothesize that the likely interaction of AuNPs with catalytic cysteine thiols conceivably led to the enhanced N-terminal cleavage efficiency. Thiophilic AuNPs perhaps achieve this via interaction with catalytic Cys1, which activates N-S acyl shift. Further mechanistic insights into the process are achieved via QM/MM analysis as explained later in this article.

SufB intein and AuNP interaction was further confirmed using UV-vis absorption spectroscopy in the wavelength ranging from 200 to 800 nm, as elaborated in the Materials and Methods section. As Mtu SufB precursor splices under native conditions, we have used Mtu SufB intein for specificity and accuracy in data analysis. SufB intein was incubated along with AuNPs (5, 10, and 20 nm) at 0.5 ppm concentration for 4 hours, and the changes in the absorption spectra were monitored.

The untreated Mtu SufB intein gave an absorption peak at 258 nm. For 5- and 20-nm AuNP treated samples, we observed a minor broadening of the absorption spectra and a redshift of 2 nm (260 nm) (Figures 4A,C). As shown in Figure 4B, for the 10-nm protein–AuNP complex, we observed a distinct broadening of absorption spectra in combination with enhanced absorption, and a significant bathochromic shift. Adsorption of proteins onto AuNPs cause the peak shift due to change in the optical properties of the sample medium. The size, aggregation state, and local dielectric environment together contribute toward broadening of absorption spectra for any NP–protein complex (Suhartono et al., 2018; Randika Perera et al., 2019). In case of 10-, 20-, and 5-nm AuNPs, we see a size-dependent broadening of the peak, suggesting that protein–AuNP complex formation is optimal the 10 nm and weaker in the latter two. Our observation for peak broadening and red shift confirms the interaction between Mtu SufB and AuNPs for all the sizes, supported by previous observations (Giri et al., 2014). The detailed mechanisms influencing minor peak broadening by the 5- and 20-nm AuNP–protein complexes are beyond the scope of this study. Next, for a quantitative determination of binding affinity and the thermodynamic parameters of AuNP–intein interaction, we performed ITC analysis.

To acquire insights into the binding thermodynamics of AuNPs with SufB intein, ITC was performed. Since Mtu SufB precursor protein gives rise to splicing and cleavage products under native conditions, ITC was conducted with purified Mtu SufB intein for precision in data analysis. The measurement of ITC relies on the fact that such interactions will be accompanied by a change in heat energy. From a single titration experiment, we can obtain information about the enthalpy of binding (ΔH) and the binding constant or the affinity (K_a). Using the equation ΔG = ΔH-TΔS, we calculated Gibb’s free energy (ΔG) and change in entropy (ΔS). The binding affinity (K_a) is calculated from the inverse of the slope of the binding curve (Supplementary Figure S4). The shape of the binding curve also depends on the unitless parameter c. A high c value indicates tight binding, and a low c value depicts low-affinity interaction (Lopez and Makhatadze, 2002). The c value was calculated using the formula c = K_a × [P] × n, where K_a is the binding constant, [P] is the total protein concentration, and n is the stoichiometry of interaction (n is obtained from software), to compare the binding affinity of AuNP of different sizes (5, 10, and 20 nm) with SufB intein.

A constant volume of AuNPs was titrated against the protein at a regular interval as highlighted in the Materials and Methods section. The reaction parameters for NP–protein interactions are shown in Supplementary Figure S4. It was observed that 10-nm AuNPs have the highest binding affinity (K_a = 2.3 × 10⁴ M⁻¹) toward SufB intein, followed by 5-nm AuNPs (K_a = 2.2 × 10³ M⁻¹) and 20-nm AuNPs (Ka = 2x10³ M⁻¹). The reaction parameters in terms of change in free energy (ΔG) were the most favorable for the 10-nm AuNPs (−5.97l kcal/mole), followed by 5-nm (−5.67l kcal/mole) and 20-nm (−5.2 kcal/mol) AuNPs (Table 1). The ΔG values, as well as the K_a values, show that overall AuNPs of all three sizes bind efficiently to the SufB intein system, additionally confirmed by the analysis of parameter c. Moreover, the binding constant values are comparable to the binding affinities of metals across various intein systems from previous studies of the literature (Table 2) (Zhang et al., 2009; Zhang et al., 2010b; Green et al., 2019). Although metals like Zn and Cu have an overall inhibitory effect upon interacting with inteins, the AuNP–Mtu SufB interaction is stimulatory for N-terminal cleavage and splicing reactions likely due to the thiophilic nature of AuNPs resulting in Cys1 activation. A thorough explanation of the said reaction is described in the forthcoming section (QM/MM analysis). Based on earlier research work, we can further predict that the AuNP–SufB intein binding interaction may be entropically driven owing to the endothermic nature of reaction (thermogram from Supplementary Figure S4), supported by a positive change in enthalpy (ΔH) as well as entropy (ΔS) (Supplementary Table S1) (Gorman and Amster, 1993; Zhang et al., 2009). Next, TEM analysis provided a visual confirmation for SufB intein adsorption on the AuNP surface.

After analyzing the affinity of SufB intein towards the gold nanoparticles, we confirmed the protein–nanoparticle interaction and intein adsorption on the nanoparticle surface by TEM analysis (Figure 5). Nanoparticles of 20 and 10 nm were considered for TEM as they displayed a significant increase in splicing and N-terminal cleavage reaction of SufB protein compared to 5-nm AuNP. After protein purification, SufB intein was allowed to interact with each of the nanoparticles, as mentioned in Materials and Methods.

In Figure 5Ai, spherical and uniformly distributed 20-nm AuNPs were observed. Approximately 2-nm protein shells were formed on the surface of 20-nm AuNP (Figures 5Aii,iii), which is absent in the case of untreated AuNPs. Likewise, protein adsorption and corona formation were noticed for 10-nm AuNPs (Figures 5Bii,iii). These results support our ITC results, revealing a tightly bound layer of SufB protein on the AuNP surface even after several washing steps. Next, we performed QM/MM analysis to gather mechanistic evidence on the AuNP effect on Mtu SufB specifically in the N-terminal cleavage site.

The intein active site formation is crucial for the initiation of the splicing reaction (Du et al., 2009; Eryilmaz et al., 2014; Mills et al., 2014; Friedel et al., 2019b). Intein folds specifically to bring together the two splice junctions and other assistive residues to form the active site. The two splice junctions (Figure 6) are, namely, N-terminal splice junction (site) (includes Gly-1 from N-extein and Cys1 from intein) and C-terminal splice junction (site) [includes terminal Asn from intein and Cys+1 from C-extein]. The first step of the splicing reaction involves the N-terminal splice site where the N-extein is linked to the intein by a thioester bond by the critical action of Cys1 (Nanda et al., 2020). Since our experimental results have shown AuNP-induced enhancement in the N-terminal cleavage reaction, the Gly-Cys system was chosen to calculate the energy barrier using QM/MM analysis, as described in Materials and Methods.

The schematic geometrical structure of the reactant and scheme diagram of reactants and tetrahedral intermediates is shown in Figure 7. The optimized geometrical coordinates of the structures as reactants and the transition state (TS) are given in the Supplementary Material (Pages S8-S12). During optimization, the dihedral angles of Gly-1, that is, 2C-5N-6C-7C and 5N-6C-7C-12N, were fixed at the values −121.4 and 156.5, respectively, as the surrounding protein is removed. The numbering for atoms is shown in Figure 7. Each frequency mode of the Gly-Cys system in TS has been given in Supplementary Table S2, where the presence of an imaginary mode represents that the system is in TS. The Gly-Cys system with Au is optimized (Pati et al., 2003), and TS was obtained with the aforementioned constraints and method (Materials and Methods section).

The energy difference of the reactants and TS provides the energy barrier, which is depicted in Table 3. In the vacuum, the energy barrier is found to be 0.26 eV in the presence of Au atom near by to the sulfur (of cysteine), which is comparatively much lower than the energy barrier value of 1.24 eV in the absence of the Au atom. For the realistic approach, we have performed the calculations in the solvent as well. We have used the vacuum-optimized structure of the system in the case of solvent because the calculations are computationally too expensive. However, a previous study has found that the difference in the energy between the vacuum optimized geometry and solvent optimized geometry is within the computational accuracy of 0.10 eV (Dearden, 2014). In our calculation (Table 3), the energy barrier in the case of solvent is quite different than that in the vacuum. With the inclusion of the solvent, the energy barrier is reduced from 1.24 to 0.85 eV in the absence of the Au atom. While in the case of Au atom–induced Gly-Cys system, the energy barrier is reduced from 0.26 to 0.10 eV. The reduction in the energy barrier due to solvent is associated with electrostatic screening as the interaction energy between the atoms is greatly reduced in the solvent. More importantly, in both the cases, that is, vacuum and solvent, we found that the induced Au atom reduces the energy barrier up to 0.98 eV (in vacuum) and 0.75 eV (in solvent), which possibly leads to an enhanced N-terminal cleavage reaction.

To elucidate the cause behind reduction in the energy barrier by Au, we investigated the electronic charge on the S (of catalytic cysteine-1), O (of carbonyl group of glycine-1), Au atoms as well as their respective nearest neighbor atoms. The Mulliken charge of the respective atoms is listed in Table 4. In this table, we have labeled the atom on the basis of our selected system as shown in Figure 7A. Table 4 shows that due to the presence of Au, the charge on other atoms, except S, is equivalent, for example, the charge on O is −0.44 (without Au) and −0.42 (with Au). There is a great decrement in the negative charge (from −0.62 to −0.28) on S, due to Au, and further, the electronic charge on the Au atom is highly negative (i.e., −0.52). The primary reason behind such changes is the lack of the π-back bonding phenomenon (Koch et al., 2019). In the π-back bonding, the electrons are channeled from the π-orbital of the ligands to the metal, in order to facilitate the bonding between ligands and the transition metal. Then the electrons from the d orbital of the transition metal are given back to the empty π* orbital of the ligand. In this process, generally, the metal atom reduces the negative charge, while the ligands enhance their negative charge. In the case of the Au atom, the d-orbital is fully filled; therefore, it does not participate in the π-back bonding process. Thus, due to the lack of π-back bonding, the Au atom contains more negative charge (−0.52), while the sulfur atom reduces the negative charge from −0.62 to −0.28. The decrement in the negative charge of the sulfur atom leads to the reduction in the repulsion between S and O atoms. The enhanced N-terminal cleavage and splicing reactions are likely due to the thiophilic nature of AuNPs that led to Cys1 activation and reduction of energy barrier in the N-cleavage site.