3.1 Divergence in SARS-CoV-2
To check the difference homologous positions of sequence of SARS-CoV-2 from SARS, we analyzed the BLOCK format  to observe the evolutionary properties like MCR, MDR, DHP and R ratio.
In case of maximum conserve ratio (MCR) (Table 1), it showed charged, uncharged polar and hydrophobic residues in SARS-CoV-2, whereas hydrophobic and neutral polar residues in SARS. In case of maximum diverse residue (MDR), it showed similar results like MCR. Dominant hetero pair also showed higher abundance of charged residues in SARS-CoV-2, but it is lacks in SARS. The R ratio (NCS:CS) showed lower value in every protein of SARS-CoV-2 than SARS.
3.2 Effect of charged residues on SARS-CoV-2 sequence
Here D, E, H, R, K took as a charged residues and C, S, T, N, Q, Y, W took as polar uncharged residues. Amino acid compositions were calculated from the non-block format whereas block format was used to calculate disorder forming residues (Dis), Zimmerman bulkiness (ZB), aliphatic index (AI), Grantham polarity (GB) and order forming residues (Ofr) etc. Is there a preference for amino acids in SARS-CoV-2 relative to SARS? To find that answer we calculate all those physicochemical properties.
Spike proteins showed higher abundance (Fig. 1a) of charged residues (except D) in SARS-CoV-2. Polar residues showed higher abundance (except C, S) in SARS. Other proteins (membrane proteins, nucleoproteins, ORF proteins) showed almost similar results, where charged residues showed positive MRA in SARS-CoV-2 (Fig. 1c, e, g). Polar and hydrophobic residues showed negative MRA in SARS-CoV-2 which means those residues have higher abundance in SARS proteins. But when we check the polarity (Fig. 1b, d, f, h) of those proteins by Grantham polarity, it showed the high value in SARS-CoV-2 than SARS. The number of disorder forming residues (Dis) has higher abundance in SARS-CoV-2 than SARS. In the other hand, order forming residues (Orf) showed negative MRA in SARS-CoV-2. Due to the latter, Zimmerman bulkiness and aliphatic index are also higher in SARS-CoV-2
GRAVY (grand average of hydropathy) is calculated by adding the hydropathy value  for each residue and dividing by the length of the protein sequence. The lower value of GRAVY indicates the hydrophilic nature of SARS-CoV-2 (Table 2).
3.3 Charged residues on surface of SARS-CoV-2
The increasing of charged residues in SARS-CoV-2 protein sequences gives a clue that they might have effect on structure. To check, those structures of protease from SARS-CoV-2 (5R81) and SARS (1P9U) have been analyzed.
Acidic residues showed similar abundance in the core of SARS-CoV-2 and SARS, but it showed higher abundance in the surface of SARS-CoV-2 (Table 3). Polar residues also showed higher abundance in the surface of SARS-CoV-2. But hydrophobic residues showed slightly low abundance in the surface of SARS-CoV-2 than SARS.
3.4 Charged residues in helix of SARS-CoV-2
Charged residues showed higher abundance in the helix of SARS-CoV-2 (Table 4). SARS-CoV-2 significantly increased the stability by stabilizing it’s helix structure. In case of SARS, charged residues are mostly present in coil rather than the helix. Polar residues also present in high number at the coil of SARS, whereas in SARS-CoV-2 polar residues are mostly present in strand. Hydrophobic residues also have higher abundance in the helix of SARS-CoV-2 and in coil of SARS.
3.5 Stability of SARS-CoV-2
Salt bridges have a huge effect on protein stability [41, 42]. Charged residues are participating in the formation of salt bridges. Normally two types of salt bridges are found in proteins . The increasing number of charged residues of SARS-CoV-2 indicates that charged residues might have an effect on salt bridge formation to gain more stability.
Results of salt bridges (Table 5) showed how it plays an important role in SARS-CoV-2 stability. The number of isolated salt bridges is high in SARS, but the energies of those salt bridges lower than SARS-CoV-2. In SARS-CoV-2, the salt bridge contributes -8.47 kcal/mol/SB energy, whereas it is -3.95 kcal/mol/SB energy in SARS. The numbers of network salt bridges are same in both SARS-CoV-2 and SARS. Both possess only one network salt bridge. But a huge energy difference identified between them. It showed -9.56 kcal/mol energy in SARS and -21.19 kcal/mol in SARS-CoV-2. It is very interesting that a single salt bridge contributes such high energy. Further investigation showed that the network salt bridge of SARS-CoV-2 is not a normal network salt bridge; it is a cyclic network salt bridge (R131-E290:K137-E290:R131-D197:K137-D197:R131-D289). ; We know that the desolvation (ΔΔGdslv) energy is always positive and the bridge (ΔΔGbrd) energy is always negative. They almost nullified each other. So, the background (ΔΔGbac) energy play an important role in gaining the total net energy (ΔΔGnet). The higher value of background energy in SARS-CoV-2 indicates that the background or surrounding environment of those salt bridges has an effect on the stability of salt bridges. So, the microenvironment of isolated and network salt bridges has been analyzed.
3.6 Effect of microenvironment residues in SARS-CoV-2
Due to the high number of isolated salt bridges (ISB) in SARS, they have more surrounding residues than SARS-CoV-2 (Table 6). But the total energies of the ISB microenvironment are high in SARS-CoV-2. SARS-CoV-2 has engineered its microenvironment for more stability. Thr 292 in SARS-CoV-2 contributes the highest energy i.e. -24.39 kj/mol. In case of network salt bridge microenvironment, SARS-CoV-2 has high number of residues and energies. Most of the microenvironment residues are present in coil followed by strand and helix.