The constant evolution of new variants of SARS-CoV-2 is a substantial challenge to people from every sector, particularly those in health care and research and development in the areas of diagnostics, prophylactics, and therapeutics development, as well as policy makers and administrators. The virus was first observed in December 2019 in China and later spread throughout the globe causing a pandemic. However, continuous mutations in the genome of the virus have created several new variants among the circulating viruses in different geographical regions worldwide. These mutations have led to increased transmissibility, antibody evasion, and severity in patients. Several research studies have cited that the spike protein of the coronavirus is responsible for interacting with human cells to penetrate inside [1,2]; however, adaptive mutations accumulated in the gene responsible for the spike protein have allowed the virus to acclimatize and escape the host immune system. A number of variants have been isolated and identified worldwide; the World Health Organization classified them as variants of concern (alpha, beta, gamma, delta, and omicron) and variants of interest (VOIs; eta, iota, kappa, and lambda) [3].

A single mutation in a gene can lead to a change of an amino acid in a protein, which can drastically affect an organism’s ability in many ways, such as transmissibility, quick adaptability, stability evading the host immune system, and pathogenicity [4]. The availability of high throughput second generation sequencing technologies like next-generation sequencing in the last decade has helped in the timely sequencing of the genomes of SARS-CoV-2 variants to identify mutations occurring among new genetic variants. Rapid sequencing technology has not only facilitated sequencing of the viral genome but also helped accelerate the development of diagnostic tools, vaccines, and therapeutics for COVID-19. Furthermore, the availability of genome sequence data and databases helped the scientific community understand the virus through the molecular, biochemical, genome, and proteome analyses of the virus. However, the continuous evolution of SARS-CoV-2 compels the scientific community to continuously strive toward new and thorough understanding of the properties of the virus variants and develop counter strategies against the virus to safeguard humankind.

The isoelectric point (pI) of a protein is crucial in determining the physicochemical properties of the protein [5]. The exposure of charged amino acids on the protein surface to solvents, hydration, and dehydration also influences the pI of a protein [6,7]. Thus, evaluation of the pI of the spike proteins of wild-type and mutated variants is quintessential in understanding the influence of the spike protein on virus behavior and the transmission rate of viruses, as well as developing prophylactic and therapeutic agents. Additionally, the roles of posttranslational modification, phosphorylation, methylation, and alkylation also influence the pI of a protein, which cannot be ignored. Similarly, the genome of SARS-CoV-2 is prone to genetic evolution or genetic shift while adapting to a human host’s microenvironment. Such mutations result in the emergence of new variants that might have different characteristics compared to its ancestral strains. Therefore, in this study, we aim to adopt an in silico method to analyze the spike protein sequences of wild-type and other variants of SARS-CoV-2 to know their physicochemical, functional, and evolutionary properties, which might help in the surveillance of SARS-CoV-2 through a systematic understanding of the continuously evolving properties as well as to develop the diagnostic tools and standardization of prophylactics and therapeutics strategies.

The current COVID-19 pandemic caused by SARS-CoV-2 has clearly demonstrated the potential of the coronavirus to continuously evolve in the wild and transmit to new species including humans with varied physicochemical and virulence properties. The world has witnessed several mutants of this virus, with “VOIs” and “variants of concern” taking several millions lives. However, the situation seems to be under control because of the availability of vaccines recently from the beginning of the year 2021. However, it is quintessential to understand the constantly mutating property of viruses to establish effective and timely health care strategies to avoid the consequential economic loss or burden and safeguard humankind. Understanding life-threatening organism such as SARS-CoV-2 is a continuous and challenging process for the health care and scientific community as well as for policy makers and administrators. There are several methods to study and understand the disease-causing agents, of which the in silico analysis method has contributed enormously to make the scientific community’s tasks easier by reducing the required effort, time, and costs in recent years. Therefore, in this study, we have made an effort to understand the variants of concerns, especially how their spike proteins spread around the globe. The analysis of the physicochemical properties of spike proteins of the wild-type and mutated variants of SARS-CoV-2 revealed interesting features of the delta variant, such as pI, molecular weight, instability index, GRAVY, and aliphatic index (Table 2).

The pI of a protein is crucial to understanding its biochemical function [23] and dependent on the dissociation and constant of the ionizable amino acid groups. The major ionizable amino acid groups present in SARS-CoV-2 are arginine, aspartate, histidine, glutamate, cysteine, and lysine, which play an important role in defining the pI of a spike protein [24-26]. However, posttranslational modification influences the pI of a protein. The mutation of P681R in the delta variant [22] changed an amino acid from histidine to arginine, which sparked our curiosity to try to understand the potential impact of the change in amino acid on the properties of the spike protein in the alpha and delta variants. Interestingly, the pKa values of both amino acids greatly differed—histidine has a value of 6.0 (low basic) and arginine has a value 12.5 (high basic). Other specific mutations observed in the delta variant were also basic or aliphatic in nature (Table 1). Thus, if this property is substantial, then that would make the protein more basic. The mutation P681H in the alpha variant did not cause substantial changes in the behavior of virus, but the mutation P681R in the delta variant did cause substantial changes in the behavior of the virus in terms of transmissibility, pathogenicity, immune evasion, and the severity of the infection [22]. Conversely, a neutral mutation was observed in the D614G variant spike protein [27,28], where an acidic amino acid (D) was replaced with neutral one (G).

To see the effect of the pI on the biochemical properties, the wild-type and variant spike proteins were evaluated by predicting the 2D gel reference map, which interestingly demonstrated grouping except the separation of the delta protein on the map (Figure 2). The results of the physicochemical properties and 2D gel analysis revealed that the pI of a protein plays an important role in the behavior of proteins, especially where high pI turns protein more positive or aliphatic. Additionally, it is interesting to know that this property of protein is used in antibody preparation—to make a protein or its subunit more immunogenic [29,30]. Hence, we suppose that this property of proteins should be taken into consideration when designing and preparing prophylactic and therapeutic agents. Similarly, a recent study on the VOIs of SARS-CoV-2 virus particles that measured pI using chemical force microscope revealed VOIs have lower surface charge and hydrophobicity than the wild type, which might have played a role in VOIs having increased transmission ability [29]. Therefore, the pI of protein and the surface charge and hydrophobicity of viral particles are important factors to consider when designing prophylactics and therapeutics for any viral diseases [4,28,30,31].

Substantial changes in the physicochemical properties of the spike protein of wild-type SARS-CoV-2 and its variants has not been observed; however, a high number of phosphorylation sites (136) was observed in the beta variant and a low number (130) was observed in the delta variant. Meanwhile, a high number of N-glycosylation sites (20) was observed in the gamma variant and a low number (16) was observed in the delta variant. The total number of disulfide bonds among the wild type and variants did not vary much; however, it ranged from 14 to 15 and the number bonds were enough to give structural stability. Overall, the physicochemical properties did not differ markedly; however, the differences found were enough to influence the change in protein behavior and, subsequently, the behavior of the virus.

The antigenicity and immunogenicity scores of wild-type SARS-CoV-2 and its variants were interesting, especially the delta variant which showed high and low scores, respectively. Comparison of pI, antigenicity, and immunogenicity was made to evaluate the significance of the data; the P values were .007 between the alpha variant and wild type, .02 between the delta variant and wild type, and .02 between the delta and alpha variants. Overall, these P values suggest that the predicted values and the antigenic and immunogenic sequences are reliable. The number of exposed B-cell and cytotoxic T-lymphocyte epitopes were low in the delta variant compared to the wild-type and other variants of SARS-CoV-2. The exposed B-cell epitope sequence for the delta variant was SLGAENSVAYSN, and the change in epitope sequence showed that mutations did have an effect on the morphology of the virus.

The evolutionary relationship among the wild type and its variants was evaluated by constructing a phylogenetic tree using protein sequences. Interestingly, we found a clear grouping of the D614G, beta, and gamma variants, with bootstrap values of 100% for beta and gamma and 93% between D614G and beta/gamma. The delta variant was close to Rousettus bat coronavirus and MERS-CoV (Figure 3) with a bootstrap value of 68%. The alpha variant was close to the wild type with an 83% bootstrap value. Interestingly, we could observe a hybrid position of the delta variant between the wild type and the MERS-CoV and Rousettus bat coronavirus. Therefore, we suppose that the delta variant might be carrying characteristics from the wild type and other wild bat coronaviruses (Figure 3). In another interesting observation, the omicron variant seems to have evolved from the D614G variant with a bootstrap value 99%, as well as having imported some characteristics from the beta and gamma variants since omicron was also seen branching from these 2 variants with a 60% bootstrap value (Figure 3).

In conclusion, our study highlights that the accumulation of adaptive mutations in SARS-CoV-2 influenced the change in pI of the spike protein and, subsequently, the behavior of the virus. The prediction and comparison of the physicochemical properties of spike proteins of the wild type and its variants revealed that the delta variant displayed unique changes compared to the wild type. Evolutionary features showed a clear separation of the wild-type, alpha, and delta spike proteins and the grouping of the D614G, beta, and gamma spike proteins. Nevertheless, the continuous evolution of new SARS-CoV-2 strains demands further systematic understanding of its variants, which would not only help in developing the improvised rapid “antigen test” market but also in developing vaccines and therapeutics. Thus, more similar studies would unravel the important biochemical properties, evolutionary history, immunological behavior, and physiological properties of viruses.