Stabilization of apoflavodoxin by replacing hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or Gln

Knowledge of protein stability principles provides a means toincrease protein stability in a rational way. Here we explorethe feasibility of stabilizing proteins by replacing solvent-exposedhydrogen-bonded charged Asp or Glu residues by the neutral isostericAsn or Gln. The rationale behind this is a previous observationthat, in some cases, neutral hydrogen bonds may be more stablethat charged ones. We identified, in the apoflavodoxin fromAnabaena PCC 7119, three surface-exposed aspartate or glutamateresidues involved in hydrogen bonding with a single partnerand we mutated them to asparagine or glutamine, respectively.The effect of the mutations on apoflavodoxin stability was measuredby both urea and temperature denaturation. We observed thatthe three mutant proteins are more stable than wild-type (onaverage 0.43 kcal/mol from urea denaturation and 2.8°C froma two-state analysis of fluorescence thermal unfolding data).At high ionic strength, where potential electrostatic repulsionsin the acidic apoflavodoxin should be masked, the three mutantsare similarly more stable (on average 0.46 kcal/mol). To ruleout further that the stabilization observed is due to removalof electrostatic repulsions in apoflavodoxin upon mutation,we analysed three control mutants and showed that, when thecharged residue mutated to a neutral one is not hydrogen bonded,there is no general stabilizing effect. Replacing hydrogen-bondedcharged Asp or Glu residues by Asn or Gln, respectively, couldbe a straightforward strategy to increase protein stability.     

A simple dual selection for functionally active mutants of Plasmodium falciparum dihydrofolate reductase with improved solubility

Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications.     

Synthesis of 6‐Kestose using an Efficient β‐Fructofuranosidase Engineered by Directed Evolution

The β‐fructofuranosidase (Ffase) from Schwanniomyces occidentalis (Ffase‐Leu196 variant) was subjected to four cycles of directed evolution to enhance the transglycosylation activity for the synthesis of β‐(2 → 6) linked fructooligosaccharides (FOS). With a 5.5‐fold improvement in fructose transferase activity over the parental type and greater selectivity for the synthesis of 6‐kestose (up to 73% of the total FOS), the mutants doubled FOS synthesis to 168 g L.⁻¹ Whilst the F523V and H510P mutations were located at the C‐terminus of the protein, mutations Q78L and I203L were associated with the acidic catalytic triad where they modified its interactions with the surrounding residues, in turn varying the hydrolase and transferase rates.     

Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position

In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.     

Stepwise transplantation of an active site loop between heat-labile enterotoxins LT-II and LT-I and characterization of the obtained hybrid toxins

Members of the cholera toxin family, including Escherichia coli heat- labile enterotoxins LT-I and LT-II, catalyze the covalent modification of intracellular proteins by transfer of ADP-ribose from NAD to a specific arginine of the target protein. The ADP-ribosylating activity of these toxins is located in the A-subunit, for which LT-I and LT-II share a 63% sequence identity. The flexible loop in LT-I, ranging from residue 47 to 56, closes over the active site cleft. Previous studies have shown that point mutations in this loop have dramatic effects on the activity of LT-I. Yet, in LT-II the sequence of the equivalent loop differs at four positions from LT-I. Therefore five mutants of the active site loop were created by a stepwise replacement of the loop sequence in LT-I with virtually all the corresponding residues in LT- II. Since we discovered that LT-II had no activity versus the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) while LT-I does, our active site mutants most likely probe the NAD binding, not the arginine binding region of the active site. The five hybrid toxins obtained (Q49A, F52N, V53T, Q49V/F52N and Q49V/F52N/V53T) show (i) great differences in holotoxin assembly efficiency; (ii) decreased cytotoxicity in Chinese hamster ovary cells; and (iii) increased in vitro enzymatic activity compared with wild type LT-I. Specifically, the three mutants containing the F52N substitution display a greater Vmax for NAD than wild type LT-I. The enzymatic activity of the V53T mutant is significantly higher than that of wild type LT-I. Apparently this subtle variation at position 53 is beneficial, in contrast to several other substitutions at position 53 which previously had been shown to be deleterious for activity. The most striking result of this study is that the active site loop of LT- I, despite great sensitivity for point mutations, can essentially be replaced by the active site loop of LT-II, yielding an active 'hybrid enzyme' as well as 'hybrid toxin'.      

Tyrp1 Mutant Variants Associated with OCA3: Computational Characterization of Protein Stability and Ligand Binding

Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions.     

Structure-activity correlations in pentachlorobenzene oxidation by engineered cytochrome P450cam

We had reported engineering of the heme monooxygenase cytochrome P450cam from Pseudomonas putida with the F87W/Y96F/L244A/V247L mutations for the oxidation of pentachlorobenzene (PeCB), a recalcitrant environmental contaminant, to pentachlorophenol. In order to provide further insights into P450 structure, function and substrate recognition, we have determined the crystal structure of this 4-mutant without a substrate and its complex with PeCB. PeCB is bound face-on to the heme, with a weak Fe--Cl interaction. One PeCB chlorine is located in the cavity generated by the L244A mutation, in striking illustration of the role of this mutation in promoting PeCB binding. The structures also show that the P450(cam) oxygen-binding groove between G248 and T252 is flexible and can tolerate significant deviations from their conformations in the wild type without loss of enzyme activity. Analysis of the PeCB binding interactions led to introduction of the T101A mutation to enable the substrate to reorient during the catalytic cycle for more efficient oxidation. The resultant 5-mutant F87W/Y96F/T101A/L244A/V247L is 3-fold more active for PeCB oxidation than the 4-mutant. Polychlorinated benzene binding by the mutants and the partitioning between substrate oxidation and non-productive (uncoupling) side reactions are correlated with the structural data.     

Effects of non-conservative changes to tyrosine 76, a key DNA binding residue of DNase I, on phosphodiester bond cleavage and DNA hydrolysis selectivity

Non-conservative changes, consisting of Y76E, Y76L, Y76Q and Y76W, have been made to tyrosine 76, one of the key DNA binding residues in DNase I. Normally Y76 inserts into the minor groove of DNA and makes an unusual, hydrophobic, stacking interaction with one of the sugars. All four mutants bind to DNA more tightly than the wild type, but cut it more slowly as assessed by Kunitz assays. This gives a rather small decrease in the specificity constants (Vmax/K(m)) for the hydrolysis of DNA, which is roughly paralleled by the loss of activity towards the non-DNA small chromophoric substrate, thymidine-3',5'-di(p- nitrophenyl)phosphate. These non-conservative mutants, therefore, show different behaviour to Y76A and Y76G, studied previously [Doherty A.J., Worrall A.F. and Connolly B.A. (1995) J: Mol. Biol., 251, 366-377]. These two mutants both bind to and cut DNA poorly, resulting in large decreases in Vmax/K(m) values. However, they showed little reduction in rates with the chromophoric substrate. It is likely that the altered side chains in the non-conservative mutants are still able to interact productively with the DNA and contribute to the observed DNA distortion that is essential for efficient catalysis. However, these mutations disrupt the active site, most probably by interference with the hydrogen bonded Y76-E78-H134 triad. H134 is a critical hydrolytic residue of DNase I that is essential for catalysis. The DNA cleavage selectivity of the Y76E, Y76L, Y76Q and Y76W mutants were little altered as compared with the wild-type enzyme as measured using the cutting patterns of a 160 base-pair Escherichia coli Tyr T promoter DNA fragment. This confirms earlier observations, with Y76F, Y76A and Y76G, that showed that this tyrosine has little role in DNA cleavage specificity.      

Effects of engineered salt bridges on the stability of subtilisin BPN'

Variants designed using PROTEUS have been produced in an attemptto engineer stabilizing salt bridges into subtilisin BPN'. Allthe mutants constructed by site-directed mutagenesis were secretedby Bacillus subtillus, except L75K. Q19E, expressed as a singlevariant and also in a double variant, Q19E/Q271E, appears toform a stabilizing salt bridge based on X-ray crystal structuredetermination and differential scanning calorimeter measurements.Although the double mutant was found to be less thermodynamicallystable than the wild-type, it did exhibit an autolytic stabilityabout two fold greater under hydrophobic conditions. Four variants,A98K, S89E, V26R and L235R, were found to be nearly identicalto wild-type in thermal stability, indicative of stable structureswithout evidence of salt bridge formation. Variants Q271E, V51Kand T164R led to structures that resulted in varying degreesof thermodynamic and autolytic instability. A computer-modelinganalysis of the PROTEUS predictions reveals that the low percentageof salt bridge formation is probably due to an overly simplisticelectrostatic model, which does not account for the geometryof the pairwise interactions.     

Rational stabilization of the C-LytA affinity tag by protein engineering

The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing to its affinity for choline and analogs, it is regularly used as an affinity tag for the purification of proteins in a single chromatographic step. In an attempt to build a robust variant against thermal denaturation, we have engineered several salt bridges on the protein surface. All the stabilizing mutations were pooled in a single variant, C-LytAm7, which contained seven changes: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displays a 7 degrees C thermal stabilization compared with the wild-type form, together with a complete reversibility upon heating and a higher kinetic stability. Moreover, the accumulation of intermediates in the unfolding of C-LytA is virtually abolished for C-LytAm7. The differences in stability become more evident when the proteins are bound to a DEAE-cellulose affinity column, as most of wild-type C-LytA is denatured at approximately 65 degrees C, whereas C-LytAm7 may stand temperatures up to 90 degrees C. Finally, the change in the isoelectric point of C-LytAm7 enhances its solubility at acidic pHs. Therefore, C-LytAm7 behaves as an improved affinity tag and supports the engineering of surface salt bridges as an effective approach for protein stabilization.     

Improvement of Oxidative and Thermostability of N-Carbamyl-D-Amino Acid Amidohydrolase by Directed Evolution

N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), whichis currently employed in the industrial production of unnaturalD-amino acid in conjunction with D-hydantoinase, has low oxidativeand thermostability. We attempted the simultaneous improvementof the oxidative and thermostability of N-carbamoylase fromAgrobacterium tumefaciens NRRL B11291 by directed evolutionusing DNA shuffling. In a second generation of evolution, thebest mutant 2S3 with improved oxidative and thermostabilitywas selected, purified and characterized. The temperature atwhich 50% of the initial activity remains after incubation for30 min was 73°C for 2S3, whereas it was 61°C for wild-typeenzyme. Treatment of wild-type enzyme with 0.2 mM hydrogen peroxidefor 30 min at 25°C resulted in a complete loss of activity,but 2S3 retained about 79% of the initial activity under thesame conditions. The K_m value of 2S3 was estimated to be similarto that of wild-type enzyme; however k_cat was decreased, leadingto a slightly reduced value of k_cat/K_m, compared with wild-typeenzyme. DNA sequence analysis revealed that six amino acid residueswere changed in 2S3 and substitutions included Q23L, V40A, H58Y,G75S, M184L and T262A. The stabilizing effects of each aminoacid residue were investigated by incorporating mutations individuallyinto wild-type enzyme. Q23L, H58Y, M184L and T262A were foundto enhance both oxidative and thermostability of the enzymeand of them, T262A showed the most significant effect. V40Aand G75S gave rise to an increase only in oxidative stability.The positions of the mutated amino acid residues were identifiedin the structure of N-carbamoylase from Agrobacterium sp. KNK712 and structural analysis of the stabilizing effects of eachamino acid substitution was also carried out.     

Mapping the folding pathway of the transmembrane protein DsbB by protein engineering

The four-helical transmembrane protein DsbB (disulfide bond reducing protein B) folds and unfolds reversibly in mixed anionic/non-ionic micelles, consisting of an unfolding intermediate I and a rate-limiting transition state (TS) between I and the denatured state D. Here, I describe the analysis of the folding behavior of 12 different alanine-scanning mutants of DsbB. For all mutants, TS is as compact as D and there is an accelerating increase in compaction as the protein proceeds to I and the native state. This unusual pattern of consolidation may reflect significant amounts of secondary structure in D, analogous to a classical folding intermediate. Unexpectedly, an increase in apolar surface area upon mutation is stabilizing whereas an increase in polar surface area is destabilizing. This effect is probably dominated by the effect of the mutations on the structure of the denatured state. I observe clear Hammond postulate behavior, in which a destabilization of I moves it closer to D. Φ-Value analysis indicates that in TS, a folding nucleus consisting of two to three residues with Φ-values of > 0.5 forms at one end of the transmembrane helices, which expands to include residues closer to the middle of the protein in I. Thus, folding proceeds from a highly polarized starting point.     

Effect of mutations on the dimer stability and the pH optimum of the human foamy virus protease

To explore the role of residues being close to the catalytic aspartates in the higher pH optimum and in the lower dimer stability of human foamy virus (HFV) protease (PR) in comparison with human immunodeficiency virus type 1 (HIV-1) protease, single (Q8R, H22L, S25T, T28D) and double (Q8R-T28D, H22L-T28D) mutants were created based on sequence alignments and on the molecular model of HFV PR. The wild-type and mutant enzymes were expressed in fusion with maltose binding protein in Escherichia coli and the fusion proteins were purified by affinity chromatography. Specificity constant of most mutants was lower, but the value of Q8R-T28D double mutant enzyme was higher than that of the wild-type HFV PR. Furthermore, urea denaturation at two pH values and pH optimum values showed an increased stability and pH optimum for most mutants. These results suggest that the mutated residues may not be responsible for the higher pH optimum of HFV PR, but they may contribute to the lower dimer stability as compared with that of HIV-1 PR.     

Reconstructing a Flavodoxin Oxidoreductase with Early Amino Acids

Primitive proteins are proposed to have utilized organic cofactors more frequently than transition metals in redox reactions. Thus, an experimental validation on whether a protein constituted solely by early amino acids and an organic cofactor can perform electron transfer activity is an urgent challenge. In this paper, by substituting “late amino acids (C, F, M, T, W, and Y)” with “early amino acids (A, L, and V)” in a flavodoxin, we constructed a flavodoxin mutant and evaluated its characteristic properties. The major results showed that: (1) The flavodoxin mutant has structural characteristics similar to wild-type protein; (2) Although the semiquinone and hydroquinone flavodoxin mutants possess lower stability than the corresponding form of wild-type flavodoxin, the redox potential of double electron reduction E_m,7 (fld) reached −360 mV, indicating that the flavodoxin mutant constituted solely by early amino acids can exert effective electron transfer activity.     

How to improve nature: study of the electrostatic properties of the surface of alpha-lactalbumin

It was recently shown that alpha-lactalbumin interacts with histones and simple models of histone proteins such as positively charged polyamino acids, suggesting that some fundamental aspects of the protein surface electrostatics may come into play. In the present work, the energies of charge-charge interaction in apo- and Ca(2+)-loaded alpha-lactalbumin were calculated using a Tanford-Kirkwood algorithm with either solvent accessibility correction or using a finite difference Poisson-Boltzmann method. The analysis revealed two major regions of alpha-lactalbumin that possessed highly unfavorable electrostatic potentials: (a) the Ca(2+)-binding loop and its neighboring residues and (b) the N-terminal region of the protein. Several individual mutants were prepared to neutralize specific individual surface acidic amino acids at both the N-terminus and Ca(2+)-binding loop of bovine alpha-lactalbumin. These mutants were characterized by intrinsic fluorescence, differential scanning microcalorimetry and circular dichroism. The structural and thermodynamic data agree in every case with the theoretical predictions, confirming that the N-terminal region is very sensitive to changes in charge. For example, desMet D14N mutation destabilizes protein and decreases its calcium affinity. On the other hand, desMet E1M and desMet D37N substitutions increase the thermal stability and calcium affinity. The Met E1Q is characterized by a marked increase in protein stability, whereas desMet E7Q and desMet E11L display a slight increase in calcium affinity and thermal stability. Examination of the unfavorable energy contributed by Glu1 and the energetically favorable consequences of neutralizing this residue suggests that nature may have made an error with bovine alpha-lactalbumin from the viewpoint of stabilizing structure and conformation.     

Analysis of the effect of accumulation of amino acid replacements on activity of 3-isopropylmalate dehydrogenase from Thermus thermophilus

A newly selected cold-adapted mutant 3-isopropylmalate dehydrogenase(IPMDH) from a random mutant library was a double mutant containingthe mutations I11V and S92F that were found in cold-adaptedmutant IPMDHs previously isolated. To elucidate the effect ofeach mutation on enzymatic activity, I11V and six multiple mutantIPMDHs were constructed and analyzed. All of the multiple mutantIPMDHs were found to be improved in catalytic activity at moderatetemperatures by increasing the k_cat with a simultaneous increaseof K_m for the coenzyme NAD⁺. k_cat was improved by a decreasein the activation enthalpy, H. The multiple mutants did notshow large reduction in thermal stability, and one of them showedenhanced thermal stability. Mutation from I11 to V was revealedto have a stabilizing effect. Mutants showed increased thermalstability when the mutation I11V was combined. This indicatesthat it is possible to construct mutants with enhanced thermalstability by combining stabilizing mutation. No additivity wasobserved for the thermodynamic properties of catalytic reactionin the multiple mutant IPMDHs, implying that the structuralchanges induced by the mutations were interacting with eachother. This indicates that careful and detailed tuning is requiredfor enhancing activity in contrast to thermal stability.     

Possible Link between Higher Transmissibility of Alpha,Kappa and Delta Variants of SARS-CoV-2 and Increased Structural Stability of Its Spike Protein and hACE2 Affinity

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak in December 2019 has caused a global pandemic. The rapid mutation rate in the virus has created alarming situations worldwide and is being attributed to the false negativity in RT-PCR tests. It has also increased the chances of reinfection and immune escape. Recently various lineages namely, B.1.1.7 (Alpha), B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.617.3 have caused rapid infection around the globe. To understand the biophysical perspective, we have performed molecular dynamic simulations of four different spikes (receptor binding domain)-hACE2 complexes, namely wildtype (WT), Alpha variant (N501Y spike mutant), Kappa (L452R, E484Q) and Delta (L452R, T478K), and compared their dynamics, binding energy and molecular interactions. Our results show that mutation has caused significant increase in the binding energy between the spike and hACE2 in Alpha and Kappa variants. In the case of Kappa and Delta variants, the mutations at L452R, T478K and E484Q increased the stability and intra-chain interactions in the spike protein, which may change the interaction ability of neutralizing antibodies to these spike variants. Further, we found that the Alpha variant had increased hydrogen interaction with Lys353 of hACE2 and more binding affinity in comparison to WT. The current study provides the biophysical basis for understanding the molecular mechanism and rationale behind the increase in the transmissivity and infectivity of the mutants compared to wild-type SARS-CoV-2.     

细胞色素P450 BM-3羟基化吲哚能力的半理性改造

为进一步改造细胞色素P450 BM-3酶对吲哚的羟基化能力,以P450 BM-3结构与功能关系的推测为指导,选择突变酶P450 BM-3 （A74G/F87V/L188Q/E435T）为父本,在可能影响P450 BM-3催化吲哚羟基化区域选择性的D168位点进行定点饱和突变,根据全细胞催化产物颜色及组成进行筛选,得到了产物组成、酶动力学性质与父本不同的两个突变酶。突变酶D168W的吲哚羟基化产物中90%是靛玉红,而另一个突变酶D168R的产物中87%是靛蓝,产物组成均不同于亲本。在催化吲哚羟基化时,D168W的k_cat与父本相当,但K_m却是父本的4.8倍,催化活力只有父本的20%;而D168R的k_cat是父本的1.9倍,K_m是父本的82%,催化活力比父本提高了1.37倍。结果表明,在E435T突变上叠加D168位氨基酸残基突变对酶的催化性质产生了单一位点突变所不具有的协同效应,对酶催化的区域选择性和催化活力都有显著影响,以致改变了催化产物组成。这种基于知识的半理性定向进化方法由于是在关键位点进行突变,因此突变目的性强、突变效果显著。     

PoPMuSiC, an algorithm for predicting protein mutant stability changes. Application to prion proteins

A novel tool for computer-aided design of single-site mutationsin proteins and peptides is presented. It proceeds by performingin silico all possible point mutations in a given protein orprotein region and estimating the stability changes with linearcombinations of database-derived potentials, whose coefficientsdepend on the solvent accessibility of the mutated residues.Upon completion, it yields a list of the most stabilizing, destabilizingor neutral mutations. This tool is applied to mouse, hamsterand human prion proteins to identify the point mutations thatare the most likely to stabilize their cellular form. The selectedmutations are essentially located in the second helix, whichpresents an intrinsic preference to form ß-structures,with the best mutations being T183     

Molecular Dynamics Studies on the Structural Characteristics for the Stability Prediction of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects the COVID-19 pandemic in the world. The spike protein of the various proteins encoded in SARS-CoV-2 binds to human ACE2, fuses, and enters human cells in the respiratory system. Spike protein, however, is highly variable, and many variants were identified continuously. In this study, Korean mutants for spike protein (D614G and D614A-C terminal domain, L455F and F456L-RBD, and Q787H-S2 domain) were investigated in patients. Because RBD in spike protein is related to direct interaction with ACE2, almost all researches were focused on the RBD region or ACE2-free whole domain region. The 3D structure for spike protein complexed with ACE2 was recently released. The stability analysis through RBD distance among each spike protein chain and the binding free energy calculation between spike protein and ACE2 were performed using MD simulation depending on mutant types in 1-, 2-, and 3-open-complex forms. D614G mutant of CT2 domain, showing to be the most prevalent in the global pandemic, showed higher stability in all open-complex forms than the wild type and other mutants. We hope this study will provide an insight into the importance of conformational fluctuation in the whole domain, although RBD is involved in the direct interaction with ACE2.