Computational Estimation of Residues Involving Resistance to the SARS-CoV-2 Main Protease Inhibitor Ensitrelvir Based on Virtual Alanine Scan of the Active Site.

Ensitrelvir is a noncovalent inhibitor of the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2. Acquisition of drug resistance in virus-derived proteins is a serious therapeutic concern, and drug resistance occurs due to amino acid mutations. In this study, we computationally constructed 24 mutants, in which one residue around the active site was replaced with alanine and performed molecular dynamics simulations to the complex of Mpro and ensitrelvir to predict the residues involved in drug resistance. We evaluated the changes in the entire protein structure and ligand configuration in each of these mutants and estimated which residues were involved in ensitrelvir recognition. This method is called a virtual alanine scan. In nine mutants (S1A, T26A, H41A, M49A, L141A, H163A, E166A, V186A, and R188A), although the entire protein structure and catalytic dyad (cysteine (Cys)145 and histidine (His)41) were not significantly moved, the ensitrelvir configuration changed. Thus, it is considered that these mutants did not recognize ensitrelvir while maintaining Mpro enzymatic activities, and Ser1, Thr26, His41, Met49, Leu141, His163, Glu166, Val186, and Arg188 may be related to ensitrelvir resistance. The ligand shift noted in M49A was similar to that observed in M49I, which has been shown to be experimentally ensitrelvir resistant. These findings suggest that our research approach can predict mutations that incite drug resistance.

Structural Impact Assessment of Cytochrome P450 2A13 Polymorphisms Using Molecular Dynamics Simulations.

One of the members of CYP, a monooxygenase, CYP2A13 is involved in the metabolism of nicotine, coumarin, and tobacco-specific nitrosamine. Genetic polymorphisms have been identified in CYP2A13, with reported loss or reduction in enzymatic activity in CYP2A13 allelic variants. This study aimed to unravel the mechanism underlying the diminished enzymatic activity of CYP2A13 variants by investigating their three-dimensional structures through molecular dynamics (MD) simulations. For each variant, MD simulations of 1000 ns were performed, and the obtained results were compared with those of the wild type. The findings indicated alterations in the interaction with heme in CYP2A13.4, .6, .8, and .9. In the case of CYP2A13.5, observable effects on the helix structure related to the interaction with the redox partner were identified. These conformational changes were sufficient to cause a decrease in enzyme activity in the variants. Our findings provide valuable insights into the molecular mechanisms associated with the diminished activity in the CYP2A13 polymorphisms.

Identification of the Most Impactful Asparagine Residues for γS-Crystallin Aggregation by Deamidation.

Crystallin aggregation in the eye lens is involved in the pathogenesis of cataracts. The aggregation is considered to be promoted by non-enzymatic post-translational modifications, such as the deamidation and stereoinversion of amino acid residues. Although in a previous study, the deamidated asparagine residues were detected in γS-crystallin in vivo, it is unclear which deamidated residues have the most impact on the aggregation under physiological conditions. In this study, we investigated the deamidation impacts of all Asn residues in γS-crystallin for the structural and aggregation properties utilizing deamidation mimetic mutants (N14D, N37D, N53D, N76D, and N143D). The structural impacts were investigated using circular dichroism analysis and molecular dynamics simulations, and the aggregation properties were analyzed by gel filtration chromatography and spectrophotometric methods. No significant structural impacts of all mutations were detected. However, the N37D mutation decreased thermal stability and changed some intermolecular hydrogen-bond formations. Aggregation analysis indicated that the superiority of the aggregation rate in each mutant varied with temperature. Deamidation at any Asn residues promoted γS-crystallin aggregation, and the deamidation at Asn37, Asn53, and Asn76 were suggested to be the most impactful in the formation of insoluble aggregations.

Functional Characterization of 12 Dihydropyrimidinase Allelic Variants in Japanese Individuals for the Prediction of 5-Fluorouracil Treatment-Related Toxicity.

The drug 5-fluorouracil (5-FU) is the first-choice chemotherapeutic agent against advanced-stage cancers. However, 10% to 30% of treated patients experience grade 3 to 4 toxicity. The deficiency of dihydropyrimidinase (DHPase), which catalyzes the second step of the 5-FU degradation pathway, is correlated with the risk of developing toxicity. Thus, genetic polymorphisms within DPYS, the DHPase-encoding gene, could potentially serve as predictors of severe 5-FU-related toxicity. We identified 12 novel DPYS variants in 3554 Japanese individuals, but the effects of these mutations on function remain unknown. In the current study, we performed in vitro enzymatic analyses of the 12 newly identified DHPase variants. Dihydrouracil or dihydro-5-FU hydrolytic ring-opening kinetic parameters, Km and Vmax , and intrinsic clearance (CLint = Vmax /Km ) of the wild-type DHPase and eight variants were measured. Five of these variants (R118Q, H295R, T418I, Y448H, and T513A) showed significantly reduced CLint compared with that in the wild-type. The parameters for the remaining four variants (V59F, D81H, T136M, and R490H) could not be determined as dihydrouracil and dihydro-5-FU hydrolytic ring-opening activity was undetectable. We also determined DHPase variant protein stability using cycloheximide and bortezomib. The mechanism underlying the observed changes in the kinetic parameters was clarified using blue-native polyacrylamide gel electrophoresis and three-dimensional structural modeling. The results suggested that the decrease or loss of DHPase enzymatic activity was due to reduced stability and oligomerization of DHPase variant proteins. Our findings support the use of DPYS polymorphisms as novel pharmacogenomic markers for predicting severe 5-FU-related toxicity in the Japanese population. SIGNIFICANCE STATEMENT: DHPase contributes to the degradation of 5-fluorouracil, and genetic polymorphisms that cause decreased activity of DHPase can cause severe toxicity. In this study, we performed functional analysis of 12 DHPase variants in the Japanese population and identified 9 genetic polymorphisms that cause reduced DHPase function. In addition, we found that the ability to oligomerize and the conformation of the active site are important for the enzymatic activity of DHPase.

Functional Characterization of 29 Cytochrome P450 4F2 Variants Identified in a Population of 8380 Japanese Subjects and Assessment of Arachidonic Acid ω-Hydroxylation.

Cytochrome P450 4F2 (CYP4F2) is an enzyme that is involved in the metabolism of arachidonic acid (AA), vitamin E and K, and xenobiotics including drugs. CYP4F2*3 polymorphism (rs2108622; c.1297G>A; p.Val433Met) has been associated with hypertension, ischemic stroke, and variation in the effectiveness of the anticoagulant drug warfarin. In this study, we characterized wild-type CYP4F2 and 28 CYP4F2 variants, including a Val433Met substitution, detected in 8380 Japanese subjects. The CYP4F2 variants were heterologously expressed in 293FT cells to measure the concentrations of CYP4F2 variant holoenzymes using carbon monoxide-reduced difference spectroscopy, where the wild type and 18 holoenzyme variants showed a peak at 450 nm. Kinetic parameters [Vmax , substrate concentration producing half of Vmax (S50 ), and intrinsic clearance (CL int ) as Vmax /S50 ] of AA ω-hydroxylation were determined for the wild type and 21 variants with enzyme activity. Compared with the wild type, two variants showed significantly decreased CL int values for AA ω-hydroxylation. The values for seven variants could not be determined because no enzymatic activity was detected at the highest substrate concentration used. Three-dimensional structural modeling was performed to determine the reason for reduced enzymatic activity of the CYP4F2 variants. Our findings contribute to a better understanding of CYP4F2 variant-associated diseases and possible future therapeutic strategies. SIGNIFICANCE STATEMENT: CYP4F2 is involved in the metabolism of arachidonic acid and vitamin K, and CYP4F2*3 polymorphisms have been associated with hypertension and variation in the effectiveness of the anticoagulant drug warfarin. This study presents a functional analysis of 28 CYP4F2 variants identified in Japanese subjects, demonstrating that seven gene polymorphisms cause loss of CYP4F2 function, and proposes structural changes that lead to altered function.

Effects of Active-Center Reduction of Plant-Type Ferredoxin on Its Structure and Dynamics: Computational Analysis Using Molecular Dynamics Simulations.

"Plant-type" ferredoxins (Fds) in the thylakoid membranes of plants, algae, and cyanobacteria possess a single [2Fe-2S] cluster in active sites and mediate light-induced electron transfer from Photosystem I reaction centers to various Fd-dependent enzymes. Structural knowledge of plant-type Fds is relatively limited to static structures, and the detailed behavior of oxidized and reduced Fds has not been fully elucidated. It is important that the investigations of the effects of active-center reduction on the structures and dynamics for elucidating electron-transfer mechanisms. In this study, model systems of oxidized and reduced Fds were constructed from the high-resolution crystal structure of Chlamydomonas reinhardtii Fd1, and three 200 ns molecular dynamics simulations were performed for each system. The force field parameters of the oxidized and reduced active centers were independently obtained using quantum chemical calculations. There were no substantial differences in the global conformations of the oxidized and reduced forms. In contrast, active-center reduction affected the hydrogen-bond network and compactness of the surrounding residues, leading to the increased flexibility of the side chain of Phe61, which is essential for the interaction between Fd and the target protein. These computational results will provide insight into the electron-transfer mechanisms in the Fds.

Functional Characterization of 40 CYP3A4 Variants by Assessing Midazolam 1'-Hydroxylation and Testosterone 6ß-Hydroxylation.

CYP3A4 is among the most abundant liver and intestinal drug-metabolizing cytochrome P450 enzymes, contributing to the metabolism of more than 30% of clinically used drugs. Therefore, interindividual variability in CYP3A4 activity is a frequent cause of reduced drug efficacy and adverse effects. In this study, we characterized wild-type CYP3A4 and 40 CYP3A4 variants, including 11 new variants, detected among 4773 Japanese individuals by assessing CYP3A4 enzymatic activities for two representative substrates (midazolam and testosterone). The reduced carbon monoxide-difference spectra of wild-type CYP3A4 and 31 CYP3A4 variants produced with our established mammalian cell expression system were determined by measuring the increase in maximum absorption at 450 nm after carbon monoxide treatment. The kinetic parameters of midazolam and testosterone hydroxylation by wild-type CYP3A4 and 29 CYP3A4 variants (K m , k cat , and catalytic efficiency) were determined, and the causes of their kinetic differences were evaluated by three-dimensional structural modeling. Our findings offer insight into the mechanism underlying interindividual differences in CYP3A4-dependent drug metabolism. Moreover, our results provide guidance for improving drug administration protocols by considering the information on CYP3A4 genetic polymorphisms. SIGNIFICANCE STATEMENT: CYP3A4 metabolizes more than 30% of clinically used drugs. Interindividual differences in drug efficacy and adverse-effect rates have been linked to ethnicity-specific differences in CYP3A4 gene variants in Asian populations, including Japanese individuals, indicating the presence of CYP3A4 polymorphisms resulting in the increased expression of loss-of-function variants. This study detected alterations in CYP3A4 activity due to amino acid substitutions by assessing the enzymatic activities of coding variants for two representative CYP3A4 substrates.

(S)-Erypoegin K, an isoflavone isolated from Erythrina poeppigiana, is a novel inhibitor of topoisomerase IIα: Induction of G2 phase arrest in human gastric cancer cells.

Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has a single chiral carbon in its structure and exists naturally as a racemic mixture. Our previous study showed (S)-erypoegin K selectively exhibits potent anti-proliferative and apoptosis-inducing activity against human leukemia HL-60 cells. To identify the target molecule of (S)-erypoegin K, we employed the human cancer cell panel analysis (termed JFCR39) coupled with a drug sensitivity database of pharmacologically well-characterized drugs for comparison using the COMPARE algorithm. (S)-erypoegin K exhibited a similar profile to that of etoposide, suggesting the molecular target for erypoegin K may be topoisomerase II (Topo II). Subsequent experiments using purified human Topo IIα established that the (S)-isomer selectively stabilizes the cleavage complex composed of double-stranded plasmid DNA and the enzyme. Moreover, (S)-erypoegin K inhibited decatenation of kinetoplast DNA. Molecular docking studies clearly indicated specific binding of the (S)-isomer to the active site of Topo IIα involving hydrogen bonds that help stabilize the cleavage complex. (S)-erypoegin K displayed potent cytotoxic activity against two human gastric cancer cells GCIY and MKN-1 with IC50 values of 0.270 and 0.327 µM, respectively, and induced enzyme activities of caspase 3 and 9. Cell cycle analysis showed marked cell cycle arrest at G2 phase in both cell lines. (S)-erypoegin K also displayed significant antitumor activity toward GCIY xenografted mice. The present study suggests (S)-erypoegin K acts as a Topo II inhibitor to block the G2/M transition of cancer cells.

Theoretical Studies on the Effect of Isomerized Aspartic Acid Residues on the Three-Dimensional Structures of Bovine Pancreatic Ribonucleases A.

Isomerized aspartic acid (Asp) residues have previously been identified in various aging tissues, and are suspected to contribute to age-related diseases. Asp-residue isomerization occurs nonenzymatically under physiological conditions, resulting in the formation of three types of isomerized Asp (i.e., L-isoAsp, D-Asp, and D-isoAsp) residues. Asp-residue isomerization often accelerates protein aggregation and insolubilization, making structural biology analyses difficult. Recently, Sakaue et al. reported the synthesis of a ribonuclease A (RNase A) in which Asp121 was artificially replaced with different isomerized Asp residues, and experimentally demonstrated that the enzymatic activities of these artificial mutants were completely lost. However, their structural features have not yet been elucidated. In the present study, the three-dimensional (3D) structures of these artificial-mutant RNases A were predicted using molecular dynamics (MD) simulations. The 3D structures of wild-type and artificial-mutant RNases A were converged by 3000-ns MD simulations. Our computational data show that the structures of the active site and the formation frequencies of the appropriate catalytic dyad structures in the artificial-mutant RNases A were quite different from wild-type RNase A. These computational findings may provide an explanation for the experimental data which show that artificial-mutant RNases A lack enzymatic activity. Herein, MD simulations have been used to evaluate the influences of isomerized Asp residues on the 3D structures of proteins.

Virtual Alanine Scan of the Main Protease Active Site in Severe Acute Respiratory Syndrome Coronavirus 2.

Recently, inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) have been proposed as potential therapeutic agents for COVID-19. Studying effects of amino acid mutations in the conformation of drug targets is necessary for anticipating drug resistance. In this study, with the structure of the SARS-CoV-2 Mpro complexed with a non-covalent inhibitor, we performed molecular dynamics (MD) simulations to determine the conformation of the complex when single amino acid residue in the active site is mutated. As a model of amino acid mutation, we constructed mutant proteins with one residue in the active site mutated to alanine. This method is called virtual alanine scan. The results of the MD simulations showed that the conformation and configuration of the ligand was changed for mutants H163A and E166A, although the structure of the whole protein and of the catalytic dyad did not change significantly, suggesting that mutations in His163 and Glu166 may be linked to drug resistance.

Molecular Mechanisms of Succinimide Formation from Aspartic Acid Residues Catalyzed by Two Water Molecules in the Aqueous Phase.

Aspartic acid (Asp) residues are prone to nonenzymatic isomerization via a succinimide (Suc) intermediate. The formation of isomerized Asp residues is considered to be associated with various age-related diseases, such as cataracts and Alzheimer's disease. In the present paper, we describe the reaction pathway of Suc residue formation from Asp residues catalyzed by two water molecules using the B3LYP/6-31+G(d,p) level of theory. Single-point energies were calculated using the MP2/6-311+G(d,p) level of theory. For these calculations, we used a model compound in which an Asp residue was capped with acetyl and methylamino groups on the N- and C-termini, respectively. In the aqueous phase, Suc residue formation from an Asp residue was roughly divided into three steps, namely, iminolization, cyclization, and dehydration, with the activation energy estimated to be 109 kJ mol-1. Some optimized geometries and reaction modes in the aqueous phase were observed that differed from those in the gas phase.

Deciphering Structural Alterations Associated with Activity Reductions of Genetic Polymorphisms in Cytochrome P450 2A6 Using Molecular Dynamics Simulations.

Cytochrome P450 (CYP) 2A6 is a monooxygenase involved in the metabolism of various endogenous and exogenous chemicals, such as nicotine and therapeutic drugs. The genetic polymorphisms in CYP2A6 are a cause of individual variation in smoking behavior and drug toxicities. The enzymatic activities of the allelic variants of CYP2A6 were analyzed in previous studies. However, the three-dimensional structures of the mutants were not investigated, and the mechanisms underlying activity reduction remain unknown. In this study, to investigate the structural changes involved in the reduction in enzymatic activities, we performed molecular dynamics simulations for ten allelic mutants of CYP2A6. For the calculated wild type structure, no significant structural changes were observed in comparison with the experimental structure. On the other hand, the mutations affected the interaction with heme, substrates, and the redox partner. In CYP2A6.44, a structural change in the substrate access channel was also observed. Those structural effects could explain the alteration of enzymatic activity caused by the mutations. The results of simulations provide useful information regarding the relationship between genotype and phenotype.

Development of Force Field Parameters for p-Carborane to Investigate the Structural Influence of Carborane Derivatives on Drug Targets by Complex Formation.

Androgen receptor (AR) has a key role in the development and progression of prostate cancer, and AR antagonists are used for its remedy. Recently, carborane derivatives, which are carbon-containing boron clusters have attracted attention as new AR ligands. Here we determined the force field parameters of 10-vertex and 12-vertex p-carborane to facilitate in silico drug design of boron clusters. Then, molecular dynamics (MD) simulations of complexes of AR-carborane derivatives were performed to evaluate the parameters and investigate the influences of carborane derivatives on the three-dimensional structure of AR. Energy profiles were obtained using quantum chemical calculations, and the force-field parameters were determined by curve fitting of the energy profiles. The results of MD simulations indicated that binding of the antagonist-BA341 changed some hydrogen-bond formations involved in the structure and location of helix 12. Those results were consistent with previously reported data. The determined parameters are also useful for refining the structure of the carborane-receptor complex obtained by docking simulations and development of new ligands with carborane cages not only for AR but also for various receptors.

Mechanisms of Deamidation of Asparagine Residues and Effects of Main-Chain Conformation on Activation Energy.

Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and by secondary structure. Information about main-chain conformation of Asn residues is necessary to accurately predict deamidation rate. In this study, the effect of main-chain conformation of Asn residues on deamidation rate was computationally investigated using molecular dynamics (MD) simulations and quantum chemical calculations. The results of MD simulations for γS-crystallin suggested that frequently deamidated Asn residues have common main-chain conformations on the N-terminal side. Based on the simulated structure, initial structures for the quantum chemical calculations were constructed and optimized geometries were obtained using the B3LYP density functional method. Structures that were frequently deamidated had a lower activation energy barrier than that of the little deamidated structure. We also showed that dihydrogen phosphate and bicarbonate ions are important catalysts for deamidation of Asn residues.

Computational Studies on Water-Catalyzed Mechanisms for Stereoinversion of Glutarimide Intermediates Formed from Glutamic Acid Residues in Aqueous Phase.

Aspartic acid (Asp) residues are prone to non-enzymatic stereoinversion, and Asp-residue stereoinversion is believed to be mediated via a succinimide (SI) intermediate. The stereoinverted Asp residues are believed to cause several age-related diseases. However, in peptides and proteins, few studies have reported the stereoinversion of glutamic acid (Glu) residues whose structures are similar to that of Asp. We previously presumed that Glu-residue stereoinversion proceeds via a glutarimide (GI) intermediate and showed that the calculated activation barriers of SI- and GI-intermediate stereoinversion are almost equivalent in the gas phase. In this study, we investigated the stereoinversion pathways of the l-GI intermediate in the aqueous phase using B3LYP density functional methods. The calculated activation barrier of l-GI-intermediate stereoinversion in the aqueous phase was approximately 36 kcal·mol-1, which was much higher than that in the gas phase. Additionally, as this activation barrier exceeded that of Asp-residue stereoinversion, it is presumed that Glu-residue stereoinversion has a lower probability of proceeding under physiological conditions than Asp-residue stereoinversion.

Influences of conformations of peptides on stereoinversions and/or isomerizations of aspartic acid residues.

Recently, non-enzymatic stereoinversions of aspartic acid (Asp) residues in proteins and peptides have been reported. Here, we performed replica exchange molecular dynamics (REMD) simulations of model peptides (exon 6, 26A-1, and 26A-2) extracted from elastin to investigate their structural features, thereby revealing the factor that influences stereoinversions. For REMD trajectories, we calculated distances between carboxyl carbon in Asp and amide nitrogen in the (n + 1) residue (CN distances). Because bond formation between carbon and nitrogen is indispensable to the formation of a succinimide intermediate the distance between them seems to play an important role in stereoinversion. Moreover, we calculated polar surface areas (PSAs) for the trajectories, finding that CN distances and PSA were different for each peptide, with the longest CN distance and smallest PSA observed for exon 6 peptide, where stereoinversion of Asp is the slowest. Although the average CN distance was shorter for exon 26A-1 peptide than for exon 26A-2 peptide, the number of conformations with CN distances <3.0 Šwas greater for exon 26A-2 peptide than for exon 26A-1 peptide. Furthermore, PSA for amide nitrogen of the (n + 1) residue was larger for exon 26A-2 peptide than for exon 26A-1 peptide. These results indicated that the flexibility of Asp and (n + 1) residues and hydrophilicity of peptides, especially in the (n + 1) residue, play important roles in the stereoinversion of Asp. This article is part of a Special Issue entitled: D-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.

Comparison of the activation energy barrier for succinimide formation from α- and ß-aspartic acid residues obtained from density functional theory calculations.

The l-α-Asp residues in peptides or proteins are prone to undergo nonenzymatic reactions to form l-ß-Asp, d-α-Asp, and d-ß-Asp residues via a succinimide five-membered ring intermediate. From these three types of isomerized aspartic acid residues, particularly d-ß-Asp has been widely detected in aging tissue. In this study, we computationally investigated the cyclization of α- and ß-Asp residues to form succinimide with dihydrogen phosphate ion as a catalyst (H2PO4-). We performed the study using B3LYP/6-31+G(d,p) density functional theory calculations. The comparison of the activation barriers of both residues is discussed. All the calculations were performed using model compounds in which an α/ß-Asp-Gly sequence is capped with acetyl and methylamino groups on the N- and C-termini, respectively. Moreover, H2PO4- catalyzes all the steps of the succinimide formation (cyclization-dehydration) acting as a proton-relay mediator. The calculated activation energy barriers for succinimide formation of α- and ß-Asp residues are 26.9 and 26.0kcalmol-1, respectively. Although it was experimentally confirmed that ß-Asp has higher stability than α-Asp, there was no clear difference between the activation barriers. Therefore, the higher stability of ß-Asp residue than α-Asp residue may be caused by an entropic effect associated with the succinimide formation.

Computational studies on the water-catalyzed stereoinversion mechanism of glutamic acid residues in peptides and proteins.

In contrast with the common belief that all the amino acid residues in higher organisms are l-forms, d-amino acid residues have been recently detected in various aging tissues. Aspartic acid (Asp) residues are known to be the most prone to stereoinvert via cyclic imide intermediate. Although the glutamic acid (Glu) is similar in chemical structure to Asp, little has been reported to detect d-Glu residues in human proteins. In this study, we investigated the mechanism of the Glu-residue stereoinversion catalyzed by water molecules using B3LYP/6-31+G(d,p) density functional theory calculations. We propose that the Glu-residue stereoinversion proceeds via a cyclic imide intermediate, i.e., glutarimide (GI). All calculations were performed by using a model compound in which a Glu residue was capped with acetyl and methylamino groups on the N- and C-termini, respectively. We found that two water molecules catalyze the three steps involved in the GI formation: iminolization, cyclization, and dehydration. The activation energy required for the Glu residue to form a GI intermediate was estimated to be 32.3 kcal mol-1 , which was higher than that of the experimental Asp-residue stereoinversion. This calculation result suggests that the Glu-residue stereoinversion is not favored under the physiological condition.

Validation of molecular force field parameters for peptides including isomerized amino acids.

Recently, stereoinversions and isomerizations of amino acid residues in the proteins of living beings have been observed. Because isomerized amino acids cause structural changes and denaturation of proteins, isomerizations of amino acid residues are suspected to cause age-related diseases. In this study, AMBER molecular force field parameters were tested by using computationally generated nonapeptides and tripeptides including stereoinverted and/or isomerized amino acid residues. Energy calculations by using density functional theory were also performed for comparison. Although the force field parameters were developed by parameter fitting for l-α-amino acids, the accuracy of the computational results for d-amino acids and ß-amino acids was comparable to those for l-α-amino acids. The conformational energies for tripeptides calculated by using density functional theory were reproduced more accurately than those for nonapeptides calculated by using the molecular mechanical force field. The evaluations were performed for the ff99SB, ff03, ff12SB, and the latest ff14SB force field parameters.

Validation of Molecular Dynamics Simulations for Prediction of Three-Dimensional Structures of Small Proteins.

Although various higher-order protein structure prediction methods have been developed, almost all of them were developed based on the three-dimensional (3D) structure information of known proteins. Here we predicted the short protein structures by molecular dynamics (MD) simulations in which only Newton's equations of motion were used and 3D structural information of known proteins was not required. To evaluate the ability of MD simulationto predict protein structures, we calculated seven short test protein (10-46 residues) in the denatured state and compared their predicted and experimental structures. The predicted structure for Trp-cage (20 residues) was close to the experimental structure by 200-ns MD simulation. For proteins shorter or longer than Trp-cage, root-mean square deviation values were larger than those for Trp-cage. However, secondary structures could be reproduced by MD simulations for proteins with 10-34 residues. Simulations by replica exchange MD were performed, but the results were similar to those from normal MD simulations. These results suggest that normal MD simulations can roughly predict short protein structures and 200-ns simulations are frequently sufficient for estimating the secondary structures of protein (approximately 20 residues). Structural prediction method using only fundamental physical laws are useful for investigating non-natural proteins, such as primitive proteins and artificial proteins for peptide-based drug delivery systems.