Roles of the respective loops at complementarity determining region on the antigen-antibody recognition

For the rational design of antibody, it is important to clarify the characteristics of the interaction between antigen and antibody. In this study, we evaluated a contribution of the respective complementarity determining region (CDR) loops on the antibody recognition of antigen by performing molecular dynamics simulations for 20 kinds of antigen-antibody complexes. Ser and Tyr showed high appearance rates at CDR loops and the sum of averaged appearance rates of Ser and Tyr was about 20⿿30% at all the loops. For example, Ser and Tyr occupied 23.9% at the light chain first loop (L1) and 23.6% at the heavy chain third loop (H3). The direct hydrogen bonds between antigen and antibody were not equally distributed over heavy and light chains. That is, about 70% of the hydrogen bonds were observed at CDRs of the heavy chain and also the direct hydrogen bond with the shortest distance mainly existed at the loops of the heavy chain for all the complexes. It was revealed from the comparison in contribution to the binding free energy among CDR loops that the heavy chain (especially at H2 and H3) had significant influence on the binding between antigen and antibody because three CDR loops of the heavy chain showed the lowest binding free energy (οG_bind) in 19 complexes out of 20. Tyr in heavy chain (especially in H2 and H3) largely contributed to οG_bind whereas Ser hardly contributed to οG_bind even if the number of the direct hydrogen bond with Ser was the fourth largest and also the appearance rate at CDR was the highest among 20 kinds of amino acid residues. The contributions ofTrp and Phe, which bear aromatic ring in the side chain, were often observed in the heavy chain although the energetic contribution of these residues was not so high as Tyr. The present computational analysis suggests that Tyr plays an outstanding role for the antigen-antibody interaction and the CDR loops of the heavy chain is critically important for antibody recognition of antigen.     

The Catalytic Mechanism of Fluoroacetate Dehalogenase: A Computational Exploration of Biological Dehalogenation

The biological dehalogenation of fluoroacetate carried out by fluoroacetate dehalogenase is discussed by using quantum mechanical/molecular mechanical (QM/MM) calculations for a whole‐enzyme model of 10 800 atoms. Substrate fluoroacetate is anchored by a hydrogen‐bonding network with water molecules and the surrounding amino acid residues of Arg105, Arg108, His149, Trp150, and Tyr212 in the active site in a similar way to haloalkane dehalogenase. Asp104 is likely to act as a nucleophile to attack the α‐carbon of fluoroacetate, resulting in the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule to the carbonyl carbon atom. The cleavage of the strong C? F bond is greatly facilitated by the hydrogen‐bonding interactions between the leaving fluorine atom and the three amino acid residues of His149, Trp150, and Tyr212. The hydrolysis of the ester intermediate is initiated by a proton transfer from the water molecule to His271 and by the simultaneous nucleophilic attack of the water molecule. The transition state and produced tetrahedral intermediate are stabilized by Asp128 and the oxyanion hole composed of Phe34 and Arg105.     

Investigation of Scrambled Ions in Tandem Mass Spectra. Part 1. Statistical Characterization

Scrambled ions have become the focus of recent investigations of peptide fragmentation. Here, an investigation of more than 390,000 high quality CID mass spectra is presented to explore the extent of scrambled ions in mass spectra and the possible fragmentation rules during scramble reactions. For the former, scrambled ions generally make up more than 10?% of mass spectra in number, although the abundances are less than 0.1 of the base peak. For the latter, relatively preferential re-opening sites were found for aliphatic residues Ala, Ile, Leu, and other residues such as Met, Gln, Ser, Phe, and Thr, whereas disfavored sites were found for basic residues Arg, Lys, and His, and Trp for both scrambled b and a ions. Similar preferential order in re-opening reaction was found in the reaction of losing internal residues when cleavage occurs at C-terminal side of 20 residues. However, when cleavage occurs at N-terminal side, Glu, Phe, and Trp become the most preferential sites. These results provide a deep insight into cleavage rules during scramble reactions for prediction of peptide mass spectra. Also, an additional investigation of whether scrambled ions could help discriminate false identifications from correct identifications was performed. Probing the number fraction of scrambled ions in falsely and correctly interpreted spectra and analyzing the correlation between scrambled ions and SEQUEST scores XCorr and Sp showed scrambled ions could at some extent help improve the discrimination in singly charged identifications, whereas no improvement was found for multiply charged results.     

Sequence composition and environment effects on residue fluctuations in protein structures

Structure fluctuations in proteins affect a broad range of cell phenomena, including stability of proteins and their fragments, allosteric transitions, and energy transfer. This study presents a statistical-thermodynamic analysis of relationship between the sequence composition and the distribution of residue fluctuations in protein-protein complexes. A one-node-per-residue elastic network model accounting for the nonhomogeneous protein mass distribution and the interatomic interactions through the renormalized inter-residue potential is developed. Two factors, a protein mass distribution and a residue environment, were found to determine the scale of residue fluctuations. Surface residues undergo larger fluctuations than core residues in agreement with experimental observations. Ranking residues over the normalized scale of fluctuations yields a distinct classification of amino acids into three groups: (i) highly fluctuating-Gly, Ala, Ser, Pro, and Asp, (ii) moderately fluctuating-Thr, Asn, Gln, Lys, Glu, Arg, Val, and Cys, and (iii) weakly fluctuating-Ile, Leu, Met, Phe, Tyr, Trp, and His. The structural instability in proteins possibly relates to the high content of the highly fluctuating residues and a deficiency of the weakly fluctuating residues in irregular secondary structure elements (loops), chameleon sequences, and disordered proteins. Strong correlation between residue fluctuations and the sequence composition of protein loops supports this hypothesis. Comparing fluctuations of binding site residues (interface residues) with other surface residues shows that, on average, the interface is more rigid than the rest of the protein surface and Gly, Ala, Ser, Cys, Leu, and Trp have a propensity to form more stable docking patches on the interface. The findings have broad implications for understanding mechanisms of protein association and stability of protein structures.     

Substrate specificity of fluoroacetate dehalogenase: an insight from crystallographic analysis, fluorescence spectroscopy, and theoretical computations

The high substrate specificity of fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with fluoroacetate was determined at 1.2 ? resolution. The orientation and conformation of bound fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild-type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the fluoroacetate- and chloroacetate-bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzyme-fluoroacetate complex. This is a stable but unreactive conformation, in which the small O-C-F angle is not suitable for the S(N)2 displacement of the F(-) ion. The cleavage of the C-F bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the C-O distance and the O-C-F angle are reasonable for the S(N) 2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer C-Cl bond; this results in an increase of the activation energy despite the weaker C-Cl bond.     

The recognition of nucleotides with model beta-hairpin receptors: investigation of critical contacts and nucleotide selectivity

[reaction: see text] We have investigated the factors that contribute to binding of ATP by a designed 12-residue beta-hairpin peptide, WKWK, and have determined its selectivity for binding to the naturally occurring nucleotide triphosphates. We have previously shown that WKWK creates an ATP binding pocket on one face of the beta-hairpin consisting of two Trp and two Lys residues. Mutation of the two Lys residues on the binding face of the beta-hairpin resulted in a lower affinity, indicating that each is involved in ATP binding and that each residue contributes approximately -1.5 kcal/mol to the energy of complexation. Replacement of either Trp residue of the ATP binding pocket with Phe or Leu destabilizes the complex formed with ATP by approximately 1 kcal/mol, indicating that both Trp residues participate in interactions with ATP. For binding to the nucleotide triphosphates, the order of binding affinity was shown to follow dTTP > GTP > ATP > CTP, with differences in binding energies spanning as much as 1.6 kcal/mol. NMR analysis demonstrates that both aromatic interactions with the Trp side chains and CH-pi interactions between the ribose protons and the Trp residues may contribute significantly to binding. The results from our model system provide useful thermodynamic information regarding protein-nucleic acid interactions that occur at the surface of a beta-sheet.     

Investigation of binding mechanism and downregulation of elacestrant for wild and L536S mutant estrogen receptor-α through molecular dynamics simulation and binding free energy analysis

The selective estrogen receptor downregulators (SERDs) are the new emerging class of drugs that are used for the treatment of endocrine resistance breast cancer. Elacestrant (ELA) is a new SERD, currently it is in phase II clinical trial. To understand the ELA–ERα interactions, the molecular docking analysis has been carried out. The ELA molecule binds with the helices H3, H5, H6, and H11 and forms important intermolecular interactions. In addition to this, the tetrahydronapthalene and phenyl rings of ELA are forming T-shaped π···π interactions with the Phe404 and Trp383 residues. Further to understand the stability and flexibility of ELA molecule in the active site of wild and mutated L536S ERα, 100ns molecular dynamics (MD) simulation was performed for both complexes. Interestingly, the MD analysis of wild complex revealed an interaction between ELA and the Asn532 of H11, which is an essential interaction for the downregulation/degradation of ERα, whereas this interaction is not observed in the mutated complex. The drug binding mechanism and H12 dynamics have been elucidated from the analysis of hydrogen bonding interactions and the secondary structure analysis. To explore the binding affinity of ELA molecule, the binding free energy and normal mode analyses were carried out. The per residue decomposition analysis also performed, which shows the contribution of individual amino acids. The principal component analysis and residue interaction network analysis were used to identify the modifications and the interaction between the residues. From the results of different analysis, the inhibition mechanism and downregulation of ERα–ELA complex has been investigated. © 2019 Wiley Periodicals, Inc.     

Three hydrophobic amino acids in <Emphasis Type="Italic">Escherichia coli</Emphasis> HscB make the greatest contribution to the stability of the HscB-IscU complex

Background  

General iron-sulfur cluster biosynthesis proceeds through assembly of a transient cluster on IscU followed by its transfer to a recipient apo-protein. The efficiency of the second step is increased by the presence of HscA and HscB, but the reason behind this is poorly understood. To shed light on the function of HscB, we began a study on the nature of its interaction with IscU. Our work suggested that the binding site of IscU is in the C-terminal domain of HscB, and two different triple alanine substitutions ([L92A, M93A, F153A] and [E97A, E100A, E104A]) involving predicted binding site residues had detrimental effects on this interaction. However, the individual contribution of each substitution to the observed effect remains to be determined as well as the possible involvement of other residues in the proposed binding site.     

Theoretical Design of Catalytic Domain of Abzyme Se-scFv2F3 by Introducing a Catalytic Triad

The single chain antibody scFv2F3 can be converted into selenium-containing Se-scFv2F3 by chemical mutation of the Ser residues. With antibody fragment 1NQB as a template, the catalytic domain of scFv2F3 was built by using homology modeling and molecular dynamics(MD) simulations. On the basis of the 3D model, we discussed the importance of Ser52 as the chemical modification site and redesigned the protein groups nearby Ser52 via introducing a catalytic triad. The following 10 ns MD results show that the des...     

The Methionine 549 and Leucine 552 Residues of Friedelin Synthase from Maytenus ilicifolia Are Important for Substrate Binding Specificity

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and β-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.     

Acyclic phenylalkanediols as substrates for the study of enzyme recognition. Regioselective acylation by porcine pancreatic lipase: a structural hypothesis for the enzymatic selectivity

The regioselective acylation of phenylalkanediols catalysed by porcine pancreatic lipase (PPL) was the reaction used for modelling different areas in the active site of the enzyme. With this aim, different racemic or prochiral (1,n)-diols, with n ranging from 2 to 6 were resolved via transesterification with vinyl acetate, and the results were explained according to microcrystalline enzyme structure. Thus, we describe a logical model for explaining the enzyme regio and stereoselectivity, based on three residues of the active site (Ser153, Phe216 and His264) which turned out to be crucial for the substrate binding and transformation.     

Tyr72 and Tyr80 are Involved in the Formation of an Active Site of a Luciferase of Copepod Metridia longa

Luciferase of copepod Metridia longa (MLuc) is a naturally secreted enzyme catalyzing the oxidative decarboxylation of coelenterazine with the emission of light. To date, three nonallelic isoforms of different lengths (17–24 kDa) for M. longa luciferase have been cloned. All the isoforms are single‐chain proteins consisting of a 17‐residue signal peptide for secretion, variable N‐terminal part and conservative C‐terminus responsible for luciferase activity. In contrast to other bioluminescent proteins containing a lot of aromatic residues which are frequently involved in light emission reaction, the C‐terminal part of MLuc contains only four Phe, two Tyr, one Trp and two His residues. To figure out whether Tyr residues influence bioluminescence, we constructed the mutants with substitution of Tyr to Phe (Y72F and Y80F). Tyrosine substitutions do not eliminate the ability of luciferase to bioluminescence albeit significantly reduce relative specific activity and change bioluminescence kinetics. In addition, the Tyr replacements have no effect on bioluminescence spectrum, thereby indicating that tyrosines are not involved in the emitter formation. However, as it was found that the intrinsic fluorescence caused by Tyr residues is quenched by a reaction substrate, coelenterazine, in concentration‐dependent manner, we infer that both tyrosine residues are located in the luciferase substrate‐binding cavity.     

Analysis of the activation mechanism of the guinea-pig Histamine H<Subscript>1</Subscript>-receptor

The Histamine H(1)-receptor (H1R), belonging to the amine receptor-class of family A of the G-protein coupled receptors (GPCRs) gets activated by agonists. The consequence is a conformational change of the receptor, which may involve the binding-pocket. So, for a good prediction of the binding-mode of an agonist, it is necessary to have knowledge about these conformational changes. Meanwhile some experimental data about the structural changes of GPCRs during activation exist. Based on homology modeling of the guinea-pig H1R (gpH1R), using the crystal structure of bovine rhodopsin as template, we performed several MD simulations with distance restraints in order to get an inactive and an active structure of the gpH1R. The calculations led to a Phe6.44/Trp6.48/Phe6.52-switch and linearization of the proline kinked transmembrane helix VI during receptor activation. Our calculations showed that the Trp6.48/Phe6.52-switch induces a conformational change in Phe6.44, which slides between transmembrane helices III and VI. Additionally we observed a hydrogen bond interaction of Ser3.39 with Asn7.45 in the inactive gpH1R, but because of a counterclockwise rotation of transmembrane helix III Ser3.39 establishes a water-mediated hydrogen bond to Asp2.50 in the active gpH1R. Additionally we simulated a possible mechanism for receptor activation with a modified LigPath-algorithm.     

Peptide Nanofibers Modified with a Protein by Using Designed Anchor Molecules Bearing Hydrophobic and Functional Moieties

Self‐assembly of peptides and proteins is a key feature of biological functions. Short amphiphilic peptides designed with a β‐sheet structure can form sophisticated nanofiber structures, and the fibers are available as nanomaterials for arranging biomolecules. Peptide FI (H‐PKFKIIEFEP‐OH) self‐assembles into nanofibers with a coiled fine structure, as reported in our previous work. We have constructed anchor molecules that have both a binding moiety for the fiber structure and a functional unit capable of capturing target molecules, with the purpose of arranging proteins on the designed peptide nanofibers. Designed anchors containing an alkyl chain as a binding unit and biotin as a functional moiety were found to bind to peptide fibers FI and F2i (H‐ALEAKFAAFEAKLA‐NH₂). The surface‐exposed biotin moiety on the fibers could capture an anti‐biotin antibody. Moreover, hydrophobic dipeptide anchor units composed of iminodiacetate connected to Phe–Phe or Ile–Ile and a peptide composed of six histidine residues connected to biotin could also connect FI peptide fibers to the anti‐biotin antibody through the chelation of Ni²⁺ ions. This strategy of using designed anchors opens a novel approach to constructing nanoscale protein arrays on peptide nanomaterials.     

Adsorption and photocatalytic decomposition of amino acids in TiO2 photocatalytic systems

The adsorption and photodecomposition of seven kinds of amino acids on a TiO2 surface were investigated by zeta potential measurements and 1H NMR spectroscopy in TiO2 aqueous suspension systems. The decomposition rates increased in the order of Phe < Ala < Asp < Trp < Asn < His < Ser. For Phe, Trp, Asn, His, and Ser, the isoelectric point (IEP) of TiO2 shifted to a lower pH with increasing decomposition rates upon adsorption on TiO2, suggesting that the effective adsorption and photocatalytic sites for these amino acids should be the basic terminal OH on the solid surface. Since the amino acids that decomposed faster than the others contain -OH (Ser), -NH (Trp, His), or -NH2 (Asn) in their side chain, they are considered to interact with the basic terminal OH groups more preferably by the side chain and are vulnerable to photocatalytic oxidation. On the other hand, Ala interacts with the acidic bridged OH on TiO2 to cause an IEP shift to a higher pH. The correlation of the surface hydroxyl groups with the photocatalysis of amino acids was verified by the use of calcined TiO2 without surface hydroxyl groups.     

Exploring the Role of C–H….π Interactions on the Structural Stability of Single Chain “All-Alpha” Proteins

C–H….π interactions are known to be important contributors to protein stability. In this study, we have analyzed the influence of C–H….π interactions in single chain “all-alpha” proteins. In the data set, a total of 181 C–H….π interactions were observed. The most prominent representatives are the interactions between aromatic C–H donor groups and aromatic π acceptors. Eighty-one percent of the C–H….π interactions between side chain to side chain and remaining19% of the C–H….π interactions were observed between side-chain to side-chain five-member aromatic ring. The donor atom contribution to C–H….π interactions was mainly from Phe, Tyr, and Trp residues. The acceptor atom contribution to C–H….π interactions was mainly from Phe, Tyr, Trp, and His. The highest percentage of C–H….π interactions were observed form Phe residue. The secondary structure preference analysis of all C–H….π interacting residues showed that Phe, Tyr, Trp, and His preferred to be in helix. Long-range C–H….π interactions are the predominant type of interactions in single chain all-alpha proteins data set. All the C–H….π interactions forming residues in the data set preferred to be in the buried region. Seventy-three percent of the donor residues and 65% of the acceptor residues are highly conserved.     

Origin of selectivity in the antibody 20F10-catalyzed Yang cyclization

The first antibody-catalyzed Yang (Norrish type II) cyclization has been achieved with antibodies that were elicited against cis- and trans-2,3-diaryloxetanes. The photocyclization of 1,4-diarylbutan-1-one produced a single stereoisomer of cis-1,2-diarylcyclobutanol with very high enantioselectivity. The background photochemical reaction in the absence of the antibody yielded only fragmentation products. The antibody 20F10-catalyzed reaction was studied in detail, exploring its selectivity, substituent effects, substrate and hapten binding, kinetic parameters and irradiation wavelength dependence. Quantum mechanical calculations suggest that the activation enthalpy of fragmentation pathway is favored by 7.9 kcal/mol over cyclization pathway. Hapten, substrate, and transition state docking studies on a homology based modeled antibody binding site indicate that the trans hapten, substrate and the cyclization transition state have similar binding modes. By contrast, the fragmentation transition state is bound in a different way, not easily accessible within the lifetime of the bound substrate excited state. Several side chain residues were identified that can act as local sensitizers to enhance the cyclization process.     

Steric factors override thermodynamic driving force in regioselectivity of proline hydroxylation by prolyl-4-hydroxylase enzymes

Prolyl-4-hydroxylase is an important nonheme iron-containing dioxygenase in humans involved in the regioselective hydroxylation of a proline residue in a peptide chain on the C(4) position. In biosystems this process is important to create collagen cross-linking and cellular responses to hypoxia. We have performed a series of density functional theory (DFT) studies into the origin of the regioselectivity of proline hydroxylation by P4H enzymes using a minimal active site model (where substrate is unhindered in the binding site) and a larger active site model that incorporates steric hindrance of the substrate by several secondary sphere aromatic residues. Our studies show that thermodynamically the most favorable hydrogen atom abstraction position of proline is from the C(5) position; hence, the small model gives a low reaction barrier and large exothermicity for this process. However, stereochemical repulsions of the substrate with aromatic residues of Tyr(140) and Trp(243) in the second coordination sphere prevent C(5) hydroxylation and make C(4) hydroxylation the dominant mechanism, despite a lesser driving force for the reaction. These studies explain the remarkable regioselectivity of proline hydroxylation by P4H enzymes and show that the regioselectivity is kinetically controlled but not thermodynamically. In addition, we calculated spectroscopic parameters and found good agreement with experimental data.     

Simultaneous analysis of photoinduced electron transfer in wild type and mutated AppAs

Photoinduced electron transfer (PET) from Tyr21 to isoalloxazine (Iso) in the excited state (Iso*) is considered to be an initial step of the photosensing function of the blue-light sensing using flavin adenine dinucleotide (BLUF) component of the anti-repressor of the photosynthetic regulation (AppA). The PET mechanism was investigated via fluorescence dynamics of AppA and Kakitani and Mataga (KM) theories as well as by molecular dynamic (MD) simulation. The local structures of both the Y21F and W104F mutant AppAs around the Iso binding sites were quite different from those of the wild type (WT) AppA. The distances between Iso and Trp104 in Y21F, and between Iso and Tyr21 in W104F were shorter by 0.06 nm and 0.02 nm, respectively, compared to the WT. The frequency factor, ν₀, in Tyr21 was 1.15-fold greater than that in Trp104. The critical distance between adiabatic and non-adiabatic PET processes, R₀, was found to be very long in the AppA Tyr21. The large values of ν₀ and R₀ for Tyr21 of AppA compared to those in a non photosensing flavoprotein, FMN binding protein (FBP), were elucidated by hydrogen bond (H bond) chain between Tyr21 and Iso through Gln63. Interaction energies among Iso*, Trp104, Tyr21 and Gln63 in WT were calculated using the semi-empirical PM3 method. The amount of the transferred charge from Trp104 to Iso* in the WT exhibited a maximum at an interaction energy of around ?20 kcal/mol, but decreased as the interaction energy (absolute value) increased.     

Conformational transition pathway of polymerase beta/DNA upon binding correct incoming substrate

The closing conformational transition of wild-type polymerase beta bound to DNA template/primer before the chemical step (nucleotidyl transfer reaction) is simulated using the stochastic difference equation (in length version, "SDEL") algorithm that approximates long-time dynamics. The order of the events and the intermediate states during pol beta's closing pathway are identified and compared to a separate study of pol beta using transition path sampling (TPS) (Radhakrishnan, R.; Schlick, T. Proc. Natl. Acad. Sci. USA 2004, 101, 5970-5975). Results highlight the cooperative and subtle conformational changes in the pol beta active site upon binding the correct substrate that may help explain DNA replication and repair fidelity. These changes involve key residues that differentiate the open from the closed conformation (Asp192, Arg258, Phe272), as well as residues contacting the DNA template/primer strand near the active site (Tyr271, Arg283, Thr292, Tyr296) and residues contacting the beta and gamma phosphates of the incoming nucleotide (Ser180, Arg183, Gly189). This study compliments experimental observations by providing detailed atomistic views of the intermediates along the polymerase closing pathway and by suggesting additional key residues that regulate events prior to or during the chemical reaction. We also show general agreement between two sampling methods (the stochastic difference equation and transition path sampling) and identify methodological challenges involved in the former method relevant to large-scale biomolecular applications. Specifically, SDEL is very quick relative to TPS for obtaining an approximate path of medium resolution and providing qualitative information on the sequence of events; however, associated free energies are likely very costly to obtain because this will require both successful further refinement of the path segments close to the bottlenecks and large computational time.