Major Transition State Research Articles

The "weak" C-H···S interaction drives enantioselectivity in cinchona alkaloid complex catalyzed thiocyanation.

The great success of asymmetric organocatalysis has made it one of the most important advancements made in chemistry in the past two decades. A significant achievement in this context is the asymmetric organocatalysis of the thiocyanation reaction. In the current study, computational studies with density functional theory have been done in order to understand an interesting experimental finding: the reversal of enantioselectivity from R to S when the electrophile is changed from b-keto ester to oxindole for the thiocyanation reaction with the cinchona alkaloid complex catalyst. The calculations reveal an unusual fact - the principal reason for the reversal is the presence of the C-H···S noncovalent interaction, which is present only in the major transition states in each of the two nucleophile cases. Only recently has it been realized that the supposedly weak C-H···S noncovalent interaction has the properties of a hydrogen bond, and the fact that this interaction is the cause of enantioselectivity has relevance, because of the large number of asymmetric transformations involving the sulphur heteroatom.

Receptor Dynamics in Molecular Recognition by Cryo-EM and Molecular Simulation.

The appropriate selection of initial receptor structure has been the "cornerstone" or foundation of successful structure-based virtual screening (SBVS), and plagued the structure-based design with a significant practical problem to determine the major physiological states or important transition states of receptors (e.g. proteins with multiple low-energy conformations and liganddependent conformational dynamics). It is well known that current SBVS methods lack the capacity to capture and characterize the intrinsic receptor flexibility with ideal cost-effectiveness. In recent years, cryoelectron microscopy (cryo-EM) has been routinely applied in the determination of biomolecular assemblies within the physiological state. In this work, we review the roles of cryo-EM and ensemble docking methods to present the intrinsically dynamic behavior of biomacromolecules, as well as the ever-improving estimation of ligand binding affinities and receptor-ligand thermodynamics. Finally, we also provide a viewpoint for further research works on modeling receptor dynamics.

Mechanistic Insights into a Chiral Phosphoric Acid-Catalyzed Asymmetric Pinacol Rearrangement.

The first catalytic enantioselective pinacol rearrangement was reported by Antilla and co-workers in 2010. The reaction was catalyzed by a chiral phosphoric acid and resulted in high levels of enantioselectivity (up to 96% ee). The present study uses density functional theory to investigate the mechanism and origins of stereoselectivity of this important reaction and to explain the difference in selectivity between different catalysts. An OH···O hydrogen bond between the intermediate indolyl alcohol and the phosphate group from the catalyst together with a CH···O hydrogen bond between the indole and the phosphate group were observed in the preferred activation mode for the stereodetermining [1,2]-aryl shift. A stronger CH···O interaction in the major transition state was found to contribute to the high levels of enantioselectivity. A more bulky catalyst (TRIP) was found to impede the formation of the key CH···O interaction, leading to lower levels of enantioselectivity.

Cinchona Alkaloid-Squaramide Catalyzed Sulfa-Michael Addition Reaction: Mode of Bifunctional Activation and Origin of Stereoinduction.

The mechanism of the enantioselective sulfa-Michael addition reaction catalyzed by a cinchona alkaloid-squaramide bifunctional organocatalyst was studied using density functional theory (DFT). Four possible modes of dual activation mechanism via hydrogen bonds were considered. Our study showed that Houk's bifunctional Brønsted acid-hydrogen bonding model, which works for cinchonidine or cinchona alkaloid-urea catalyzed sulfa-Michael addition reactions, also applies to the catalytic system under investigation. In addition, we examined the origin of the stereoselectivity by identifying stereocontrolling transition states. Distortion-interaction analysis revealed that attractive interaction between the substrates and catalyst in the C-S bond forming transition state is the key reason for stereoinduction in this catalytic reaction. Noncovalent interaction (NCI) analysis showed that a series of more favorable cooperative noncovalent interactions, namely, hydrogen bond, π-stacking, and C-H···π interaction and C-H···F interactions, in the major R-inducing transition state. The predicted enantiometric excess is in good accord with the observed value.

Human DNMT1 transition state structure.

Human DNA methyltransferase 1 (DNMT1) maintains the epigenetic state of DNA by replicating CpG methylation signatures from parent to daughter strands, producing heritable methylation patterns through cell divisions. The proposed catalytic mechanism of DNMT1 involves nucleophilic attack of Cys(1226) to cytosine (Cyt) C6, methyl transfer from S-adenosyl-l-methionine (SAM) to Cyt C5, and proton abstraction from C5 to form methylated CpG in DNA. Here, we report the subangstrom geometric and electrostatic structure of the major transition state (TS) of the reaction catalyzed by human DNMT1. Experimental kinetic isotope effects were used to guide quantum mechanical calculations to solve the TS structure. Methyl transfer occurs after Cys(1226) attack to Cyt C6, and the methyl transfer step is chemically rate-limiting for DNMT1. Electrostatic potential maps were compared for the TS and ground states, providing the electronic basis for interactions between the protein and reactants at the TS. Understanding the TS of DNMT1 demonstrates the possibility of using similar analysis to gain subangstrom geometric insight into the complex reactions of epigenetic modifications.

Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945A strains

Bacterial natural genetic competence - well studied in Bacillus subtilis - enables cells to take up and integrate extracellularly supplied DNA into their own genome. However, little is known about competence development and its regulation in other members of the genus, although DNA uptake machineries are routinely encoded. Auxotrophic Bacillus licheniformis 9945A derivatives, obtained from repeated rounds of random mutagenesis, were long known to develop natural competence. Inspection of the colony morphology and extracellular enzyme secretion of two of these derivatives, M28 and M18, suggested that regulator genes are collaterally hit. M28 emerged as a 14 bp deletion mutant concomitantly displaying a shift in the reading frame of degS that encodes the sensor histidine kinase, which is part of the molecular switch that directs cells to genetic competence, the synthesis of extracellular enzymes or biofilm formation, while for M18, sequencing of the suspected gene revealed a 375 bp deletion in abrB, encoding the major transition state regulator. With respect to colony morphology, enzyme secretion and competence development, both of the mutations, when newly generated on the wild-type B. licheniformis 9945A genetic background, resulted in phenotypes resembling M28 and M18, respectively. All of the known naturally competent B. licheniformis representatives, hitherto thoroughly investigated in this regard, carry mutations in regulator genes, and hence genetic competence observed in domesticated strains supposedly results from deregulation.

Evidence That Gene Activation and Silencing during Stem Cell Differentiation Requires a Transcriptionally Paused Intermediate State

A surprising portion of both mammalian and Drosophila genomes are transcriptionally paused, undergoing initiation without elongation. We tested the hypothesis that transcriptional pausing is an obligate transition state between definitive activation and silencing as human embryonic stem cells (hESCs) change state from pluripotency to mesoderm. Chromatin immunoprecipitation for trimethyl lysine 4 on histone H3 (ChIP-Chip) was used to analyze transcriptional initiation, and 3′ transcript arrays were used to determine transcript elongation. Pluripotent and mesodermal cells had equivalent fractions of the genome in active and paused transcriptional states (∼48% each), with ∼4% definitively silenced (neither initiation nor elongation). Differentiation to mesoderm changed the transcriptional state of 12% of the genome, with roughly equal numbers of genes moving toward activation or silencing. Interestingly, almost all loci (98–99%) changing transcriptional state do so either by entering or exiting the paused state. A majority of these transitions involve either loss of initiation, as genes specifying alternate lineages are archived, or gain of initiation, in anticipation of future full-length expression. The addition of chromatin dynamics permitted much earlier predictions of final cell fate compared to sole use of conventional transcript arrays. These findings indicate that the paused state may be the major transition state for genes changing expression during differentiation, and implicate control of transcriptional elongation as a key checkpoint in lineage specification.

Converting structural information into an allosteric-energy-based picture for elongation factor Tu activation by the ribosome

The crucial process of aminoacyl-tRNA delivery to the ribosome is energized by the GTPase reaction of the elongation factor Tu (EF-Tu). Advances in the elucidation of the structure of the EF-Tu/ribosome complex provide the rare opportunity of gaining a detailed understanding of the activation process of this system. Here, we use quantitative simulation approaches and reproduce the energetics of the GTPase reaction of EF-Tu with and without the ribosome and with several key mutants. Our study provides a novel insight into the activation process. It is found that the critical H84 residue is not likely to behave as a general base but rather contributes to an allosteric effect, which includes a major transition state stabilization by the electrostatic effect of the P loop and other regions of the protein. Our findings have general relevance to GTPase activation, including the processes that control signal transduction.

Partially Folded States of Mutant Ubiquitin in Mild Denaturing Conditions

Conformational change of ubiquitin variant with valine to alanine mutation at sequence position 26 was studied by varying solvent pH. Fluorescence emission spectra indicated that this variant ubiquitin has some residual structures in acidic and basic solution as compared to denaturant-induced unfolded state. Far-UV circular dichroic spectra indicated that the base-denatured state had more secondary structure than the acid-denatured state. Near-UV circular dichroic spectra indicated that the aromatic side-chains were in the relatively more rigid environment in the base-denatured state than those in the acid-denatured state. Although it appears that the more tertiary structure present in the base-denatured state, refolding reactions measured by stopped-flow fluorescence device suggest that both the acid- and base-denatured states occur before the major folding transition state. The acid- and base-denatured states are considered to reflect the early event of protein folding process.

Reaction Mechanisms of C2Cl3 + NO2 via Nitro and Nitrite Adducts

The atmospherically and environmentally important reaction of chlorinated vinyl radical with nitrogen dioxide (C 2Cl 3 + NO 2) is investigated by step-scan time-resolved Fourier transform infrared emission spectroscopy and electronic structure calculations. Vibrationally excited products of CO, NO, Cl 2CO, and NO 2 are observed in the IR emission spectra. Geometries of the major intermediates and transition states along the potential energy surface are optimized at the B3LYP/6-311G(d) level, and their energies are refined at the CCSD(T)/6-311+G(d) level. The reaction mechanisms are characterized to be barrierless addition-elimination via nitro (C 2Cl 3-NO 2) and nitrite (C 2Cl 3-ONO) adducts. Four energetically accessible reaction routes are revealed, i.e., the decomposition of the nitrite adduct forming C 2Cl 3O + NO and its sequential dissociation to CO + NO + CCl 3, the elimination of ClNO from the nitrite adduct leading to ClNO + Cl 2CCO, the Cl-atom shift of the nitrite adduct followed by the decomposition to CCl 3CO + NO, and the O-atom shift of the nitro adduct followed by C-C bond cleavage forming ClCNO + Cl 2CO. In competition with these reactive fluxes, the back-decomposition of nitro or nitrite adducts leads to the prompt formation of vibrationally excited NO 2 and the long-lived reaction adducts facilitate the vibrational energy transfer. Moreover, the product channels and mechanisms of the C 2Cl 3 + NO 2 reaction are compared with the C 2H 3 + NO 2 reaction to explore the effect of chlorine substitution. It is found that the two reactions mainly differ in the initial addition preferentially by the N-attack forming nitro adducts (only N-attack is plausible for the C 2H 3 + NO 2 reaction) or the O-attack forming nitrite adducts (O-attack is slightly more favorable and N-attack is also plausible for the C 2Cl 3 + NO 2 reaction). The addition selectivity can be fundamentally correlated to the variation of the charge density of the end carbon atom of the double bond induced by chlorine substitution due to the electron-withdrawing effect of chlorine groups.

Origin of Stereoselectivity in the Reduction of a Planar Oxacarbenium

The Kishi reduction of a planar oxacarbenium was investigated theoretically. The high diastereoselectivity for hydride transfer to the oxacarbenium intermediate is attributed to the conformation of the transition state that places the allyl side chain in an equatorial position in the major transition state and axial position in the minor. The minor transition state is destabilized by a 1,3-diaxial strain between the attacking hydride and the syn allyl side chain.

How general is the nucleation-condensation mechanism?

We investigate the structures of the major folding transition states of nine proteins by correlation of published Phi-values with inter-residue contact maps. Combined with previous studies on six proteins, the analysis suggests that at least 10 of the 15 small globular proteins fold via a nucleation-condensation mechanism with a concurrent build-up of secondary and tertiary structure contacts, but a structural consolidation that is clearly nonuniformly distributed over the molecule and most intense in a single structural region suggesting the occurrence of a single folding nucleus. However, on average helix- and sheet-forming residues show somewhat larger Phi-values in the major transition state, suggesting that secondary structure formation is one important driving force in the nucleation-condensation in many proteins and that secondary-structure forming residues tend to be more prominent in folding nuclei. We synthesize the combined information on these 10 of 15 proteins into a unified nucleation-condensation mechanism which also accounts for effects described by the framework, hydrophobic collapse, zipper, and funnel models.

Improved Stability of the Jun-Fos Activator Protein-1 Coiled Coil Motif: A STOPPED-FLOW CIRCULAR DICHROISM KINETIC ANALYSIS

Two c-Jun leucine zipper variants that bind with high affinity to c-Fos have been selected using semirational design combined with protein-fragment complementation assays (JunW) or phage display selection (JunW(Ph1)). Enriched winners differ from each other in only two of ten semi-randomized positions, with DeltaT(m) values of 28 degrees C and 37 degrees C over wild-type. cFos-JunW, cFos-JunW(Ph1), and two intermediate mutants (cFos-JunW(Q21R) and cFos-JunW(E23K)) display biphasic kinetics in the folding direction, indicating the existence of a folding intermediate. The first reaction phase is fast and concentration-dependent, showing that the intermediate is readily populated and dimeric. The second phase is independent of concentration and is exponential. In contrast, in the unfolding direction, all molecules display two-state kinetics. Collectively this implies a transition state between unfolded helices and dimeric intermediate that is readily traversed in both directions. We demonstrate that the added stability of cFos-JunW(Ph1) relative to cFos-JunW is achieved via a combination of kinetic rate changes; cFos-JunW(E23K) has an increased initial dimerization rate, prior to the major transition state barrier while cFos-JunW(Q21R) displays a decreased unfolding rate. The former implies that improved hydrophobic burial and helix-stabilizing mutations exert their effect on the initial, rapid, monomer-collision event. In contrast, electrostatic interactions exert their effect late in the folding pathway. Although our focus is the leucine zipper region of the oncogenic transcription factor Activator Protein-1, coiled coils are ubiquitous and highly specific in their recognition of partners. Consequently, generating kinetic-based rules to predict and engineer their stability is of major significance in peptide-based drug design and nano-biotechnology.

The Structure of the Major Transition State for Folding of an FF Domain from Experiment and Simulation

We have analysed the transition state of folding of the four-helix FF domain from HYPA/FBP11 by high-resolution experiment and simulation as part of a continuing effort to understand the principles of folding and the refinement of predictive methods. The major transition state for folding was subjected to a Phi-value analysis utilising 50 mutants. The transition state contained a nucleus for folding centred around the end of helix 1 (H1) and the beginning of helix 2 (H2). Secondary structure in this region was fully formed (PhiF=0.9-1) and tertiary interactions were well developed. Interactions in the distal part of the native structure were weak (PhiF=0-0.2). The hydrophobic core and other parts of the protein displayed intermediate Phi-values, suggesting that interactions coalesce as the end of H1 and beginning of H2 are in the process of being formed. The distribution of Phi-values resembled that of barnase, which folds via an intermediate, rather than that of CI2 which folds by a concerted nucleation-condensation mechanism. The overall picture of the transition state structure identified in molecular dynamics simulations is in qualitative agreement, with the turn connecting H1 and H2 being formed, a loosened core, and H4 partially unfolded and detached from the core. There are some differences in the details and interpretation of specific Phi-values.

A Computational Analysis of the Ring-Opening Polymerization of rac-Lactide Initiated by Single-Site β-Diketiminate Metal Complexes: Defining the Mechanistic Pathway and the Origin of Stereocontrol

The ring-opening polymerization of rac-lactide at a beta-diketiminate magnesium center, [HC{CMeN-2,6-(i)Pr(2)C(6)H(3)}(2)]Mg(OMe)(THF), has been investigated using a B3-LYP density functional procedure employing three different layers of basis set: 6-311G(3d) at the Mg center, 6-31G(d) for both the ligand skeleton and the monomer, and a STO-3G basis set at the bulky 2,6-diisopropylphenyl substituents. By studying the consecutive ring-opening of two lactide molecules, clear conclusions are drawn regarding both the mechanism of ring-opening and the origin of heterotactic stereocontrol observed with such initiators. Polymerization proceeds via two major transition states, an observation applicable to other coordinative initiator systems, with the highest energy transition state dictating the stereochemistry of monomer insertion. In the beta-diketiminate magnesium system, a detailed examination of the rate-limiting second transition state geometries reveals that heterotactic poly(lactic acid) arises via the minimization of several steric interactions, possibly reinforced by an attractive CH...pi interaction.

PII: S0969-2126(01)00700-6

PII: S0969-2126(01)00700-6

Mutational analysis of hydrogen bonding residues in the BPTI folding pathway

Nine BPTI variants with replacements that remove one or more hydrogen bonds from the native protein were constructed, and the folding pathways of these proteins were determined by isolating and identifying the disulfide-bonded intermediates that accumulated during unfolding and refolding. The forward and reverse rate constants for the individual steps in the folding pathways for each protein were measured, providing a detailed description of the energetic effects of the substitutions. The native forms of eight of the nine variants were measurably destabilized, by 1–7 kcal/mol (1 cal = 4.184 J), with an average effect of 1.6 kcal/mol per hydrogen bond removed. The folding pathways for the variants were found to be similar to that previously described for the wild-type protein, with the kinetically preferred mechanism involving intramolecular rearrangements of intermediates with two disulfide bonds. Some of the substitutions, however, significantly destabilized the major intermediates and broadened the distribution of species with one or two disulfide bonds, thus identifying residues that play important roles in stabilizing the normal intermediates and defining specificity in the folding process. The kinetic data also suggest that one residue, Asn43, may play a distinctive role in defining the BPTI folding mechanism. Replacement of this residue with either Gly or Ala appeared to stabilize the major transition states for folding and unfolding. In the native protein, the side-chain of Asn43 participates directly in the hydrogen bonding pattern of the central β-sheet, and the kinetic behavior of the Asn43 variants suggests that the major energy barriers in folding and unfolding may be due in part to the steric constraints imposed by this structural element, together with those imposed by the chemical transition states for thiol-disulfide exchange.

The major transition state in folding need not involve the immobilization of side chains.

During protein folding in which few, if any, definable kinetic intermediates are observable, the nature of the transition state is central to understanding the course of the reaction. Current experimental data does not distinguish the relative contributions of side chain immobilization and dehydration phenomena to the major rate-limiting transition state whereas this distinction is central to theoretical models that attempt to simulate the behavior of proteins during folding. Renaturation of the small proteinase inhibitor cystatin under oxidizing versus reducing conditions is the first experimental case in which these processes can be studied independently. Using this example, we show that sidechain immobilization occurs downstream of the major folding transition state. A consequence of this is the existence of states with disordered side chains, which are distinct from kinetic protein folding intermediates and which lie within the folded state free energy well.

Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the β(3–4) hairpin contained a particularly high degree of such side-chain interactions but it lacked β-structure in two of the three denatured ensembles: β(3–4) was the only portion of the β-structure to contain significant residual structure in the denatured state. The two principal α-helices (α1 and α2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in α1 and α2, as well as fluctuating tertiary contacts in the β(3–4) region, and between α1 and β(3–4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between β(3–4) and α1 assisted in the refolding of α1. This contact-assisted helix formation was confirmed in fragment simulations of β(3–4) and α1 alone and complexed, and, as such, α1 and β(3–4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both β(3–4) and α1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further β-strands pack. The β-structure condenses about β(3–4), while α1 aids in stabilizing β(3–4) and maintaining its orientation. The resulting β-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the β-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.

Analysis methods for comparison of multiple molecular dynamics trajectories: applications to protein unfolding pathways and denatured ensembles

In molecular dynamics simulations of protein unfolding, the pathway of one protein molecule is studied at a time. In contrast, experimental denaturation studies sample from large ensembles of molecules passing from the native to unfolded state. If reasonable comparisons with experiment are to be made, then the generality of the simulations needs to be confirmed by performing multiple unfolding simulations. Given that protein unfolding trajectories are very complicated functions of the proteins and the environment, comparing different trajectories, even under the same conditions, is not straightforward. Several methods are presented here that attempt to accomplish this task at different levels of complexity. The simpler methods are geometry based and make use of the root-mean-squared deviations between structures, while the more complicated methods are based on the time variation of the various properties of the system during the unfolding process. These methods are applied to multiple simulations of three different proteins, bovine pancreatic trypsin inhibitor, chymotrypsin inhibitor 2, and barnase. In general, for these three proteins protein unfolding proceeded via expansion of the core and fraying of secondary structure to yield the major transition state. Once past the transition state, the trajectories for a given protein diverged as the protein lost further secondary and tertiary structure by a variety of mechanisms. Although the unfolding pathways diverged, similar conformations were populated in the denatured state even when the unfolding occurred via different pathways. The multitude of different pathways leading to the denatured state agrees with the funnel description of protein folding. Although the pathways differed in conformational space, the physical properties of the conformations were often similar, highlighting the danger of assuming that similar observed properties imply similar conformations. In fact, there may be many different “conformational pathways” of unfolding that fit within a preferred “property space pathway”.